Novel Nanomaterials for Protein Analysis

CHAPTER

2

NOVEL NANOMATERIALS FOR PROTEIN ANALYSIS

Pengyuan Yang*, Liming Wei*,† Key Laboratory of Medical Epigenetics and Metabolism, Institutes of Biomedical Sciences & Department of Chemistry & The Fifth People’s Hospital of Shanghai, Fudan University, Shanghai, China* Shanghai Songjiang District Central Hospital, Shanghai, China†

INTRODUCTION As the product of gene transcription in biology, protein has become an important role due to its pertinence to functional genomics. The analysis of protein is focused on the identification and quantification to unravel protein structures, functions, localizations, interactions, and posttranslational modifications in the complicated samples. With the development of novel nanomaterials, a category of powerful platforms was established for protein analysis. Here, we will summarize recent progresses in the application of novel nanomaterials for protein analysis.

PROTEIN ANALYSIS As the central cellular components in biological networks, proteins play an important role in diverse biological functions including cytoskeletal building blocks, biochemical reactions, enzymatic catalysis, immune response, or transcription factors affecting gene expression [1–5]. Protein has become a key factor to explore the biological sciences and a source of potential biomarkers and drug targets [6–8]. Therefore, protein analysis at a system level has come into existence to narrow the existing gaps between discovery sciences and the associated clinical applications. Proteomics has attracted considerable interest in discerning the global biological processes. Originally put forwarded in 1995 by Marc Wilkins, proteomics is the science that explores the dynamic of proteins in response to physiological, pathological, and environmental influences [9]. The first dimension structure of protein is its amino acid sequence, providing a link between proteins and their coding genes via the genetic code [10, 11]. The divergence of protein expression level in different phenotypes might support the discovery of important regulator factors, potential biomarkers, biological mechanisms, and even drug targets. Moreover, the spatial partition of proteins at a subcellular level provides information regarding protein function, interactions, and cellular mechanisms [12]. Furthermore, as an essential cellular regulatory mechanism, posttranslational modifications (PTMs) can alter the activities of protein targets [13], and the cumulative effect of PTMs in biology can regulate the large signaling pathways and networks in cell. Finally, the activities and functions of proteins are further modulated by Novel Nanomaterials for Biomedical, Environmental and Energy Applications. https://doi.org/10.1016/B978-0-12-814497-8.00002-3 Copyright # 2019 Elsevier Inc. All rights reserved.

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CHAPTER 2 NOVEL NANOMATERIALS FOR PROTEIN ANALYSIS

interacting proteins in metabolic and signaling pathways [14]. Consequently, proteomics aims to characterizing the whole proteome profile and further explore the biological mechanism and clinical applications.

CHALLENGES IN PROTEIN ANALYSIS With the rapidly growing field of protein analysis, proteomics has embedded in several fields in biology, including disease pathogenesis, biomarker discovery, and pharmacology of drugs. However, the analysis of whole proteins in cell or biology species presents several challenges. First, the large-scale protein analysis of proteome is more complicated. Due to the alternative splicing, mutation, and integral chemical modifications of mRNA used for protein expression, highly complex proteins are expressed in biology samples [15, 16]. As a result, it is estimated that a single gene can generate up to 10–20 proteoforms [17]. Additionally, there are approximately 430 PTMs that contribute to the complexity of proteins [18]. Second, the high dynamic range difference of expressed proteins hampers the detection of low-abundance protein. Due to responses to activation, turnover, downregulation, conformation, or localization, proteins are expressed in different abundance in biological samples. In a blood sample, more than 10,000 different proteins are expressed with a dynamic range of nine orders of magnitude. Consequently, proteins at concentrations of 10
12–10
15 M usually coexist with those at concentrations of 10
3–10
4 M, making detection of low-abundance proteins extremely difficult [19]. Meanwhile, compared with gene analysis with tools such as polymerase chain reaction (PCR) for the amplification of a single nucleic acid molecule in a complex biological sample up to the limit of detection by modern instruments, detection of ultralow-abundance proteins remains a great challenge with the absence of such tool in protein analysis. Therefore, development of ultrasensitive, robust, and high-throughput analytic techniques that can rapidly detect proteins in biological sample is in urgent need in the protein analysis.

NANOMATERIALS FOR PROTEIN ANALYSIS Various nanomaterials can be categorized into carbon-based or silicon-based nanomaterials, metallic nanomaterials, metal oxide nanomaterials, semiconductor nanomaterials, and polymer nanomaterials according to its texture [20]. The application of nanomaterials in biology has been of great interest in recent years due to the chemically tunable sizes and unique physical (e.g., electronic, photonic, and magnetic) properties and has been applied in various biological frontier including imaging [21], biosensing [22], target drug/gene delivery [23], biological conjugates, and biomacromolecule surface recognition [24]. A novel technical platform known as nanoproteomics utilizes the advantages of nanotechnology to help tackle those aforementioned challenges presented in protein analysis such as protein complexity and dynamic range. Due to large surface-to-volume ratio and chemical functional group on the surface, nanomaterials as sorbent materials have been extensively applied in the sample pretreatment of proteins, such as pretreatment of low-abundance proteins or peptides, specific posttranslational modification of proteins or peptides, and immobilization of enzyme to increase the efficiency of digestion. Meanwhile, the electric and optical properties of nanomaterials can also support it as matrix substrate or detection probe for enhancing protein detection sensitivity. Furthermore, nanomaterials also place the important role in the protein quantification as labeling probe. In summary, the unique physical and chemical properties

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39

of nanomaterials, such as high surface-to-volume ratios, variable surface functionality, and adjustable size or structure, offer several advantages in nanoproteomics including low sample consumption, high-throughput capability, short assay time, and sensitive detection. Herein, we summarize the recent nanomaterials that are applied in various protein analyses.

NANOMATERIALS FOR THE SAMPLE PREPARATION OF PROTEIN ANALYSIS In a living organism, tens or thousands of proteins are expressed in a dynamic range of ten orders of magnitude. Meanwhile, there are hundreds of modifications decorated on proteins posttranslationally for different biological functions. Thus, to promote the efficiency for protein identification and characterization, more efficient sample pretreatment methods were developed using novel nanomaterials. Here, various novel nanomaterials are reviewed in the sample preparation of protein analysis.

PRETREATMENT OF LOW ABUNDANCE PROTEINS OR PEPTIDES Mass spectrometry-based proteomics analysis using soft ionization techniques such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) has become a major player in protein profiling search due to capabilities of these techniques to perform protein analysis with high throughput and excellent sensitivity [25, 26]. However, the identification of low-abundance proteins has encountered some challenges including the inevitable sample loss and coconcentration with the contaminants in protein analysis. Thus, an efficient pretreatment step prior to MS analysis can aid identification of low-abundance proteins. Nanomaterials have been employed for pretreatments of low-abundance proteins or peptides. Among the diverse classes of nanomaterials, silica-based nanomaterials [27, 28], carbon-based nanomaterials [29, 30], and polymer nanomaterials are the most commonly used composites that offer several advantages in pretreatment of low-abundance proteins or peptides, including low sample consumption, ultralow detection, and short assay time (Fig. 2.1).

CNTs, chitosan/MWCNTs, MWCNTs@Fe3O4-MIPs, etc.

Fe3O4@mSiO2 Fe3O4@nSiO2@mSiO2 Fe3O4@nSiO2@mSiO2-C8 etc.

C8-modified GO@mSiO2, G@SiO2@PMMA, GO/rGO-Fe3O4, Fe3O4@SiO2@GO, etc.

Fe3O4@SiO2-C8, Fe3O4-NH2C8,Fe3O4@C-C8, etc.

NDs

HSBF, C60-f-MSs SBA-15

MCM-41

Silica-based

Carbon-based nanomaterials

ZnO(TiO2)@PMMA, Fe3O4@SiO2@PMMA, polymer brush nanosponge, etc.

Polymer-based

FIG. 2.1 Schematic illustration of low-abundance proteins or peptide enrichment for MS detection using diverse nanomaterials.

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CHAPTER 2 NOVEL NANOMATERIALS FOR PROTEIN ANALYSIS

Silica-based nanomaterials Silica has remarkable properties such as large specific surface area, hydrophilic and hydrophobic surface function, making it a promising material for the enrichment of trace proteins or peptides. Nanozeolite beta (b-X, X ¼ Si/Al ratio) and silicalite-1 (S-1) nanocrystals were applied for the first time in the enrichment and identification of trace peptides and proteins [27]. Nanozeolites with a medium or high Si/Al ratio exhibited best features on the enrichment of tryptic myoglobin digests with no apparent discrimination. Furthermore, with the high specific surface areas and ordered porous structures with narrow size distribution, mesoporous materials have opened up new applications to adsorb or capture large biomolecules through the interaction with the functional groups of pore and surface. As wellordered hexagonal mesoporous silicate molecular sieves, SBA-15 were used for the capturing proteins and also as a multifunctional nanoreactor where digests of proteins can be done. After the evaluation of the protein adsorption isotherms for standard proteins, SBA-15 exhibited efficient adsorption. Meanwhile, due to the digestion process was reacted inside the channels, sample loss was decreased compared with normal procedure [28]. Furthermore, a novel mesoporous silica particles with critical ˚ can perform selective extraction of pore sizes with a narrow pore size distribution centering at 20.5 A endogenous peptides (from 1 to 12 kDa) from human plasma and exclusion of untargeted proteins at the same time [31]. Considering the pH conditions of endogenous peptides, highly ordered mesoporous nanoparticles MCM-41 were modified with strong cation-exchange and anion-exchange groups to the enrichment of endogenous peptides at natural pH condition [32]. Then, combined enrichment with three kinds of materials (SCX-MCM-41, SAX-MCM-41, and unmodified MCM-41) and online 2-D nano-LC/MS/MS analysis, a large-scale identification of endogenous peptides from mouse liver extract was reported. To enhance the enrichment efficiency, the multifunctional magnetic materials combining with mesoporous silica and iron oxides were fabricated with highly ordered pores, special surface features, and superparamagnetic properties [33, 34]. For instance, the novel microspheres Fe3O4@nSiO2@mSiO2 composed of the Fe3O4@nSiO2 core and a perpendicularly aligned mesoporous SiO2 shell were prepared, and a 100-fold increase of S/N ratio of standard peptides was observed. To directly prepare a mesoporous SiO2 shell on the magnetic microspheres (Fe3O4@mSiO2 spheres), nonporous silica composites were dissolved in the alkaline solution with the template cetyltrimethylammonium bromide (CTAB), and upon completion, the template interacted with silicate species was removed by ammonium nitrate ethanol solution [34].

Carbon-based nanomaterials Carbon-based nanomaterials have many promising applications due to their unique properties such as good chemical stability and high specific surface area. Additionally, this surface modification of this type of material can be easily achieved and has been extensively studied. Consequently, carbon-based nanomaterials have also been widely functionalized as novel adsorbents for sample pretreatment in protein analysis. The adsorption mechanism relies on the affinity between the proteins or peptides, and carbon nanomaterials stem from hydrophobic interactions and sometimes π-stacking interactions between the materials and aromatic residues such as histidine and tryptophan. Carbon nanomaterials, such as carbon nanotubes, fullerenes (buckyballs), graphene, and nanodiamond, can be applied for the pretreatment of low-abundance proteins or peptides from biological samples. In order to improve the specific enrichment and dispersity of carbon nanomaterials in aqueous solution, some modification or functionalization of carbon nanomaterials has been fabricated and employed to improve their performance.

NANOMATERIALS FOR THE SAMPLE PREPARATION OF PROTEIN ANALYSIS

41

The carbon nanotubes (CNTs), including single-walled nanotubes (SWNTs) and multiwalled nanotubes (MWNTs), could be readily modified via covalent or noncovalent functionalization. Chen et al. pretreated CNTs with citric acid to obtain CNTs with anionic surfaces, which could be used to selectively concentrated proteins or peptides with opposite charge by varying the pH of the sample solution [29]. Meanwhile, with the assistance of its electronic properties, peptides adsorbed on CNTs can be analyzed by MALDI-MS without the need of any extra MALDI matrix [30]. Guo et al. derivatized the surface of CNTs using 2,5-dihydroxybenzoyl hydrazine, which introduced phenolic hydroxyl and phenyl groups [35]. Combined mechanical and electronic properties of CNTs and a source of protons by the derivatized molecules, the functionalized CNTs could be placed as assisted matrix for the MALDI-MS analysis, on which the detection could be enhanced about 10–100 times. Meanwhile, 2521 endogenous peptides have successfully been identified from human plasma assisted by CNTs. For the specific isolation of hemoglobins, a chitosan/MWCNT composite has been fabricated by cross-link. The novel composite exhibited good enrichment properties than conventional chitosan particles with micrometer-range dimension including faster adsorption equilibrium and higher adsorption capacity [36]. Furthermore, to enhance the enrichment specificity, a novel magnetic MWCNTs coated with a layer of molecularly imprinted polymers (MWCNTs@Fe3O4-MIPs) were synthesized for specific enrichment of some template proteins [37]. C60 fullerene and its derivatives have become more popular in biological applications due to their unique properties including well-defined structure and strong absorption in the UV region. A star-like water-soluble fullerene derivative, hexa(sulfonbutyl)fullerene (C60[(CH2)4SO3]6, HSBF), was used for the enrichment of positively charged species through ion interaction and hydrophobic interaction [38]. With strong absorbance in the UV range, proteins or peptides enriched using fullerene derivatives can be deposited onto a target as a mixture and analyzed by MALDI-MS. Fullerene (C60)-functionalized silica gel has been fabricated for the pretreatment of peptides and other biomolecules [39, 40]. Further, polymerization of C60 on the surfaces of Fe3O4@SiO2 magnetic nanoparticles (MNPs) (designated C60-f-MS) was synthesized and successfully applied for the enrichment of low-concentration tryptic cytochrome C with good salt tolerance capacity (100 mM CaCl2)[41]. As some commercial C8/C18-functionalized SiO2 beads have been applied for the desalting of proteins or peptides, the alkyl group-functionalized MNPs were designed for the pretreatment of proteins/peptides in biological samples through hydrophobic interaction [42–44]. In the initial study, C8 groups were successfully immobilized on the surface of magnetic silica microsphere with the routine silanization using chloro(dimethyl)octylsilane [42]. In the following work, alkyl groups were simply anchored on the outside of amine-functionalized MNPs (Fe3O4-NH2). To improve the dispersibility and magnetic response, C8 group was loaded on magnetic carbonaceous polysaccharide (Fe3O4@C) MNPs. Due to the hydrophilic groups of carbonaceous polysaccharide bonded outside the spheres, the hydrophilicity and stability of the novel Fe3O4@C-C8 spheres were improved in aqueous systems. These well-dispersed Fe3O4@C-C8 spheres also have a strong magnetic response, in which the spheres can be collected within 2 s [44]. Owing to the excellent stability, biocompatibility, and unbleachable fluorescing from point defects, nanodiamond (ND) has emerged as a new material for applications in nanoproteomics [45, 46]. Chang et al. successfully utilized the carboxylated/oxidized-NDs for protein enrichment [47]. Notably, since diamonds are optically transparent up to the UV region, the enriched proteins or peptides can be directly analyzed by MALDI-MS without eluted from NDs. Yang et al. established the on-plate enrichment procedure to peptides by detonation NDs (dNDs), and the detection sensitivity using these

42

CHAPTER 2 NOVEL NANOMATERIALS FOR PROTEIN ANALYSIS

materials was enhanced by more than two orders of magnitude. Meanwhile, dNDs could be used for the enrichment of peptides in highly diluted and complex sample solutions. As dNDs were prepared in extreme environment during detonation process, dNDs have a variety of surface functional groups. As a result, enrichment using this material can be achieved in a wide pH range [48]. As the new generation of carbon materials, graphene (G) is a single carbon atom thick and twodimensional honeycomb crystal lattice, and this material has recently attracted much attention in biological application [49]. Owing to an ultrahigh specific surface area (theoretical value 2630 m2 g
1) and the large delocalized p-electron system, graphene has a strong affinity to biomolecule with carbonbased ring structures. G and GO were firstly fixed on different silica NPs through covalent bonding or electrostatic interactions [50–52]. Graphene-bound silica exhibited good desalting of protein samples for MALDI-TOF MS analysis with good enrichment features (recoveries of protein ranged from 82.4% to 94.7%). To simplify the enrichment process, Fe3O4 particles were covalently attached onto the GO sheets. The prepared Fe3O4-GO composites can be applied for immobilization and pretreatment of low-abundance proteins or peptides that have abundant hydrophilic and hydrophobic groups with high loading capacity [53]. It’s noteworthy that Fe3O4@SiO2@graphene microspheres with perfect core-shell structure were fabricated and could be reusable for protein enrichment [54]. Recently, to improve the hydrophilicity of magnetic-functionalized graphene, Zhang et al. established a Fe3O4chitosan@graphene (Fe3O4-CS@G) for the pretreatment of low-abundance proteins [55].

Polymer nanomaterials With the development of biopharmaceuticals, the composite of polymers and nanoparticles has been widely used as drug delivery vehicles for protection and slow release. Due to the flexible properties and activated functional groups, polymer nanomaterials have drawn attention on sample preparation for protein or peptides analysis. As poly(methyl methacrylate) (PMMA) possesses a large number of hydrophilic and hydrophobic groups, it has potential promise for fabricated a series of PMMA-coated nanoparticles for the pretreatment of proteins or peptides. Yang and coworkers prepared CaCO3@PMMA with a destructible CaCO3 core, which can be dissolved with acetic acid after pretreatment and desalination of proteins or peptides. The proteins or peptides enriched on PMMA were released into solution for MALDI-MS analysis [56]. Moreover, PMMA shell was polymerized on the ZnO and TiO2 NPs by virtue of the inherent free radicals for the pretreatment of proteins or peptides [57–59]. This synthesis method could make stable ZnO and TiO2 cores, and the resulting PMMA nanobeads are dispersive in solution. Due to the small diameters of the ZnO@PMMA NPs and TiO2@PMMA NPs, enrichment experiments using these nanoparticles could be directly spotted on the MALDI plate without an eluting step. Good enrichment features (limit of detection (LOD) of tryptic bovine serum albumin (BSA) peptides, 100 aM) and high salt tolerance (1 nM L
1 BSA tryptic peptides in NaCl (6.2 M), saturated NH4HCO3 (2.6 M), or 1 M urea aqueous solution) were observed. PMMA was functionalized on Fe3O4@SiO2 MNPs with the assistance of 3methacryloxypropyltrimethoxysilane (MPS), a polymerizable silane coupling agent [60]. On gold substrate, a novel polymer brush-like nanosponge was developed by 70% poly(N-isopropylacrylamide) (PNIPAAM) and 30% poly(methacrylic acid) (PMAA), in which the structure could be adjusted with different pH condition and realized the separation and enrichment of peptides/proteins [61]. In a later study, a cationic polymer brush nanosponge was also reported for the selective extraction and enrichment of acidic proteins and peptides [62].

NANOMATERIALS FOR THE SAMPLE PREPARATION OF PROTEIN ANALYSIS

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PRETREATMENT OF PHOSPHORYLATED PROTEINS OR PEPTIDES Protein phosphorylation is a vital PTM that dynamically modulates numerous biological processes controlled by the protein kinases and phosphatases [63, 64]. Phosphorylation places important roles in many biological processes, including the cell signaling networks and communication, gene expression, protein synthesis, and cell division and apoptosis [65, 66]. Thus, phosphorylated proteins or peptides are very important for illuminating the specific signaling pathways and finding new targets for the diagnosis and therapy of diseases. MS-based proteomics is a powerful tool for the identification of phosphorylated proteins or peptides. However, due to the low abundance and worse ionization efficiency of phosphorylated proteins or peptides in MS analysis compared with their unphosphorylated parts, sample pretreatment of phosphorylated proteins or peptides prior to MS is necessary for their effective detection. Based on ionic interaction, metal ion-immobilized nanoparticles and nanostructure metal oxides were fabricated and applied for the specific pretreatment of phosphorylated proteins or peptides. Nanomaterial-based metal ion-immobilized particles focus on the application of new metal ions, the introduction of new chelating ligands, and the fabrication of novel nanomaterial substrates. Different novel nanomaterials for the pretreatment of phosphorylated proteins or peptides are summarized as follows.

Metal ion-immobilized composites Generally, the phosphate groups show typical Lewis-base properties in a wide pH range. Thus, the phosphate groups on the phosphorylated proteins/peptides can interact with metal cations by electrostatic interaction or/and chelation. New metal ions, new chelating ligands, and novel nanomaterial substrates have been developed for the immobilized metal ion affinity chromatography (IMAC) techniques in the phosphorylated protein analysis (Table 2.1).

Metal ions Metal ions including Al3+, Cu2+, Fe3+, Zn2+, Ti4+, and Zr4+ have been utilized in IMAC strategies (Table 2.1). As the trivalent/tetravalent ions of the stable rare earth can strongly interact with phosphate group, rare earth metal materials have attracted interest by researchers in the biomedical frontiers several years ago. Therefore, rare earth ions such as Ga3+ and Ce4+ have also been used as immobilized metal ion in IMAC strategy for the phosphoprotein or phosphopeptide isolation. With iminodiacetic acid (IDA) as a chelating ligand, Ce4+ was immobilized onto magnetic silica microspheres (Fe3O4@SiO2) and shown better enrichment capacity than the Fe3O4@SiO2-IDA-Fe (III) [81]. Further, Ni et al. coated poly(vinylphosphonic acid) (PVPA) as a chelating ligand for the immobilization of Ce(IV) onto the Fe3O4@SiO2. As Ce4+ has unique properties such as good dispensability in solutions and high abundance after coating, the Fe3O4@SiO2@PVPA-Ce(IV) showed good sensitivity and specificity for the enrichment of phosphorylated peptides from biological samples [82]. Moreover, other rare earth ions including La3+, Ho3+, and Er3+ have been fixed on PVPA spheres for the pretreatment of phosphorylated proteins [83].

Chelating ligand and matrix supports The traditional ligands including IDA and nitrilotriacetic acid (NTA) was applied for immobilizing metal ions (e.g., Fe3+ and Ga3+) [67, 68, 70]. The immobilized metal ions with only one IDA ligand may be easily lost during sample loading and washing. As the chelating ligand, NTA was much better

44

Table 2.1 Nanomaterials for Pretreatment of Phosphorylated Proteins or Peptides Based on IMAC

Fe (III) Al (III) Ga (III) Ti(IV)

Zr (IV)

Zn(II)

Ce (IV)

Type of Matrix Fe3O4@SiO2-IDA-M

Chelating Ligand

Characterization of Matrix

Selectivity (BSA/α/ β-Casin)

Sensitivity

References

50:1

–

[67]

GLYMO-IDA NTA, IDA

d ¼ 370 nm Ms ¼ 68.2 emu g
1 d ¼ 15 nm –

– –

5 fmol –

[68] [69]

Fe3O4@SiO2-EDAS-SANTA-Mn+ MCNC@PMAA@PEG MP Fe3O4-ATP-Mn+

EDAS-SA-NTA

d ¼ 50 nm

–

[70]

Fe3O4@PD-Mn+

Dopamine

500:1

SiO2@graphene

1000:1

Fe3O4@SiO2-PO3-Mn+

Dihydroimidazole group –PO3-OH

d ¼ 70 nm

100:1

Fe3O4@C-PO3-Mn+

–PO3-OH

d ¼ 300 nm

25:1

Fe3O4@p(VPA-EDMA-1) Mn+

–PO3-OH

50:1

Fe3O4@SiO2-AsO3-Mn+

Arsenate group

Fe3O4-GAPT-Mn+

Glutaric acid

d ¼ 400–600 nm (20 nm of polymer shell) Ms ¼ 29 emu g
1 d ¼ 30 nm Ms ¼ 39.1 emu g
1 d ¼ 4–6 nm

50 fmol tryptic digest of α/β-casein 0.5 nM tryptic digest of β-casein 3 amol tryptic digest of β-casein 5 fmol tryptic digest of β-casein 40 pM tryptic digest of β-casein 50 fmol tryptic digest of β-casein 0.8 pmol tryptic digest of β-casein –

CoFe2O4-GAPT-Mn+ Fe3O4@SiO2-IDA-Mn+

Glutaric acid GLYMO-IDA

Fe3O4@SiO2@PVPA-Mn+

–PO3-OH

n+

GLYMO-IDA

Fe3O4@SiO2-IDA-Mn+ Ni-NTA/IDA-Mn+

500:1 ATP

d ¼ 150 nm

d ¼ 9.96  1.03 nm d ¼ 370 nm Ms ¼ 68.2 emu g
1 d ¼ 500 nm Ms ¼ 50.5 emu g
1

5000:1

100:1

10 fmol tryptic digest of β-casein

99:1 (mass ratio)

50:1

[71] [72] [73] [74] [75] [76] [77]

[78] [79]

1.15 μM β-casein

[80] [81]

5 nM β-casein

[82]

CHAPTER 2 NOVEL NANOMATERIALS FOR PROTEIN ANALYSIS

Metal ion

NANOMATERIALS FOR THE SAMPLE PREPARATION OF PROTEIN ANALYSIS

45

than IDA due to the optimized enrichment efficiency with four or six coordinate number. Meanwhile, 2
some novel chelating ligands including arsenate (–AsO2
3 ), phosphate (–PO3 ), adenosine triphosphate (ATP), and dopamine were developed to enhance the coordination force [78, 84]. Moreover, to further increase the coordination capacity and simplify the pretreatment process, various silica-based, carbonbased, polymer-based, and magnetic-based matrix supports have been utilized to incorporate these ligands. To enhance the stability and specificity for the enrichment of multiply phosphorylated peptides, an easy and cost-effective method to prepared modified arsenate groups (As) on silica-based and magnetic silica nanoparticle matrix materials was developed, respectively [78, 84] (Fig. 2.2A). The zirconium arsenate-modified magnetic nanoparticles (ZrAs-Fe3O4@SiO2) and ZrAs-silica nanoparticles show many potential applications in clinical research. Meanwhile, based on an octahedral coordination model with phosphate groups, phosphate ligands for metal ion immobilization were established on porous silicon wafer and silica nanoparticles by the chemical reaction between –NH2 and POCl [75, 85] (Fig. 2.2B). To improve the performance of phosphate-group ligand-functionalized matrix, with the assisted polymeric matrices, Fe3O4@SiO2@PEG-Ti4+ affinity microspheres were developed obtained through polymerization of monomers carrying reactive groups (e.g., epoxy and amino) (Fig. 2.2C) [86]. Fe3O4@SiO2@PEG-Ti4+ from previous discussion has the following benefits including simple isolation during experimental steps due to its magnetic core, minimal nonspecific protein adsorption due to PEG chains, and high abundance of Ti4+ on the surface. Hence, other polymer-related ligands were developed for the immobilization of metal ion with good selectivity, high recovery, and an ultralow detection limit, such as MCNC@PMAA@p(EGMP)-Ti4+ composite and Fe3O4@SiO2@ (HA/CS)10-Ti4+ [71, 87]. Other novel ligands also place important roles for the improvement of the specificity, sensitivity, and enrichment capacity for phosphorylation [72]. With the help of adenosine triphosphate (ATP) molecules, Ti4+ was grafted onto Fe3O4 nanoparticles with high sensitivity (3 amol β-casein). Due to easily polymerized onto a variety of substrate, dopamine was used for the immobilization of Ti4+ ions through the strong chelation. The prepared Fe3O4@ polydopamine (PD)-Ti4+ exhibited good specificity (molar ratio of tryptic BSA/β-casein up to 500:1) and high sensitivity (2 fmol tryptic β-casein) [73]. Additionally, the unique hydrophilicity of dopamine also allows the resulting nanoparticles to have excellent water dispersibility and reduced nonspecific binding. Another exceptional novel affinity ligand is dipicolylamine (DPA), which formed Zn-DPA coordination complex on the surface of superparamagnetic NPs (4.6  0.7 nm) via glutaric acid (hereafter referred to as GAPT) [79], as illustrated in Fig. 2.2D. Attributed to the super small size, the reduced nonspecific adsorption due to the PEG chains, and the high abundance of Zn2+, the prepared Fe3O4-GAPT-Zn NPs (4–6 nm) gave outstanding specificity (mass ratio of BSA/β-casein up to 99:1). Significantly, the Fe3O4-GAPT-Zn NPs could perform specific enrichment of phosphorylated proteins in human plasma and showed good compatibility with the top-down proteomics technique for the analysis of phosphorylated protein from a swine heart tissue extract. Recently, the author designed and synthesized the cobalt ferrite (CoFe2O4) NPs (9.96  1.03 nm) as the core instead of Fe3O4 for the preparation of the CoFe2O4-GAPT-Zn NPs, which showed a stronger magnetic response and better reproducibility for intact phosphorylated protein enrichment than the first generation Fe3O4-GAPT-Zn NPs [80].

46

Representative works in the pretreatment of phosphorylated proteins/peptides. (A) Zirconium arsenate-modified magnetic nanoparticles (ZrAsFe3O4@SiO2) (Copyright 2011, Springer-Verlag), (B) ZrP-functionalized mesoporous silica microspheres (Copyright #2011, Royal Society of Chemistry), (C) Fe3O4@SiO2@PEG-Ti4+ affinity microspheres microspheres (Copyright 2012, Royal Society of Chemistry), and (D) functionalized Fe3O4-GAPT-Zn NPs (Copyright 2015, American Chemical Society).

CHAPTER 2 NOVEL NANOMATERIALS FOR PROTEIN ANALYSIS

FIG. 2.2

NANOMATERIALS FOR THE SAMPLE PREPARATION OF PROTEIN ANALYSIS

47

Metal oxide based nanomaterials Metal oxide (MO) nanoparticles or core/shell nanocomposites are promising materials for the selective phosphorylation enrichment, which also used metal ions as affinity sites to phosphorylated proteins/ peptides. Different with IMAC materials, in which metal ions was chelated with ligand on the surface of matrix, the metal ions from metal oxide-based nanomaterials bind to their neighbor oxygen ions via strong chemical bonds. Some metal oxides are amphoteric, for example, TiO2 with pKa1 ¼ 4.4 and pKa2 ¼ 7.7, which can capture phosphorylation proteins/peptides at low pH and then be eluted at high pH based on the reversible Lewis acid-base actions [88].

Pure metal oxide nanomaterials Most of pure metal oxides in their common form have been used for the specific enrichment of phosphorylated proteins or peptides including TiO2 [89], ZrO2 [90], CeO2 [91], NiO [92], Fe2O3 [93], HfO2 [94], Al2O3 [95], Ga2O3 [96], ZnO [97], SnO2 [98], La2O3 [99], and Ta2O3 [100]. In an evaluation test, the authors observed that ZrO2 has greater specificity to single phosphorylated peptides, while TiO2 favored multiply phosphorylated peptides [90]. CeO2 and Ce-Zr composite, which exhibited dual properties in phosphopeptide enrichment, work as a sorbent for phosphorylated peptides and a catalyst for dephosphorylation [101]. In MS spectrum, the signal of phosphorylated peptides treated with CeO2 showed a characteristic mass loss of
80 Da, which may be useful for the direct identification of phosphorylated peptides. Other metal nanomaterials including Fe3O4, NiFe2O4, ZnFe2O4, and NiZnFe2O4 have shown distinct specificity to mono- and multiply phosphorylated peptides [102]. Recently, with the unique properties including high surface area, stable skeleton, and size exclusion effect (with pore sizes typically between 2 and 50 nm), mesoporous metal oxides showed an even higher efficiency and loading capacity for binding phosphate groups than micro- and nanoparticles [91, 103]. For example, with a high binding capacity due to its surface area, mesoporous TiO2 has been used as a special adsorbent for phosphorylated peptides. It has the advantage of both the specific affinity with TiO2 and the size exclusion mechanism enabled by the mesoporous structure. Meanwhile, with the highly hydrophilic outer surface of each cluster, nonspecific binding of many hydrophobic proteins or peptides can be mitigated [104]. Further, superparamagnetic iron oxide core allows the material-target conjugate isolation from the solution using an external magnet after selective adsorption [105].

Magnetic core based MOs Magnetic core-based MOs usually have core-shell structures. A variety of metal oxides (TiO2 [106], ZrO2 [107], Al2O3 [95], ZnO [79], Ta2O5 [108], Nb2O5 [109], Ga2O3 [96], SnO2 [110], and CeO2 [91]) can be functionalized on the outside of magnetic cores. Deng et al. developed a series of synthetic procedure for the preparation of metal oxide-coated MNPs (Fe3O4@MxOy) with a well-defined core-shell structure [95, 100, 106, 107, 110]. A solvothermal and polymerization reaction was applied for the preparation of Fe3O4 microspheres and the carbon layer, respectively. Finally, metal oxide was prehydrolyzed and adsorbed onto the Fe3O4@C microspheres, by a sol-gel process and calcination. TiO2, ZrO2, Al2O3, and Ga2O3 were successfully coated on Fe3O4 MNPs using this procedure [110]. To prepare new metal oxide-coated MNPs with high surface area and porous structure, either sol-gel method or liquid-phase deposition method was applied for the immobilization of metal oxide on magnetic hollow mesoporous silica sphere for the enrichment of phosphorylated peptides [91]. Wang et al. coated a highly crystalline mesoporous TiO2 shell via sol-gel method followed by a hydrothermal process [104].

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CHAPTER 2 NOVEL NANOMATERIALS FOR PROTEIN ANALYSIS

The mesoporous Fe3O4@mTiO2 microspheres possess a well-defined core/shell structure, large pore volume (0.45 cm3 g
1), high specific surface area (167.1 m2 g
1), and high magnetic susceptibility. Due to the purity and high density of the TiO2 mesoporous shells, the Fe3O4@mTiO2 microspheres showed over 10 times as that of the Fe3O4@TiO2 microspheres (20 mg g
1). Considering the improvement of the specificity and sensitivity and the mitigation of “shadow effect”, Ni et al. fabricated the Fe3O4@fTiO2 multifunctional microspheres with flowerlike hierarchical meso-/macroporous TiO2 shells [111]. Compared with the former materials with macroporous shell (denoted as Fe3O4@pTiO2), Fe3O4@fTiO2 exhibited excellent specificity and selectivity for the enrichment of phosphorylated peptides. With chemical stability, catalysis, and coordination, some rare earth (RE) composites have also been immobilized on the outside of magnetic core as novel affinity absorbents for the pretreatment of phosphorylated peptides. The Fe3O4@REPO4 microspheres (RE ¼ La and Y) were synthesized via precipitation and ion-exchange method [112, 113]. 2678 unique phosphorylated peptides had been identified by Fe3O4@hYPO4 microspheres from the tryptic digested mouse brain. Furthermore, other RE-based materials were also applied for the enrichment and detection of phosphorylated peptides, such as magnetic Fe3O4@LaxSiyO5, γ-Fe2O3@REVO4 (RE ¼ Sm, Dy, and Ho), and γ-Fe2O3@xNH4FyLuF3 [114–116].

Other nanomaterials-based MOs To increase the dispersibility of MOs in aqueous solution, carbon-based nanomaterials have been utilized as matrices for supporting MOs. Recently, TiO2/CNTs composites and TiO2/graphene composites are the most attractive materials of choice [117, 118]. With assisted by graphene, a graphene-titania platform (G-TiO2) was established by sol-gel method and applied for detecting phosphorylated peptides in HeLa cells. Notably, with the UV absorbance of graphene and titania, G-TiO2 can be utilized as a substrate for the direct MS analysis of phosphorylated peptides and simplified experimental workflow [119]. To improve affinity sites, G@PD@TiO2 composite was fabricated by coating PD on graphene for the deposition of TiO2 nanoparticles [120]. With the assistance of PD, the G@PD@TiO2 possesses a large amount of TiO2 and good dispersibility and showed good enrichment features, with which 334 phosphopeptides can be identified from mouse brain tissue. With its unique properties, the modifiable surface of silica is an ideal feature for the fabrication of MO-based nanomaterials. For instance, with large surface area and appropriate Lewis acidity of the TiO2- and ZrO2-modified mesoporous silica composites, they exhibited better enrichment performance than commercial available TiO2 particles [103, 121]. Furthermore, to overcome the “shadow effect” in some mesoporous materials with long channels, the macroporous silica with large and open pores has also been applied for the MOs coating to facilitate efficient mass transport. The Al2O3 and TiO2 onto the macroporous ordered silica foam (MOSF) were fabricated and used as a multifunctional reactor with enzyme immobilization and the substrate for the subsequently pretreatment of phosphorylated peptides [122]. Subsequently, Deng et al. designed the synthesis of TiO2-modified hierarchically ordered macro-/mesoporous silica composites [123]. Besides metal ion-immobilized nanoparticles and MO-modified nanomaterials, other nanomaterials have been successfully fabricated for the specific enrichment of phosphorylated proteins/peptides. For instance, amine-based nanomaterials are expected to capture phosphorylated proteins/peptides by the ionic interaction controlled by the pH of buffers. With the various styles of amine groups, some molecules have been successfully functionalized on the surface of nanomaterials (Table 2.2).

Type

Chelating Molecules

Fe3O4@NH2 nanoparticles

1,6-Hexane-diamine

C60fullerene@aminopropylsilica Fe3O4@SiO2-NH2

Amino-propylsilica

Characterization of Matrix

Selectivity (BSA/α/ β-Casin)

Sensitivity

References

d ¼ 370 nm Ms ¼ 68.2 emu g
1 d ¼ 3–5 μm

50:1

–

[124]

–

2.2 μM β-casein

[125]

d ¼ 15 nm

–

5 fmol

[126]

Fe3O4@SiO2@PEI Diamond nanoparticles@PA

N0 -[3-(trimethoxysilyl)propyl]-diethylenetriamine and tetramethylorthosilicate Polyethylenimine (PEI) Polyarginine(PA)

– d ¼ 50 nm

– –

[127] [128]

Graphene@guanidyl

1,6-Hexanediamine

–

500:1

Fe3O4@PGMA

Poly(glycidyl methacrylate) (PGMA)

d ¼ 150 nm

5000:1

Fe3O4@SiO2@PGMAguanidyl

3-Guanidopropyl triethoxysilane

d ¼ 360 nm

500:1

– 50 fmol tryptic digest of α/βcasein 0.5 nM tryptic digest of β-casein 3 amol tryptic digest of β-casein 5 fmol tryptic digest of β-casein

[129]

[130]

[131]

NANOMATERIALS FOR THE SAMPLE PREPARATION OF PROTEIN ANALYSIS

Table 2.2 Nanomaterials for Pretreatment of Phosphorylated Proteins/Peptides Based on Amine Group

49

50

CHAPTER 2 NOVEL NANOMATERIALS FOR PROTEIN ANALYSIS

The typical 1,6-hexanediamine-functionalized magnetic nanoparticles (Fe3O4@NH2) can capture phosphorylated peptides in the 1% TFA buffer, and the peptides can be eluted in the eluting buffer containing 5% NH3H2O [124]. To increase the abundance of amino group on the composite surface, some polymerized amino molecules were coated on the surface of magnetic particles by sol-gel method and polymerization reaction. Fe3O4@SiO2@ polyethylenimine (PEI) carrying abundant amine groups can capture phosphorylated peptides by forming bidentate complexes [127]. Through stable “covalentlike” interaction with phosphate groups, guanidine-related nanomaterials was synthesized and applied for the enrichment of phosphorylated peptides for direct MALDI-MS analysis. With the affinity mechanism similar to the MOs, the magnetic metal-organic frameworks (mMOFs) have also been synthesized via electrostatic self-assembly between positively charged MIL-101(Fe) and the negatively charged Fe3O4 magnetic nanoparticles (MNPs) [132]. With highly specific surface area, excellent magnetic property, and well-defined structures, Fe3O4/MIL-101(Fe) composite exhibited good specificity, recovery, and application performance for real samples.

PRETREATMENT OF GLYCOPROTEINS/GLYCOPEPTIDES Protein glycosylation plays important factors in various cellular processes, such as gene expression, cell adhesion, signal transduction, and immune responses. Due to its close related with disease biomarkers, glycosylation contains much information that has been extensively studied using proteomics tools. For example, some glycosylated proteins were recognized by FDA as cancer biomarkers, including human chorionic gonadotropin-β, α-fetoprotein, carcinoembryonic antigen, prostate-specific antigen, human epidermal growth factor receptor 2, and mucins, which are clinically applied in diagnosis, monitoring, or selected therapies [133]. In glycosylation protein analysis, the complex process requires the identification of the peptide sequence and the glycosylation site and the characterization of glycan composition and the structure. The routine strategy of the glycosylated protein analysis is to perform enzymatic digestion followed by glycosylated peptide sequencing through mass spectrometry, which has been widely applied in glycoproteomics research. However, due to the low abundant and low ionization efficiency of glycosylated proteins/peptides and the microheterogeneity of each glycosylation site, rapid and effective pretreatment strategies before MS analysis are urgently needed for glycoproteomics. Currently, some effective pretreatments of glycosylated proteins/peptides are realized based on three aspects: (a) hydrophilic interaction, (b) chemical reaction, and (c) lectin affinity. Recently, with the growing attention on glycosylation, various functionalized nanomaterials based on the above mentioned principles have been synthesized for the pretreatment of glycosylated proteins/peptides.

Hydrophilic-interaction based pretreatment There are various materials that utilize the principles of hydrophilic interaction, such as siloxane, zwitterionic (ZIC)-based, amide-based, and carbohydrate-modified materials. Based on the hydrophilicity of the glycan moiety, some hydrophilic group-modified nanomaterials have been developed for the pretreatment of glycosylated proteins or peptides with the advantages including good reproducibility, high selectivity, and nonirreversible alterations of glycosylated proteins/peptides (Table 2.3). Meanwhile, hydrophilic interaction-based method utilizes high composition of organic solvent as reaction buffer, making the workflow compatible with MS analysis. Glycosylated proteins or peptides contain abundant hydroxyl and carboxylic acid groups, especially in negatively charged sialylated and sulfated glycosylated peptides, which may interact with positive ions via electrostatic interaction. For instance,

51

NANOMATERIALS FOR THE SAMPLE PREPARATION OF PROTEIN ANALYSIS

Table 2.3 Nanomaterials for Pretreatment of Glycosylated Proteins/Peptides Based on HILIC Interaction Type Silica Fe3O4@ NH2

Functional Moiety Silica hydroxyl Amine

Phenolic mesoporous polymers

Amine

Amine-functionalized mesoporous silica nanoparticles (SBA-15APTES) MIL-101(Cr)NH2@PAMAM Magnetic particlecarbohydrate conjugates Fe3O4@SiO2@PEGmaltose

Amine

Highly cross-linked chitosan microspheres (CSMs) PAM-g-allosex@SiO2 Cross-linked CD-MOFs (LCD-MOFs) GO-Fe3O4/SiO2/ AuNWs/L-Cys magG/PDA/Au/L-Cys

Amine

Characterization of Support

Real Sample

References

d ¼ 1.7 μm

CD44 fusion protein Mouse uterine luminal fluid Human serum

[134]

[136]

Human serum

[137]

– MP-70000-220, BET surface area (548 m2 g
1), mesopore size (13 nm) The content of –NH2 in the SBA-15-NH2 was 45.55  10
5 mol g
1 Surface area, 778.11 m2 g
1

Carbohydrate

Carbohydrate

Carbohydrate

Size, Fe3O4 core (240 nm); silica shell (6 nm); PEG shell (20 nm); Ms, 35.9 emu g
1 Surface area and pore size, 191 m2 g
1 and 10 nm

Carbohydrate

–

γ-Cyclodextrin

Cubic particle size, 200–300 nm –

Poly(MBAAm-coMAA)@L-Cys MIL-101(NH2)@AuCys Water-soluble zwitterionic Au NCs Histidine bonded silica (HBS) Carbonized Mil-101 (Cr)

Amino acid (cysteine) Amino acid (cysteine) Amino acid (cysteine) Amino acid (cysteine) Amino acid (cysteine) Amino acid (histidine) Size-exclusion effect

SiO2-RAFT@PMSA

Zwitterionic

[135]

[138] Human sera

[139]

–

[140]

Rat brain

[141]

HeLa S3 cell lysate Mouse liver

[142] [143] [144]

Au nanoparticles was 60 nm

Mouse liver proteins Human serum

d ¼ 200 nm

Human plasma

[146]

Surface areas, 240.4 m2 g
1

HeLa cell lysate Mouse liver

[147]

Average diameter 2.0 nm The surface coverage of histidine was 0.9 μmol m
2 BET surface area of 531.88 m2 g
1; pore diameter, 3.03 nm

Human serum digest Human serum

Mouse liver glycoproteome

[145]

[148] [149] [150]

[151]

Continued

52

CHAPTER 2 NOVEL NANOMATERIALS FOR PROTEIN ANALYSIS

Table 2.3 Nanomaterials for Pretreatment of Glycosylated Proteins/Peptides Based on HILIC Interaction—cont’d Type

Functional Moiety

Fe3O4@SiO2@PMSA

Zwitterionic

TCP-SMs Fe3O4@PDA@ZrSO3H CS@PGMA@IDATi4+ Titanium dioxide

Zwitterionic Zwitterionic

Characterization of Support

Real Sample

References

Mouse liver

[152]

Mouse liver Human serum

[153] [154]

Ti4+

Size, Fe3O4 core 280 nm; silica shell 10 nm, PMSA layer 60 nm d ¼ 50
100 nm Ms 26.4 emu g
1; pore size, 3.2 nm d ¼ 20
250 nm

Mouse liver

[155]

TiO2

d ¼ 5 μm

[156]

MCNC@PMAA@AgNPs

Ag+

[157]

Graphitic carbon nitride (g-C3N4)-

Triazine ring and N–H

–

[158]

Magnetic graphene functionalized with metal-organic frameworks (MOFs) (MG@Zn-MOFs)

Triazine ring and N–H

Size, magnetic colloid nanocrystal cluster (MCNC) core 308 nm; PMAA shell 36 nm; Ag NP shell 10–30 nm; Ms, 12.1 emu g
1 Surface area of 10.6 m2 g
1 and a pore volume of 0.03 m3 g
1 Size, Fe3O4 core 200 nm; ZIF-8 crystals 30 nm (BET) surface area 322 m2 g
1, pore ˚ 10.6 A

HeLa cell membranes Rat serum

Human serum

[159]

the bare silica phase with silanol surface groups is a type of hydrophilic material, which can concentrate the glycan chain of glycosylated proteins or peptides in the hydration layer around the silica particles via electrostatic forces and hydrogen bonding [134]. Based on the hydrophilic interaction, some novel nanomaterials have been fabricated for the selective enrichment of glycosylated proteins or peptides from complex sample.

Amide group based hydrophilic interaction Generally, the amide stationary phase is currently the most frequently used HILIC material for separating glycans and glycopeptides, as shown in a recent survey by Zauner et al. [160]. Therefore, the amide group was applied for the preparation of absorbent to glycosylated proteins/peptides. For example, with the hydrophilic and ionic interaction between amino group and glycan structure, aminefunctionalized Fe3O4 MNPs could be directly applied for the enrichment of glycosylated peptides, especially on the sialic acid/sulfate residue of glycosylated peptides [135]. Khoo et al. prepared and used amine-functionalized Fe3O4 MNPs for the identification of glycosylated peptides from mouse uterine luminal fluid [135]. The percentage of glycoprotein identification increased from 70% using Sepharose CL-4B to 77% using NH2-Fe3O4 MNPs. From the obtained data, NH2-functionalized

NANOMATERIALS FOR THE SAMPLE PREPARATION OF PROTEIN ANALYSIS

53

nanoparticles performed favorably against the Sepharose CL-4B enrichment method selected for direct comparative evaluation and, in fact, against any other nonselective glycopeptide enrichment. Dai et al. proposed a new template-free strategy for the fabrication of phenolic mesoporous polymers (MPs) for highly selective glycosylated peptide enrichment (Fig. 2.3A). The resulting MPs showed a number of attractive features, including high surface areas (613 m2 g
1), large accessible porosities (pore volume, 0.53 cm3 g
1), nitrogen-containing functionalities, and intrinsic hydrophilic skeletons, that facilitate highly specific and efficient enrichment with excellent sensitivity of glycosylated peptides from minute

FIG. 2.3 Different nanomaterials applied in the pretreatment of glycosylated proteins/peptides. (A) Phenolic mesoporous polymers (MPs) (Copyright 2015, American Chemical Society), (B) Fe3O4@SiO2@PEG-maltose MNPs (Copyright 2012, Royal Society of Chemistry), (C) PAM-g-allosex@SiO2 (Copyright 2016, American Chemical Society), (D) Fe3O4@SiO2@PMSA (Copyright 2014, Royal Society of Chemistry), (E) Fe3O4@PDA@Zr-SO3H (Copyright 2017, Elsevier B.V.), (F) poly(MBAAm-co-MAA)@L-Cys (Copyright 2016, American Chemical Society), (G) MIL-101(NH2)@Au-Cys (Copyright 2017, American Chemical Society), (H) Fe3O4@L-Cys (Copyright 2017, Springer-Verlag GmbH Germany), (I) MCNC@PMAA@Ag-NPs (Copyright 2012, Royal Society of Chemistry), (J) zirconia-coated mesoporous silica (ZrO2/MPS) (Copyright 2012, Royal Society of Chemistry), (K) magnetic graphene functionalized with metal-organic frameworks (MOFs) (MG@Zn-MOFs) (Copyright 2016, American Chemical Society), and (L) graphitic carbon nitride (g-C3N4) (Copyright 2017, American Chemical Society).

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CHAPTER 2 NOVEL NANOMATERIALS FOR PROTEIN ANALYSIS

amounts of biological samples, where they identified 169 unique glycopeptides and 92 glycoproteins in 5 μL human serum. Thus, it opens up new opportunities for the development of glycosylated peptides enrichment [136]. Further, Gao et al. synthesized an amino-functionalized metal-organic framework (MOF) MIL-101(Cr)-NH2 whose surface is grafted with a hydrophilic dendrimer poly(amidoamine) (PAMAM) for N-glycopeptide enrichment based on hydrophilic interaction [138]. The selected substrate MOF MIL-101(Cr) possesses high surface area that provides support for peptide adsorption. In addition, the MOF displayed a good hydrophilic property after being modified with amino groups. The MIL-101(Cr)-NH2 material showed to have low detection limit (1 nm standard glycoprotein horseradish peroxidase digestion) and good selectivity when the concentration of nonglycosylated peptides was 100-fold higher than the target N-glycopeptides.

Carbohydrate-based ligands Carbohydrate-based ligands have emerged as specialized hydrophilic interaction materials, and these carbohydrates include monosaccharides, polysaccharides [139], cellulose, chitosan [141], and cyclodextrins in the stationary phase. These carbohydrates are often linked on the stationary phase via click chemistry, which is a chemical approach to quickly and reliably join small precursors for a desired conjugated moiety (Fig. 2.3B and C). Zou et al. fabricated hybrid Fe3O4@SiO2@PEG-Maltose MNPs to achieve large binding capacity by conjugation on the PEG molecules (Fig. 2.3B) [140]. This strategy utilizes the terminal hydroxyl group of the PEG molecule, which can be chemically modified to azido functionality for further functionalization via click chemistry. The existence of PEG molecule provides enhanced water solubility for the nanoparticles in aqueous solutions. The availability of high abundance of hydroxyl groups on maltose groups can form complex hydrogen-bonding networks for cooperative binding with the oligosaccharide of glycosylated proteins/peptides via multivalent hydrophilic interactions. As a result, enhancement of the sensitivity and higher sequence coverage for glycosylated proteins/peptides were achieved. To enrich trace sialylated glycosylated peptides from highly complex samples, a novel sialylated receptor, allose units were integrated into a polyacrylamide chain and generated a saccharide-responsive smart copolymer (SRSC) according to the saccharide-saccharide interactions in life systems [142]. This design significantly improves the selectivity of sialylated acid binding (500-fold BSA) and exhibits high-performance enrichment capacity. In Hela S3 cell lysate, more than 200 sialylated glycosylated peptides with 180 unique sialylated glycosylation sites were successfully characterized from 50 μg protein samples pretreated by PAM-g-allosex@SiO2 in two replicated experiments (Fig. 2.3C).

Zwitterionic molecules functionalized nanomaterials Zwitterionic molecule-functionalized materials are well-known super-hydrophilic and environmental materials. Generally, zwitterionic groups on nanoparticles can bind water molecules more strongly via electrostatically induced hydration, in comparison with water retention materials based on hydrogenbonding induced hydration. Thus, zwitterionic groups enable the formation of a layer of water molecules, which greatly contributes to hydrophilic interaction. Recently, additional hydrophilic interaction novel nanomaterials were prepared for glycosylated peptides enrichment, such as the zwitterionic polymer and “thiol-ene” cysteine (TE-Cys) bound to different supports (Fig. 2.3D and E). The thick zwitterionic polymer shell grants the nanomaterials not only with excellent hydrophilic surface properties but also with abundant zwitterionic functional sites on the polymer chains for high detection sensitivity (0.1 fmol), large binding capacity for glycosylated peptides (100 mg g
1), and high enrichment

NANOMATERIALS FOR THE SAMPLE PREPARATION OF PROTEIN ANALYSIS

55

efficiency (above 73.6%). As a promising zwitterionic monomer, cysteine can contain both positive and negative charges simultaneously at suitable pH and is capable of enhancing the efficiency of glycosylated peptides enrichment via hydrophilic interaction (Fig. 2.3F–H). Cysteine was functionalized on the surface of different nanomaterials via Au or other functional group [145, 147, 161]. Zhang et al. synthesized a novel zwitterionic-hydrophilic material named poly (N,N-methylenebisacrylamidecomethacrylic acid)@L-Cys (poly(MBAAm-co-MAA)@L-Cys), via the combination of distillation precipitation polymerization and “thiol-ene” click reaction (Fig. 2.3F) [146]. To attain high enrichment sensitivity and capacity for the pretreatment of glycosylated proteins/peptides, a novel ultrathin Au nanowire-assisted zwitterionic-hydrophilic magnetic graphene oxide (GO-Fe3O4/SiO2/AuNWs/L-Cys) was synthesized with combined properties of individual components including the good biocompatibility of GO, strong magnetic responses of Fe3O4, large surface area of ultrathin Au nanowires, and finally excellent hydrophilicity of L-Cys via four simple and rapid synthetic steps [144]. Due to its one-dimensional structure, the ultrathin Au nanowires were easily grafted with an abundant amount of L-Cys, which attributed to the excellent enrichment performance for glycosylated proteins/peptides, including low detection limit (10 fmol of human Ig G and 26 glycosylated peptides) and large binding capacity (150 mg g
1). In addition to conventional hydrophilic interaction, several other variations for this technique have also recently emerged, such as metal-based hydrophilic nanomaterials or carbon-based nanomaterials, for the enrichment of glycosylated proteins/peptides (Fig. 2.3I–L). Owing to the multivalent interaction between glycosylated peptides and silver nanoparticles (Ag-NPs), an Ag-NP functional shell was coated on the outside of a magnetic core via a PMAA interim layer (Fig. 2.3I) [157]. Meanwhile, TiO2 NPs can also be applied for the specific enrichment of sialic acid (SA)-containing glycosylated peptides via multipoint binding, and a protocol for the comprehensive analysis of SA-containing glycosylated peptides was established. In a combination of filter-aided sample preparation (FASP), TiO2 enrichment, multiple enzyme digestion, and high pH reversed-phase peptide separation, an optimized strategy was used for the establishment of SA glycosylation database in human plasma, in which a total of 982 glycosylation sites were identified in 413 proteins [156]. Furthermore, a shell of zeolitic imidazolate framework coated on magnetic graphene (MG@Zn-MOFs) (Fig. 2.3K) and a novel graphitic carbon nitride (g-C3N4) were also prepared and applied for the pretreatment of low-abundance glycosylated proteins/peptides and SA-containing glycosylated peptides (Fig. 2.3L) [158, 159].

Chemistry based pretreatment As the cis-diol group on the carbohydrate is present in glycan structures, pretreatment strategy was developed for the enrichment of glycosylated proteins or peptides without discrimination among different glycan structures. The chemical reactions involved in glycosylated proteins/peptides pretreatment include boronic acid chemistry, hydrazide chemistry, reductive amination chemistry, oxime click chemistry, coordination chemistry, and alkyne click chemistry. Lu et al. has given the detailed review to their respective mechanism and applications [162]. In the following section, the nanomaterials developed for the boronic acid chemistry and hydrazide chemistry method were discussed and summarized.

Boronic acid chemistry based nanomaterials Boronic acid can be reacted with the cis-diol group on the sugar moieties by producing five- or sixmembered cyclic esters in nonaqueous or basic aqueous media [163]. Importantly, this covalent cyclic ester is reversible in an acidic pH, and therefore, this mechanistic feature has been exploited to isolate

56

CHAPTER 2 NOVEL NANOMATERIALS FOR PROTEIN ANALYSIS

glycosylated proteins/peptides. Since boronic acid is stable, readily available, and their reactivity has been extensively studied, incorporation of these molecules onto solid supports is relatively simple, especially nanomaterials. Magnetic nanoparticles (MNPs) have become an ideal support for the functionalization of boronic acid groups for pretreatment of glycosylated proteins/peptides before MS analysis as the resulting nanomaterials can be easily isolated via an external magnet. Aminophenylboronic acid (APBA)functionalized MNPs (Fe3O4@SiO2-CHO-APBA) were synthesized through multistep covalent modification [164]. To simplify the multistep chemical procedure, the same authors first prepared the amine-functionalized MNPs (Fe3O4-NH2), followed by synthesis of the phenylboronic acidfunctionalized disperse core-shell MNPs (Fe3O4@SiO2@VPBA) using polymerization reaction and sol-gel method [165]. To further improve the abundance of boronic acid functionalized on MNPs, Au or polymer was also used to synthesize core-shell structure including Fe3O4@C@Au [166], Fe3O4@SiO2@Au [167], dendrimeric boronic acid-functionalized MNPs [168], and Fe3O4@P (AAPBA-co-monomer) NPs [169]. Due to the large amount of the functionalization group of the above nanomaterial, the boronic acid-functionalized magnetic nanoparticles exhibited significantly enhanced binding strength for glycosylated proteins. For example, the dendrimeric boronic acid-functionalized MNPs gave the dissociation constants of 10
5–10
6 M for glycoproteins, which was three to four orders of magnitude higher than the affinity associated with single boronic acid binding [168]. Besides MNPs, various novel nanomaterials have also been applied as solid supports in boronic acid chemistry-based approaches, and these nanomaterials exhibited remarkable performance for the pretreatment of glycosylated proteins/peptides owing to their unique nanostructures. For instance, boronic acid-functionalized FDU-12 (FDU-12-GA) and MCM-41 were synthesized for the enrichment of glycosylated peptides [170, 171]. The detection limit of glycopeptides had two orders of magnitude improvement by the prepared boronic acid-functionalized FDU-12 (FDU-12-GA) compared with traditional analysis. With the combination of the size exclusion effect of MCM-41 and the selectivity of boronic acid chemistry, this material was used in glycopeptidome research with excellent selectivity (glycopeptide/nonglycopeptide analyses at molar ratios of 1:100), ultralow sensitivity (femtomolelevel detection limitation), good binding capacity (40 mg g
1), and high recovery of glycopeptides (up to 88.10%). With the assisted alkyl linker, aminophenylboronic acid (APBA) was functionalized on the surface of detonation nanodiamond (dND), and then, the specific enrichment capacity of this material was as high as 350 mg of glycoprotein per gram of dND [172]. To enhance the dispersibility and the amount of boronic acid group on the surface of dND, Yang et al. functionalized dNDs with APBA assisted by poly-L-lysine and poly(ethyleneglycol)-diglycolic acid (PEG) with good dispersibility and plentiful boronic acid functional groups [173]. Meanwhile, boronic acid has also been functionalized on MALDI plate with various supports, including Au NPs and gold-coated silicon wafer for the on-plate enrichment of glycosylated peptides [174].

Hydrazide chemistry based nanomaterials The hydrazide chemistry-based method, another important type of N-glycoproteome enrichment strategy, was first developed by Zhang et al. [175]. The carbohydrates were firstly oxidized to aldehydes and then covalented with hydrazide groups on the support. The proteolysis step was followed to remove the nonglycosylated peptides and release the N-linked glycopeptides from the support with PNGase F for the identification of glycosylated peptides and glycosylation site. Initially, resins or gels were

NANOMATERIALS FOR THE SAMPLE PREPARATION OF PROTEIN ANALYSIS

57

applied for the immobilization of hydrazine, but these materials are difficult to be dispersed and separated, and the enrichment efficiency is low. To make the enrichment method easier and more efficient, some materials including magnetic nanoparticles and Au NPs were exploited to be modified with hydrazine groups for specific pretreatment of glycosylated proteins/peptides from biological samples [176]. Among the progress, hydrazide groups were grafted on the outside of superparamagnetic silica particles, and the coupling capacity was about 36 mg of total glycosylated protein mass per gram particles [177]. To develop a simple and rapid glycosylated protein/peptide isolation method by modifying magnetic particles with hydrazine chemistry, a hydrazine-functionalized magnetic particles (HFMP) was prepared by four different routes [178]. HFCA derivatized from adipic dihydrazide and the carboxyl-silanized magnetic particles exhibited good enrichment capacity (130  5.3 mg bovine fetuin per gram particles) and best specificity for capturing of glycoprotein. A longer spacer assisted for functionalization of hydrazine on solid supports may also decrease the steric effect and hence good capture efficiency. Further, a new type of hydrazide-functionalized core-shell magnetic nanocomposite, Fe3O4@poly(methacrylic hydrazide) (Fe3O4@PMAH), was fabricated to develop a more sensitive enrichment approach for in-depth glycoproteome research, which showed more than five times enrichment capacity compared with hydrazide resin for enrichment of standard glycopeptides [171].

Lectin affinity-based pretreatment Lectins can be specifically bind to carbohydrate residues of glycoproteins or glycolipids and have been used in isolation of biomolecules [179]. Lectin binding to the target glycan structure follows several factors including multivalency, water- and metal-stabilized structures adjacent to the binding site, and the molecular geometry. Although lectins have weak and variable affinities for glycosylated proteins (mM to μM), the lectin-based pretreatment method is highly specific. To enhance the binding affinity, binding and separation protocols to special lectin have been optimized. More than 400 lectins have been discovered from a variety of species including viruses, bacteria, plants, and animals. With the high specificity and good biocompatibility, lectin has been used for capturing specific glycan structural motifs. Generally, lectins are grafted on agarose, sepharose, silica particles, or monolith as the stationary phase of chromatography column [180]. For example, concanavalin A (Con A) is the most widely used lectin and has been employed for glycosylated protein/peptide enrichment from biological samples with specific binding to oligomannosidic, hybrid, and diantennary N-glycans by forming a boronic acid-sugar-Con A bond in a sandwich structure using. Using methyl α-D-mannopyranoside, Con A was immobilized on APBA-functionalized MNPs, which was applied for the pretreatment of glycosylated proteins. From human hepatocellular carcinoma cell line, a total of 184 glycosylated sites were detected within 172 different glycosylated peptides corresponding to 101 glycosylated proteins [181]. To improve the effectiveness of glycoprotein capture, a silica-based Con A (SiO2-ODex Con A) was synthesized to enrich glycosylated proteins/peptides using oxidized dextran as the spacer, which reduced the complexity of the synthesis and provided microenvironment to maintain the spatial configuration of Con A molecules [182]. After pretreated with SiO2-ODex Con A material from human serum, the biological samples were identified with a total of 109 glycosylation sites corresponding to 50 glycoproteins in one fraction by LC-MS/MS characterization. In contrast, 86 glycosylation sites from 45 glycoproteins have been reported in human serum using the multilectin column [183].

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NANOMATERIALS FOR PROTEIN IDENTIFICATION As the connection of cell physiology and genetics, proteins provide a window into complex cellular regulatory networks. Thus the high-throughput identification of proteins in complex samples is vital in biology. Prior to protein identification and characterization by MS analysis, well-defined and reproducible peptides should be obtained by the specific enzymatic reaction in a proper time. The conventional in-solution digestion is limited by time-consuming digestion procedure, autodigestion of enzyme, sample loss during concentration, and artificial modifications, severely affecting the efficiency and the result of the identification of proteins. Therefore, developing novel digestion methods was necessary to achieve highly efficient proteolysis for the identification of proteins. Besides the conventional procedure for the identification of proteins or peptides by digestion, matrix-assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS) is another technology for generating molecular maps within the tissue section by the combination of the identification of proteins and peptides with their spatial localization [184]. Therefore, MALDI-IMS is an unbiased approach to search for potential disease-related proteins and peptides (complementing immunohistochemistry). In imaging application, matrix is coated onto the whole tissue sections, and the mass spectra signals of targets molecules are acquired and analyzed into two- or three-dimensional sections. To enhance the molecule weight field analyzed by MALDI-MSI and its sensitivity, various matrices have been developed to increase the MS signals. With the development of nanoproteomics, nanomaterials have play important roles in the above demands including enzyme immobilization and MALDI-MSI matrix.

ENZYME IMMOBILIZATION To develop more efficient digestion procedures, the immobilized-enzyme reactors have recently attracted considerable interest. Immobilized-enzyme reactors have several advantages for proteolysis in protein analysis, including a low amount of autodigestion, a larger surface and interface area for digestion, a rapid proteolysis, and the good recovery of samples. The result from experiments using these immobilized-enzyme reactors has a higher sequence coverage than that obtained using conventional in-solution digestion [185]. Immobilization of enzyme requires a stationary phase/support onto which enzyme is fixed via covalent linkage, adsorption, affinity binding, or entrapment/encapsulation [186]. In recent years, various types of nanomaterials have been employed in enzyme immobilization, such as nanoparticles, nanofibers, and mesoporous materials [187]. Nanomaterials have several advantages such as large surface areas, tunable pore sizes that can be tailored to the dimensions of proteins, readily functionalized surfaces, multiple sites for interaction or attachment, and reduced mass transfer limitation. Consequently, researchers have used nanomaterials to prepare various forms with different stationary phases for a high amount of enzyme and ultimately offering enzyme-linked materials with long-term stability and enhanced proteolytic efficiency.

Magnetic nanoparticles Magnetic nanoparticles (MNPs) can be easily dispersed in solution and recovered by a simple magnet, and these materials mitigate the shortcoming of general nanoparticles used as carriers of enzyme, such as forming clumps and sonication with ultrasonic waves for temporary dispersion and separation by centrifugation or membrane. In most cases, enzyme was immobilized on the surface of MNPs via covalent bonding via cross-linker groups including epoxide groups [188, 189], aldehyde groups [190],

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carboxylic groups [191], and terminal amine group [192]. With the assistance of the trypsinimmobilized MNPs, the protein digestion could be completed in a short time (5 min), without any complicated reduction and alkylation procedures. Meanwhile, in accordance with the requirement for the optimal catalytic activity and enzymatic stability, the mild reaction condition was considered for the immobilization of enzyme. Activated by N-hydroxysuccinimide (NHS)-1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDAC) or NHS-1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), the carboxylic groups on the surface of the MNPs can be bonded with the amino groups on enzyme in a mild condition [193]. To comprehensively study the general functionalization strategies for MNPs, Li et al. compared the properties of the immobilized trypsin on the MNPs with different terminal groups, such as terminal amine (–NH2), epoxy (–COC), carboxylic (–COOH), aldehyde (–CHO), thiol (–SH), and maleimide (–PMPI) groups [191]. In their investigation, the highest activity per gram of MNP was obtained on –COOH and –SH surfaces, followed by –PMPI, –NH2, and –CHO surfaces. The trypsin immobilized on the –SH surface shows efficient proteolysis to the cysteine-containing proteins. To enhance the loading amount of enzyme and decrease the steric hindrance between immobilized enzyme and proteins, a new type of approach was developed for the immobilization of enzyme on magnetic nanoparticles by a hairy non-cross-linked polymer chain [194]. The non-cross-linked hairy polymer chains have numerous binding sites and place as scaffolds to support three-dimensional enzyme immobilization. Meanwhile, as no cross-linkage is present between these polymer chains, proteins could penetrate through this material, thus leading to the improvement of the immobilized-enzyme efficiency. The physical adsorption method was normally applied for the immobilization of enzyme to decrease the loss of enzyme activity. Enzyme can be immobilized on support by ionic interactions, hydrogen-bonding networks, van der Waals forces, hydrophobic interaction, etc. As enzyme absorption does not require additional reagents, it has the advantage that this method does not affect the biological activity of enzyme. For instance, the enzyme could be bound to the MNPs via Cu2+ chelation [195, 196]. The magnetic microspheres were first modified with tetraethylorthosilicate (TEOS) and then reacted with GLYMO-IDA, formed by the reaction of GLYMO and iminodiacetic acid (IDA). Subsequently, the copper ion and trypsin were introduced onto the surface. To increase the detection sensitivity of small volume sample, the prepared microspheres were packed into the microchannel by a strong magnetic field to make an on-chip enzymatic microreactor. The proteolysis of a fraction of protein extracted from rat liver could be completed within 5 min, and 23 unique peptides corresponding to seven proteins were identified [196].

Nanoporous or mesoporous materials Based on the former MNPs, immobilization of enzyme can only occur on the surfaces. In fact, a larger surface available on the solid support will provide higher abundance of the enzyme. Therefore, porous supports are favored for the conjugation of enzymes and offer attractive loading capacity. Generally, enzymes can be immobilized into the mesoporous channels by weak physical adsorptions, such as hydrogen bonding, hydrophobic attraction, or electrostatic interaction, or by covalently linkage inside the mesoporous channels on reactive groups such as aldehyde, epoxide, and thiol groups in the nanomaterials such as SBA-15, FDU-12, and MCM-41. To achieve rapid digestion, high sequence coverage, and sensitivity in protein identification, Yang and coworkers reported protein digestion placing in the nanoreactor channels of mesoporous silica SBA-15 and evaluated the performance by MS analysis. After incubation of 10 min, eight peptides that cover 58% of the myoglobin sequence with an intense

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signal (signal/noise ratio >70) were identified using the mesoporous silica-based nanoreactors. In comparison, the conventional overnight in-solution digestion of proteins generated only three peptides with 27% sequence coverage [197]. Enzyme immobilized on mesoporous structures can provide stable enzyme adsorption with high efficiency. Terracciano et al. found that the well-ordered porous array of SBA-15 (6 nm) demonstrated the greatest adsorption capacity of enzyme in the host frameworks (1 min). Meanwhile, compared with the unmodified SBA-15 with the same pore diameters, the amine-functionalized SBA-15 showed higher digestion efficiency and the proteolytic efficiency and exhibited 1000 times faster than that of using conventional in-solution method. With the confinement from molecular crowding in mesoporous pores, the catalytic activity of protein and enzyme can be both stabilized and induced order-of-magnitude enhancements. Although the adsorption of trypsin on the functionalized SBA-15 via electrostatic interaction is simple, the risk of trypsin leaching out is a concern. For covalent immobilized enzyme in the channels of mesoporous silica SBA-15, Bein et al. developed the azide-functionalized SBA-15 that reacted with acetylene-modified trypsin in a copper (I) using the “click reaction.” The resulted immobilized trypsin showed retention of enzyme activity and stability without desorption under the experimental conditions [198]. For the mesoporous material-based enzyme reactor, the proper pore size is very vital to encapsulate the enzyme inside the channels of mesoporous material for enzymatic catalysis. Meanwhile, the efficiency of size-dependent separation using mesoporous materials is based on the ratio of between pore size and the size of proteins. Thus, if the pore size is suitable for to the enzyme, the enzyme would adsorbed on the internal surface, and then the proteins smaller than the pore size will be captured in the pore channel, which is subsequently digested, while proteins larger than the pore size will be excluded. This has been applied for the development of a size-selective digestion of low-molecularweight protein method. Zou et al. covalently immobilized trypsin on the thiol-functionalized SBA15 with the pore size of ca. 5.7 nm. The materials could exclude the BSA, which has an approximate dimension of 5.0  7.0  7.0 nm3, and capture low-molecular-weight proteins including cytochrome C, lysozyme, and myoglobin and allow these small molecular weight proteins to be enzymatically digested by the immobilized trypsin [199]. By this size-selective digestion, the low-molecular-weight proteins will be separated and proteolyzed in one step, thus having the potential for the rapid proteolysis and online LC-MS analysis of low-molecular-weight proteins from complex biological samples.

Carbon based nanomaterials Owing to the high surface area, high electric conductivity, mechanical strength, and chemical stability, carbon-based nanomaterials are emerging materials that are functionalized for separation science and bioconjugation. These include carbon nanotubes, nanodiamonds, and graphene oxide. Enzyme loading capacity and efficiency of the nanomaterial are usually higher when compared with the enzyme reactors prepared from larger particles. Hereby, these have attracted attention in the immobilization of enzyme. Both single-walled carbon nanotubes (SWNT) and multiwalled carbon nanotubes (MWNT) have been used for immobilization of protein/enzyme by physical adsorption and covalent bonds. Yang and coworkers reported a carbon-based nanomaterial in which trypsin was immobilized on carbon nanotubes assisted by dendrimer spacers [200]. Further, they fabricated a trypsin-immobilized polyaniline (PA)-coated Fe3O4/carbon nanotube composite, in which CNTs can increase its specific surface area and dispersibility. Additionally, polyaniline serving as the “outer clothing” was introduced into the composite to protect the nanostructures and to accommodate trypsin [201]. The obtained

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trypsin-immobilized PA/Fe3O4/CNT has been coupled with MALDI-TOF MS for the digestion and peptide mapping of bovine serum albumin (BSA), myoglobin, and lysozyme with the corresponding amino acid sequence coverage of 46%, 81%, and 63%, respectively. As a nanomaterial member of carbon allotropes, nanodiamond has a specific chemical stability and biocompatibility. Detonation nanodiamonds (dNDs) have a large surface area and a variety of surface functional groups, which can be utilized in immobilization of enzyme. After activated by carbodiimide and treated with NHS, the carboxylated dNDs was functionalized with trypsin by covalent bonds, and subsequently used for the proteolysis of myoglobin. These materials produced superior sequence coverage as compared with that obtained with in-solution digestion or commercial Poroszyme® beads [202]. Meanwhile, the enzymatic performance immobilized on dNDs was also investigated by the proteolysis of standard proteins, including the pH tolerance, temperature, immobilized time, and nanoparticle concentrations. The optimized nanoparticles/nanomaterials with immobilized enzymes often show four advantages than the native enzymes: (i) wider temperature tolerance range, (ii) broader working pH, (iii) greater thermostability, and (iv) reusability. The high surface-to-volume ratio and chemical, electrical, and optical properties of nanoparticles or nanomaterials can increase enzyme loading and affect the diffusion of immobilized enzymes. Possessing specific structures, graphene oxide is an ideal nanomaterial with large surface area for the immobilization of enzyme. With the help of the abundant hydrophilic groups, the immobilized GO exhibits well dispersibility in aqueous solution. Jiang et al. functionalized GO sheets with aminefunctionalized Fe3O4 nanoparticles via covalent bond [203]. Trypsin was following immobilized on the prepared material via stacking and hydrogen bonding for efficient microwave-assisted proteolysis. The novel trypsin-immobilized nanocomposite was successfully employed for the digestion of bovine serum albumin and myoglobin with the 275 μg mg
1 of the enzyme immobilization amount. As the enzyme-functionalized GO is well dispersed in aqueous solution, these materials can find a wide range of applications in the fabrication of microchip bioreactors for highly efficient proteolysis. The feasibility and performance of the novel GO-based microfluidic bioreactors were demonstrated by the proteolysis of standard proteins, and the digestion time was significantly reduced to 5 s. Recently, Yang immobilized trypsin onto the GO surface assisted to fabricated bioreactors for efficient microwaveassisted proteolysis [204]. The use of spacer molecules for the immobilization of trypsin on graphene oxide proved to be beneficial. For instance, dendrimer was applied as spacer for the functionalization of trypsin onto GO nanosheets on which the loading amount of trypsin was calculated to be about 649  20 mg g
1, and an efficient digestion of standard proteins could be obtained within 15 min [205].

Other nanomaterials There are other nanomaterials that either are underdeveloped or show promise in bioconjugates, such as gold nanoparticles, metal-organic frameworks (MOFs), and nanoporous alumina. Due to their unique physical and chemical properties, gold nanoparticles (Au NPs) have also attracted interest for enzyme immobilization. Yang et al. reported a microreactor established by entrapping trypsin in a microchip coated with a gold nanoparticle network for the efficient online proteolysis of low-abundance proteins and complex extracts of the mouse macrophages. The PDDA/Au NP-assembled microchip was fabricated via a layer-by-layer electrostatic binding of PDDA and Au NPs. Here, charge-stabilized Au NPs provide a mild microenvironment similar to that of enzymes in native states and thus offer a large specific surface area for enzyme binding. The maximum proteolytic rate of the immobilized trypsin was

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400 mM min
1 (mg of enzyme)
1, and low-abundance proteins down to femtomole could be proteolyzed. Using this enzymatic microreactor, proteins isolated from the mouse macrophages were efficient digested, and 497 proteins were identified by 2-D-HPLC-ESI-MS/MS analysis. Due to their extraordinary surface areas, high porosity, and high functional tenability, MOFs have also become a promising type of porous materials for the biological conjugation [206].

LASER DESORPTION IONIZATION MASS SPECTROMETRY IMAGING (LDI-IMS) AND MATRIX Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS) have played important roles in the proteomics. ESI-MS can easily couple with online pre-MS separation, such as high-performance liquid chromatography or capillary electrophoresis. Meanwhile, MALDI-MS is a robust platform for the identification of proteins due to ease of sample preparation and tolerance of salts. In MALDI-MS, low-molecular-weight and energyabsorbing matrices are used over the target samples or tissue sections to minimize sample damage from laser irradiation. The suitability of an organic compound as matrix is determined by the type of the laser and its emission wave length. The most popular of matrices for MALDI-MS with a N2 laser at 337 nm include 2,5-dihydroxybenzoic acid (DHB), a-cyano-4-hydroxycinnamic acid (CHCA), and sinapinic acid (SA). Although the above matrices are essential in the energy relay process from the laser and transfer it to crystallized target molecules, these matrix molecules are ionized and generate significance background signals of small matrix ion suppressing and hindering small-molecule analysis. Although a homogeneous mixture is assumed to be spotted on the MALDI plate, “hot spots” exist on the plate with traditional matrix and thus limited the record of uniform signal of LDI-IMS. Therefore, nanomaterials of various sizes, shapes, and compositions have been tested for their applicability in the MS matrix for analysis of targets in aqueous sample or tissue section to solve the limitation of traditional matrices [207]. Nanomaterials have been applied and designed as efficient assisted matrices for LDI-MS with their unique properties including large surface area, absorption in the ultraviolet region (220–370 nm), and abundance functional group. Compared with the traditional organic matrices, nanomaterial-assisted LDI has some advantages including the accessible detection of the low-molecule-weight regions (<700 Da) with a background-free mass spectrum, the excellent shot-to-shot reproducibility, the enhancement of signal intensity, and the ability of pretreatment of analytes from complex samples. Nanomaterials including carbon-based and metal-based materials have been successfully applied as the assisted matrices in LDI analysis.

Carbon-based nanomaterials Carbon-based nanomaterials including carbon nanotube (CNTs), fullerenes, nanodiamond, and graphene have been used as promising matrices for LDI to analyze proteins, peptides, and corresponding small molecules. CNT was firstly applied as an effective MALDI matrix for small molecules in 2003 [208]. In the related study, oxidized CNT was reported to be a matrix substance for the analysis of small molecules, oligosaccharides, peptides, and insulin [209, 210]. Due to the oxidation process, many impurities and surface functionalization were removed on CNTs. The oxidized CNT matrix thus exhibits excellent reproducibility of peak intensity within and between sample spots. Additionally, intensity and signal-to-noise ratios of analytes are much improved over the simple CNTs. Furthermore, CNTs can be

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functionalized to be both the adsorbent in solid-phase extraction and the matrix for LDI-MS for the analysis of small molecules including β-Arg, propranolol, and Leu-Tyr. The detection limit for small molecules improved 10–100-fold when the samples were pretreated by CNTs [211]. With the high thermal conductivity and transparent in near-UV region, detonation diamond has also gained attention as the matrix for the MALDI- or imaging MS. Detonation nanodiamond with the diameter from 5 to 10 nm can be deposited as a thin layer on MALDI plate to enhance the ionization of compounds based on the combined characteristics of physisorption and chemisorption on the detonation nanodiamond surfaces. Furthermore, Wang et al. prepared the sample using three partially separated layers formed by matrix, diamond nanoparticles, and analyte solutions. This method compensated the difference in ionization efficiency between carbohydrates and proteins and allowed the direct analysis of carbohydrates from protein mixtures [212]. With subbandgap absorption and heat confinement inside the wires, the boron-doped diamond nanowires (BDD NWs) were applied as the inorganic substrate for matrix-free laser desorption/ionization mass spectrometry (LDI-MS) analysis of small molecules, on which the limit of detection (LOD) of verapamil was 200 zmol μL
1 [213]. With the excellent optical and electric properties, graphene has been acted as an excellent matrix for LDI-MS [214]. Compared with traditional organic matrix, graphene exhibited a high desorption/ ionization efficiency for nonpolar compounds. Meanwhile, graphene as matrix could avoid fragmentation of analytes and offer good reproducibility and salt tolerance. Min et al., prepared various types of different surface-immobilized carbon materials utilizing graphene oxide (GO), reduced GO (rGO), multiwalled CNT (MWCNT), and aminated MWCNT for comparison of LDI efficiency. GO/ MWCNT-NH2 and rGO/MWCNT-NH2 double-layer platforms showed excellent performance for the analysis of enzyme activity, small molecules, and mouse brain tissue mass imaging because of negligible interference, high salt tolerance, mechanical durability, and homogenous signal intensities across regions [215]. To enhance the ionization efficiency and sensitivity of LDI-MS, the composites of graphene combined with other materials were developed, such as graphene coated with mesoporous silica (G@SiO2) and graphene oxide embedded sol-gel (GOSG) film [216, 217].

Metal-based nanomaterials Metal-based nanomaterials are also important materials that can be used as highly efficient matrices for the biological molecule analysis by LDI-MS, such as Au NPs [218], silver nanoparticles [219], TiO2 [220], zinc oxide (ZnO) nanowires [221], platinum nanoflowers [222], ZrO2 [223], and BaTiO3 nanoparticles [224]. These nanomaterials provide several advantages that include a large surface-areato-volume ratio, strong absorption in UV spectral range, high stability, and operability. Due to the straightforward sample preparation, high ionization coefficiency, excellent stability, and biocompatibility, Au NPs have achieved the high attention as potential inorganic matrix for the analysis of small molecules by LDI-MS. Russell et al. firstly applied size-selected (2–10 nm) Au NPs for the MALDI analysis of peptides. However, with the production of Au cluster peaks, the application of Au NPs was limited in LDI-MS [225]. To solve the above problem, various modified Au NPs were fabricated and used as matrices for LDI-MS. Citrate-capped Au NPs served as matrix for the determination of biomolecules including neutral steroids and carbohydrate. To avoid the poor reproducibility of Au NPs homogeneously distributed on the plate, the on-chip patterned Au NP microarray modified with a nanoscale silicate coating layer by layer was prepared and applied for analysis of small and medium compounds with LDI-TOF MS, which could be performed over many cycles with good reproducibility spot to spot [218]. With tree- or bouquet-like morphology with distinct physicochemical properties,

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flowerlike Au NPs were applied to improve LDI-MS by enhancing the ionization of peptides in the mass range up to 2500 Da and providing in parallel an effective internal calibration of mass spectra. Thus, flowerlike Au NPs, with gold monoisotopicity and excellent chemical stability, have a promising feature for the quantitative and qualitative analysis of putative biomarkers in diagnosis and therapy of diseases. TiO2 nanomaterials exhibit strong UV absorption characteristics with the 3.2 eV band gap of bulk anatase and the capability to rapidly transmit energy to analytes. Therefore, it was potentially applied as matrices for the analysis of peptides or small proteins by LDI-MS. TiO2 nanocrystals (TiO2 nanoparticles (TiO2 NPs)), TiO2 nanotubes (TiO2 NTs), and TiO2 prolate nanospheroids (TiO2 PNSs) with different shapes and sizes were investigated for the quantitative analysis of peptides or small proteins (steroid hormones, amino acids, and saccharides). It showed that all TiO2 nanocrystals, regardless of their size and shape, could be used as substrates for the efficiency ionization of steroid hormones, amino acids, and saccharides. Meanwhile, the larger nanocrystals, TiO2 PNSs and TiO2 NTs, exhibit the best reproducibility having potential applications for the quantification of small molecules [226]. Boukherroub et al. reported a method for analysis of peptides and small molecules by using surfaceassisted laser desorption-ionization mass spectrometry (SALDI-MS) coated with a titanium oxide (TiO2) nanotube layer based on electrochemical anodization. Compared with the classical MALDIMS, the detection limit was improved 10-fold for the analysis of tyrosine kinase inhibitor and verapamil with the prepared TiO2 nanotube substrate [227]. Moreover, other TiO2 nanocomposites including nylon nanoweb with TiO2 nanoparticles and surface-modified BaTiO3 nanoparticles were also prepared as matrix of LDI-MS. The novel matrix can spread on the target plate homogeneously using a nylon nanoweb as a solid scaffold. With leucine-enkephalin (555.6 g mol
1) and cyclic citrullinated peptide (1668 g mol
1) as model analytes, the intensities of mass peaks and the S/N ratio of mass spectra exhibited to be far more improved than that observed with the TiO2 nanoparticles without nanowebs [228]. Nowadays, owing to the large surface area, tunable pore sizes, high porosity, and UV-range absorption, metal-organic frameworks (MOFs) have been used as matrix for LDI-MS. In the series MOFs, the cage-type MIL-100(Fe) gives a good stability of MS intensity and a good response with the polycyclic aromatic hydrocarbons (PAHs) [229]. Further, they applied MIL-100(Fe) as matrices for the LID-MS analysis of polar carbohydrates (glucose; sucrose; galactose; lactose; fructose; maltose; and a-, b-, and g-cyclodextrins (a-, b-, and g-CD)) with good reproducibility and low background interferences [230]. Combined the merits of MOFs with magnetic particles, magnetic nanocomposites coated with ZIF-8 (Fe3O4@ZIF-8 MNCs) were fabricated and used as pretreatment probe and matrix for the LDI-MS analysis of small molecules [231]. Recently, Huang et al. prepared carbonized MOFs (cMIL-53 and cCYCU-3) and applied these materials for the analysis of carbohydrates, peptides, PAHs, and phenolic acids with better MS response than the pure MOFs and nanoporous carbons as matrix [232].

NANOMATERIALS FOR THE QUANTIFICATION OF PROTEIN Proteins are vital biomolecules in living organisms and are closely related to the discovery of biomarkers in diseases. Meanwhile, the quantitative expression of proteins and their interacting partners may help systematic analysis of biological processes. Hence, highly efficient determination of absolute protein amounts and the quantification of differentially expressed proteins are one of the key subjects in

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proteomics analysis. Despite great advancements in the tools for the detection and quantification of proteins, the analysis of low-abundance proteins still faces great challenges. Nanomaterials with desired properties have been optimized for detection and quantification of low-abundance proteins. Herein, recent advances using different nanomaterials for the detection and quantification of proteins in an organism were summarized, with particular emphasis on the outlook for different nanomaterials applied for the quantitative protein analysis, as well as possibilities and limitation of nanomaterials in this rapidly growing field.

NANOMATERIALS FOR PROTEIN MICROARRAY In the systematic analysis of biological processes, the quantitative expression of proteins and their interacting proteins is a vital component in proteomics research. The differential expression of proteins, suggested by quantitative proteomics, reveals fruitful information regarding the changes between the normal state and the disease state. As a promising technique, protein microarrays are able to directly measure changes in the proteome. Moreover, the protein microarray can composite, quantify, and characterize proteins or proteoforms in the proteome arising from posttranslational modifications, splicing events, and interactions [232]. However, the sensitive and reliable detection of protein microarray needs to be improved to meet the increasing demands in studying the large number of proteins. To quantify target proteins and the disease biomarkers in biological samples, various biosensors have been developed [233]. Additionally, label-based systems like fluorescence, chemiluminescence, and radioisotope have witnessed substantial advancements. More recently, nanomaterials such as gold nanoparticles, quantum dots, dye-doped nanoparticles, and silica nanoparticles are employed for protein quantification studies. To reduce the impediment of labeling reaction in label-based systems, label-free detection techniques, including surface plasmon resonance (SPR), nanocomposite and microcantilevers have also advanced in measuring the inherent properties of target molecules, such as mass, dielectric, or optical properties. With unique properties, nanomaterials have extended surface for immobilization of biomolecules resulting in increased binding properties and enhanced detection sensitivity. Therefore, nanomaterials are developed and incorporated in several novel detection platforms discussed in this section.

Gold nanoparticles (GNPs) With a number of unique physical and chemical properties, gold nanoparticles (Au NPs) have drawn attention as ideal candidates for sensitive detection of biomolecules. These nanoparticles are known to have excellent, electronic properties, optoelectronic property, chemical stability, high quantum efficiency in a wide range of wavelengths, and excellent biocompatibility. Moreover, these properties of Au NPs can be easily adjusted by their size, shape, interparticle distance, and the solvent and temperature of the surrounding environment. Therefore, Au NPs have been employed for the development of novel detection methods with high sensitivity, stability, and reliability [233]. As feasible alternatives to fluorophore-based probes, Au NP conjugates have been used to scatter light and enhance the sensitivity of the assay with the amplification by electroless metal deposition. Recognition antibodies functionalized on gold NPs were applied as a tool for multiplexed detection of tumor markers for lung cancer with increased sensitivity on protein microarray. Using this method, detection of biomarkers in serum samples could reach to a concentration of <1 ng mL
1 in 1 h [234]. Based on an adsorption-controlled kinetic model, semi-quantitative sandwich nanoparticle

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immunoassays were developed utilizing a platform that includes antibody conjugated with gold nanoparticles, whose signal is further enhanced by silver nucleation. Target analyte could be detected at a concentration as low as 0.1 μg cm
3 (4 ng total) [235]. Utilizing the reduction of HAuCl4 by NH2OH and self-catalysis of Au NPs, a colorimetric method for the ultrasensitive detection of prostate-specific antigen (PSA) using a novel biotin-polyethylene glycol Au NPs probes is reported [236]. The signal of Au NPs can be amplified by introducing HAuCl4 –NH2OH solutions, in which Au NPs catalyze the reduction of Au3+ by NH2OH to form larger Au NPs. PSA and nucleic acid targets (microRNAs) from a single sample can be detected with low detection concentration (50 fM for nucleic acid targets and 1 μg L
1 for the PSA target). This method shows a potential in the microarray for biological samples with high sensitivity, selectivity, and simplicity. In lectin microarray, microbe-gold nanoparticle conjugates are applied as labels to monitor specific affinity of microbes with lectins, followed by a resonant light scattering (RLS) enhancement step based on the electroless deposition of silver onto the gold particles [237]. Using the same detection principle, a microarray format for the detection of carbohydratelectin or glycoprotein-lectin interactions by gold nanoparticle probes has also been developed by Wang et al. Compared with the conventional fluorescence-based microarray format, the RLS-based microarray format exhibits lower detection limit for both carbohydrate and lectin. However, this assay is only applicable to specific carbohydrate motifs rather than the entire glycome. Furthermore, Au NPs have also been used as signal enhancers for Raman scattering-based detection of biomolecules [238, 239]. For example, in a sandwich-type SERS immunoassay, the primary antibody was attached to Au NPs, which were immobilized on a substrate. After being exposed to a solution containing PSA, the immunoassay was interacted with the secondary antibody conjugated to Au NPs with Rhodamine 6G dye. The sensitivity of the novel immunoassays has been drastically enhanced in the SERS spectra with the detection limit of this immunoassay as low as  1 ng L
1 for PSA in human serum [240]. Wang et al. established a sensitive peptides microarray-based SERS assay using biotinylated anti-phosphoserine antibodies, which were tagged with avidin-conjugated Au NPs, followed by silver deposition for Raman signal enhancement to analyze the kinase function and inhibition [241]. The result demonstrates that the H89 inhibitor has highly selective inhibition to protein kinase A.

Quantum dots With a wide spectral band gap and stable light properties, quantum dots (QDs) are fabricated by a semiconductor fluorescent core coated with another semiconductor shell. The common QDs are composed of CdSe core covered by a ZnS shell. Upon UV excitation, this kind of QDs emits light ( 20–25 nm fwhm), which is adjusted with the size of the CdSe core. Meanwhile, the colloidal chemistry is well studied that allows the synthesis of five to six spectrally distinct colors of emission across the visible spectrum, making them excellent labels for multiplexed, high-throughput screening. QDs exhibit bright fluorescence (20–100 times higher) [242], excellent photostability against photobleaching (50–1000 times) [243], multifluorescence excitation, and greater quantum yield. Hence, with these advantages, QDs have diverse applications in diagnostic imaging [244], detection of cancer biomarkers [245], direct probing of human serum [246], and tumor biopsy analysis [246, 247]. With above advantages, QDs are applied to label peptides, protein, or oligonucleotides for the sensitive detection of microarrays [248]. In the potential Alzheimer’s disease biomarker screening research, the performance of QDs and the fluorescent dye Alexa 647 were compared. The QDs were shown as more effective reporters in the microarray, in which the limit of detection of apolipoprotein E by QDs was five times more sensitive than that of Alexa microarray [249]. Zhukov et al. innovatively

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established the streptavidin QD-biotinylated detector antibody model, in which six cancer-specific cytokines were successfully detected down to picomolar [246]. Moreover, after pegylation, streptavidinconjugated and pegylated QDs have improved sensitivity and specificity compared with their nonpegylated form in assays analyzing cellular protein extracts [250]. Combining the planar and suspension microarray, Jia et al. fabricated a sensitive and selective microbead array for multiplexed detection of three lung cancer biomarkers including carcinoembryonic antigen (CEA), fragments of cytokeratin 19 (CYFRA21-1), and neuron-specific enolase (NSE). QDs functioned as fluorescent signal amplifiers that result in low detection limit (CEA, 0.19 ng/mL; CYFRA21-1, 0.97 ng/mL; NSE, 0.37 ng/mL; and S/N ¼ 3). These probes can also be used directly with serum [251]. Recently, Sukhanova et al. designed a novel multiplexed suspension immunoassay system based on QD-encoded microspheres for simultaneous detection of three lung cancer markers in the human bronchoalveolar lavage fluid (BALF). The system showed almost identical results utilizing Luminex xMAP® assay [252]. However, the QDs conjugation and detection steps make it difficult to establish the best labeling technology and widespread application. To extend the QDs applied in the biology, several novel QD styles have been fabricated and applied for the rapid and high-throughput biological application, such as CdSeTe/CdS QDs [253], QD-encoded hydrogel microparticles [254], and Mn-doped ZnS QDs [255].

Other nanomaterials Due to the distinct electric and spectroscopic properties with strong and characteristic resonance Raman signatures, single-walled carbon nanotubes (SWNTs) have been applied as a promising label for SERS-based protein detection [256]. SWNTs possess unique properties such as enormous Raman scattering cross section (10
21 cm2 sr
1 molecule
1) and simple and tunable spectra. Dai et al. functionalized macromolecular SWNTs with specific Raman-labeled antibodies as multicolor microarray slide. The SWNT tags enable strong Raman intensity and decrease the detection limitation of protein down to 1 fM, which is three orders of magnitude more sensitive than conventional fluorescence [257]. Recently, Strano et al. developed a sensitive and selective label-free protein detection platform on the coupling of aptamer-anchor polymers to semiconducting SWNT near-infrared (nIR) emitters [258]. The selectivity for specific protein was realized via synthetic DNA aptamers adhered to SWNTs. On the platform, the single secreted proteins are from organisms including Escherichia coli, HEK 293, and Pichia pastoris cells in real time. Combined with the selectivity provided by aptamers, SWNTs are a promising nanomaterial for the long-term optical monitoring of specific protein from single cells over long timescales. Meanwhile, other carbon-based nanomaterials are also prepared as biosensors for the detection of biomarkers [259]. Dye-doped silica nanoparticles (NPs) are a type of silica nanoparticles that trap a large quantity of fluorescent dye molecules inside the silica matrix. This design leads to an increase of the quantum yield of the fluorophores, thus enhancing its brightness [260]. Silica NPs are known for their minimal toxicity, biocompatibility, environment-friendliness, easy modification and high recovery rate, and resistance to microbial growth. Therefore, entrapment of dye molecules in silica NPs enables selectively labeling on some important sample such as cancer cells, bacteria, and specific targets [261]. Rhodamine B isothiocyanate-doped silica NPs were synthesized using the St€ober method, and these molecules have an average fluorescence intensity that is 4000 times higher than conventional organic dye molecules. The novel dye-doped silica NPs were also used for the sensitive detection of reverse-phase protein microarrays [262]. Fluorescein-doped silica NPs modified with glycans were

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CHAPTER 2 NOVEL NANOMATERIALS FOR PROTEIN ANALYSIS

used as labels for screening carbohydrate targets on lectin super microarray. Compared with other traditional techniques, the rapid and high-throughput nature of the described lectin super microarrays, in conjunction with the versatility of the nanoparticle-based glycan labeling technique, provides a robust and unique approach for quantitative glycan profiling in a large library [263]. These novel platforms open new opportunities for fundamental research in glycobiology and for the development of novel diagnostic tools.

NANOMATERIALS FOR INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY (ICP-MS) As an element-specific detector, inductively coupled plasma-mass spectrometry (ICP-MS) has the advantages including low detection limits, good tolerance to sample matrix, a wide range of linear response, high sensitivity, and the ability of multiplex detection. Coupled with the specificity of the antigen-antibody assay, ICP-MS-based immunoassay has become a revolutionary technique for biomolecule quantification including protein [264–266], DNA [267, 268] and RNA [269, 270], living cells [271, 272], and bacteria [273, 274] with perfect sensitivity and excellent accuracy. Naturally, some special heteroatoms including metal isotopic (e.g., iron, zinc, and copper) and nonmetal atoms (e.g., phosphorus, sulfur, and selenium) present in biomolecules, which can be directly quantification by ICP-MS. However, the absence of metal atoms, in addition to existing challenges such as high detection limits and signal interference of the endogenous phosphorous and sulfur tags, limits the ICP-MS development. To overcome it, several types of element tags, including metal chelates, metal-containing compounds, and polymer-based elemental tags, have been applied for the quantification targets by ICPMS. For example, the metal-containing NPs were applied as element tags to increase their sensitivity in ICP-MS. Here, we summarized the application of metal nanomaterials in the quantification of proteins by ICP-MS. With the specificity of the antibody, the ICP-MS-based immunoassay does not require chromatographic separation and then easily reduces the effect of complex biological matrix. Zhang et al. firstly developed the ICP-MS immunoassay with DTTA chelate labeling workflow for the quantification of the thyroid-stimulating hormone (TSH) in serum [275]. To improve specific identification and signal amplification of ICP-MS immunoassay, different types of metal nanoparticles were utilized including Ag NPs, Au NPs, PbS NPs, quantum dots, metal-doped silica NPs, and upconversion NPs (Table 2.4). With its unique properties, Au NPs are the most used nanomaterials in the quantification of proteins by the ICP-MS. Baranov et al. applied Au cluster (<2 nm in diameter)-labeled antibodies combined with ICP-MS to quantify the target proteins. This method can detect target proteins at a concentration of 0.1–0.5 ng mL
1 with a linear response to protein concentration over three orders of magnitude [277]. Yan et al. applied aptamer-modified Au NPs and antibody-modified silver NPs as specific element tags for quantification of cytochrome C and insulin, respectively [282]. Combining the magnetic microparticle separation and ICP-MS detection, the ultrasensitive method for simultaneous determination of different target proteins was established (Fig. 2.4). The method provides a promising simultaneous determination of two or more low-abundance proteins from complex sample via multielement tags. Moreover, Lv et al. established an immunoassay with the signal amplification of catalytic silver deposition on immunogold tags to detect low-abundance proteins such as human carcinoembryonic antigen in serum with low limit of detection of 0.03 ng mL
1 [283]. Sanz-Medel et al. developed a novel Mn-doped ZnS QD-based immunoassay platform for highly sensitive detection of cancer biomarkers.

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NANOMATERIALS FOR THE QUANTIFICATION OF PROTEIN

Table 2.4 Analytic Performance of ICP-MS-Based Methods With Elemental Tags for the Quantification of Protein Analytic Method ICP-MS with Au NP labeling ICP-MS with Au cluster labeling

Au NP amplification-ICP-MS

Single-particle mode ICP-MS with Au NP labeling ICP-MS with nanogold labels

ICP-MS with aptamermodified Au NPs Au NPs (immunogold-silver amplification)-ICP-MS ICP-MS with Au NP labeling ICP-MS with Au NP labeling, Cs/FITC-doped MMNPS ICP-MS with Au NP labeling ICP-MS with CdSe QD labeling

ICP-MS with CdTe QDs labeling

ICP-MS with CdSe/ZnS QDs labeling ICP-MS with Mn-doped ZnS QDs labeling

Analyte Rabbit antiIgG IgG

Linear Ranges 0.8–50 ng mL


1

LOD 0.4 ng mL

References
1

[276] [277]

0.05–1 nM

3 ng mL
1 (filter immunoassay) 0.075 ng mL
1 (protein sepharose A immunoassay) 0.22 ng mL
1 0.0023 ng mL
1 10 pM

0.0018–18 nM

0.5 pM

[279]

0.3–10 ng mL
1

0.1 ng mL
1

[280]

62.5–2000 pg mL
1

28.7 pg mL
1

[281]

0.1–20 nM 0.2–40 nM 0.07–1000 ng mL
1

50 pM 110 pM 0.03 ng mL
1

[282] [282] [283]

HIV-p24 CA19-9

7.5–75 pg mL
1 1.5–90 units mL
1

1.49 pg mL
1 0.02 units mL
1

[284] [285]

Caspase-3 AFP Human IgG

1–200 ng mL
1 0.005–2.0 ng mL
1 0.2–10 ng mL
1 (Cd signal) 0.5–10 ng mL
1 (Cd signal)

0.42 ng mL
1 1.85 pg mL
1 0.058 ng mL
1 (Cd signal) 0.097 ng mL
1 (Cd signal) 8 ng mL
1

[286] [287] [288]

AFP HCG Human a-thrombin Human serum albumin Rabbitantihuman IgG Vascular endothelial growth factor Cytochrome c Insulin CEA

Human serum albumin Transferrin Human IgG Transferrin PSA

10
3–104 pg mL
1

[278]

[289]

20 ng mL
1 30 ng mL
1 0.18 ng mL
1

[290]

0.026 fg mL
1

[255]

Continued

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CHAPTER 2 NOVEL NANOMATERIALS FOR PROTEIN ANALYSIS

Table 2.4 Analytic Performance of ICP-MS-Based Methods With Elemental Tags for the Quantification of Protein—cont’d Analytic Method

Analyte

Linear Ranges

LOD

References

ICP-MS with CeO2-deposited mesoporous silica nanoparticles and MNPs ICP-MS with Y, Cd, Au-doped silica NPs and Cs-doped MMNPs

CEA

10
3–5 ng mL
1

0.36 pg mL
1

[291]

AFP Neuronspecific enolase (NSE) c-reactive protein (CRP) PSA

2–200 ng mL
1 2–200 ng mL
1

0.37 ng mL
1 0.36 ng mL
1

[292]

200–104 ng mL
1

77 ng mL
1

ICP-MS with TiO2 NPs labeling ICP-MS with PbS NPs labeling ICP-MS with NaYF4:Yb,Er upconversion nanoparticle (UCNP) labeling

1.16 fg mL
1

[293]

CEA

0.2–50 ng mL
1

0.058 ng mL
1

[294]

AFP

0.5–35 ng mL
1

0.22 ng mL
1

[295]

The amplification of signal ICP-MS was achieved by the deposition of gold ions on the Mn-doped ZnS QD tags, on which gold ions might be reduced to gold. The sensitivity of detection of prostate-specific antigen (PSA) is at the low attog mL
1 level. Another highly sensitive immunoassay based on singleparticle mode detected by ICP-MS in a time-resolved analysis (TRA) mode with Au NPs serving as model tags was developed. Based on the single-particle mode, the limit of detection of the ICP-MS immunoanalysis of rabbit-antihuman IgG was nearly one order of magnitude lower than traditional immunoassays based on integral mode [280]. With the development of different metal nanomaterials, the ICP-MS immunoassay has expended the quantification of low-abundance targets in complex sample. However, signal amplification, the elimination of matrix, and high-throughput detection should be taken for progress. Therefore, the new labeling tags for the absolute quantification of protein in complex sample by ICP-MS could be focused on high sensitivity, selectivity, and efficiency of the labeling process and multiplex tags for the proteome analysis and the determination to specific sites or modification.

SUMMARY AND PROSPECT The development of nanomaterials is attributed to the promising progress in the sample preparation, identification, and quantification of protein. Various kinds of nanomaterials possess unique properties including simple separation, high sensitivity, larger capacity, electrophonic, thermal, and mechanical properties. Meanwhile, these materials modified by various kinds of functional groups extend the different applications including pretreatment of low-abundance proteins or peptides and posttranslation

SUMMARY AND PROSPECT

71

Proteins

Surface-activated MMPs Block

1% BSA

Antibody−AgNPs Aptamer−AuNPs

Heat Dissociation

Magnetic separation ICPMS

FIG. 2.4 Schematic diagram showing the principle of the developed immunoassay and aptamer-based bioassay for determining two proteins by combining Au NPs and Ag NPs label, magnetic microparticle separation, and ICP-MS detection. Target proteins were immobilized on the surface of activated MMPs by glutaric dialdehyde coupling, followed by adding BSA to block the free activated sites. Aptamer-modified Au NPs and antibody-modified Ag NPs were added as probes to react with the corresponding protein. The protein-probe complex was separated from the sample mixture under a magnetic field. After washing, the protein-probe complex was heat dissociated (at 90°C). The MMPs were removed, and the Au NPs and Ag NPs in the solution were analyzed by ICP-MS (Copyright 2011, Royal Society of Chemistry).

modification proteins or peptides, immobilization of enzyme, supporting matrix for MS or IMS analysis, and the relative or absolute quantification of proteins. Nowadays, the pretreatment techniques and approaches developed for protein analysis have achieved enormous progress. Facing with complexity of biological samples, proteins possessing vital biological and medical functions are those relatively low-abundance proteins and having posttranslational modifications. For efficient enrichment of low-abundance proteins, silica-based, carbon-based, polymer-based, and metal-based nanomaterials have developed. However, the high sensitivity and 100% recovery could not realize in this field. Thus, the high adsorption and recovery method based on nanomaterials are needed for the pretreatment of low-abundance proteins in complex sample in the future. Meanwhile, to enhance the efficiency profile of low-abundance protein with low concentration in huge volume, the online enrichment methods combined with MS should be developed.

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CHAPTER 2 NOVEL NANOMATERIALS FOR PROTEIN ANALYSIS

At present, facing with the single-cell analysis, several groups have set up methods to analyze the proteome of hundreds of cell sample [296]. Nanomaterials assisted for the cell pretreatment will make a promising development in the field. For the specific pretreatment of PTM proteins or peptides, the design and synthesis of new nanomaterials with functional groups have gained increasing attention in recent years. With various kinds of PTM in biology, most of nanomaterials focused on the two major forms of protein PTM, namely, phosphorylation and glycosylation. Because of the hydrophilic group of glycosylation and phosphorylation, two of them can be enriched by some pretreatment nanomaterials on time, which would give chance to the simultaneous characterization of cross talk of them. However, in the specific pretreatment method, both selectivity and sensitivity are indispensable. The ideal pretreatment method for PTM enrichment is to reach a balance between selectivity and sensitivity, which present high demand to the fabrication and design of nanomaterials. Meanwhile, the challenges of revealing other PTMs still demand the development of new approach and novel nanomaterials for the enrichment and identification of them. For the efficient identification of proteins in biological sample, nanomaterials combined with MS have made a stride in the deep profiling of the proteome. The immobilization of enzyme on different nanomaterials has shown improved proteolysis resulting higher sequence coverage and long-term enzyme stability, in addition to possible reusability of the nanomaterials. Recently, coupling the enzymeimmobilized nanomaterials with MS and/or LC is applicable to the development of automated, high-throughput proteomics analyses. With the demand of deep profile of proteome and PTM analysis, novel pretreatment methods of online sample extraction, digestion, enrichment, and separation shed light on designing and synthesizing new materials. Additionally, these novel nanomaterials with unique properties, namely, large surface area, strong ultraviolet absorption, and special optical properties, have enabled these materials to be ideal matrices for the analysis of different kinds of targets (including small molecules, peptides, and proteins) and imaging mass spectrometry analysis. These nanomaterialbased matrices have empowered a more robust mass spectrometry-based analysis. The biological samples pretreated by nanomaterials are more homogenous than traditional methods and thus eliminate the presence of “sweet spots". The experimental workflow also reduces background signals in the low mass region. However, the inorganic matrices capable of analyzing high-molecular-weight proteins are limited. Additionally, internal standard in the imaging mass spectrometry is need for the quantification of biological molecules for MS imaging. In the systematic analysis of biological processes, relative and absolute quantification provides vital information regarding the relationship among proteins, protein families, and complexes. In recent years, protein microarray offers many avenues to simultaneously analyze multiple samples to achieve relative quantitation of proteins for potential use in diagnosis, prognosis of diseases, and possible therapeutic efficacy. To improve the sensitivity and quality of microarray data, different nanoparticle amplifiers such as Au NPs, QDs, CNTs, and dye-doped silica NPs have been developed to bind target of interest. In the future, standardization efforts concerning experimental design of the nanoparticle amplifier and the assay itself and quality controls are stringently required to compare the results produced in different laboratories. Meanwhile, the novel nanoparticles that possess good stability in aqueous and high ionic solution will benefit for the reliable and reproducibility of protein microarray data. As nanomaterials have great potential in the field of label-free assays, new nanomaterials are being developed by studying the surface chemistry of nanoparticle. Finally, the development of multifunctional nanomaterials will extend their future utility as signal amplifiers in protein microarrays. ICP-MS has drawn more attention with the development of labeling strategies and nanomaterial tags for the quantification

REFERENCES

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proteins, nucleic acid, cells, bacteria, and viruses. In the future, the ICP-MS assisted by nanomaterials will continue to evolve in the design and fabrication of novel tags. Meanwhile, to keep pace with proteome, the accuracy of the quantification for multiplex detection in complex samples should be ensured in the ICP-MS with labeling tags. Furthermore, for the application of ICP-MS in the quantification of a smaller size organism, novel element tags are needed to enhance the synergy of ICP-MS and other technologies including luminescence imaging and molecular MS. With the development of novel element tags, ICP-MS will continue to play an important technique in the further clinical research.

ACKNOWLEDGMENT This work is supported by Special Project on Precision Medicine under the National Key R&D Program (SQ2017YFSF090210), National Key Research and Development Program of China (2016YFA0501300, 2017YFA0505100, and 2017YFA0505001), National NSF (21305018, 31570825, and 31600665), RFDP (20130071140007), Shanghai Pujiang Program (18PJD002) and Open Foundation of Shanghai Key Laboratory of Forensic Medicine (KF1404). We would like to thank Zhijie Wu for critical reading of this manuscript.

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