Bionanoconjugation for Proteomics applications — An overview

JBA-06816; No of Pages 19 Biotechnology Advances xxx (2014) xxx–xxx

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Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Bionanoconjugation for Proteomics applications — An overview João Pinto da Costa a,1, Rui Oliveira-Silva a,1, Ana Luísa Daniel-da-Silva b,⁎, Rui Vitorino a,⁎ a b

Mass Spectrometry Centre, QOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal CICECO, Aveiro Institute of Nanotechnology, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

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Article history: Received 17 December 2013 Received in revised form 15 March 2014 Accepted 26 April 2014 Available online xxxx

Formed as an interdisciplinary domain on the basis of Human Genome Project, Proteomics aims at the large-scale study of proteins. The enthusiasm that resulted from obtaining the complete human genetic information has, however, been chastened by the realization that this information contributes little to the comprehension and knowledge of the expressed proteins. In the wake of this realization, the Human Proteome Project (HUPO) was founded, which is a global, collaborative initiative, aiming at the complete characterization of the proteins of all protein-coding genes. Nonetheless, the rapid detection of these molecules in complex biological samples under conditions considered to be of clinical relevance is extremely difficult, requiring the development of very sensitive, robust, reproducible and high throughput platforms. Nanoproteomics has emerged as a feasible, promising option, offering short assay times, low sample consumption, ultralow detection and high throughput capacity. Additionally, the successful synthesis of biomolecules and nanoparticle hybrids yields systems which often exhibit new or improved features. Herein, we overview the recent advances in bioconjugation at the nanolevel and, specifically, their application in Proteomics, discussing not only the merits and prospects of Proteomics, but also present day limitations. © 2014 Elsevier Inc. All rights reserved.

Keywords: Nanoproteomics Bioconjugates Nanoparticles and nanomaterials

Contents Introduction . . . . . . . . . . . . . . . Concepts and early steps . . . . . . . . . General applications of nanomaterials . . . Within the scope of Proteomics . . . . . . General considerations . . . . . . . . . Enzyme immobilization and digestion . . Enrichment of low abundance proteins PTM enrichment . . . . . . . . . . Bridging the gap — linkage . . . . . . . . Physical adsorption . . . . . . . . . . Crosslinking . . . . . . . . . . . . . Zero-length crosslinkers . . . . . . Homobifunctional crosslinkers . . . . Heterobifunctional crosslinkers . . . Protecting groups during crosslinking Challenges . . . . . . . . . . . . . . . . An outlook for the next decade . . . . . . Acknowledgments . . . . . . . . . . . . References . . . . . . . . . . . . . . . .

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⁎ Corresponding authors at: Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal. Tel.: +351 234 370 700. E-mail addresses: [email protected] (A.L. Daniel-da-Silva), [email protected] (R. Vitorino). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.biotechadv.2014.04.013 0734-9750/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: da Costa JP, et al, Bionanoconjugation for Proteomics applications — An overview, Biotechnol Adv (2014), http:// dx.doi.org/10.1016/j.biotechadv.2014.04.013

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Introduction Within Nanotechnology, a new field of research has emerged that has drawn increasing interest in the past few years. Nanobiotechnology — or Bionanotechnology — can be defined as a field standing at the intersection between nanomaterials and biotechnology (Bagchi et al., 2012; Gazit, 2007), and, generally, it is considered as having two basic goals (Sapsford et al., 2013): a) using the intrinsic properties — such as catalytic, structural and specific binding properties — of biomolecules in the assembly of hybrid materials that show new or improved characteristics and b) the utilization of the unique properties of the nanomaterials (NMs) within a biological setting, such as using nanoparticles (NPs) for in vivo imaging (Gonçalves et al., 2012) or localized drug delivery (Hartmann et al., 2013). In Table 1, we provide an overview of some of the nanobioconjugates already developed. It should be noted, however, that, due to the recent explosion of interest in these compounds, such description could not but be invidious. Magnetic nanoparticles (MNPs), such as magnetite (Fe3O4) and maghemite (γ-Fe2O3), are the most commonly used nanomaterials, offering controllable size and the ability to be externally manipulated (Santhosh and Ulrih, 2013). For example, Bystrzejewski and co-workers successfully controlled the diameter and magnetic properties of carbon-encapsulated iron nanoparticles (Bystrzejewski et al., 2013) while Li et al. (2007b) were able to coat Fe3O4 particles with alumina by controlling the formation thin carbon layers resorting to the hydrothermal reaction of glucose. Additionally, such particles can be easily functionalized with other components. For instance, different poly(amino acids) have been used for the direct surface modification of MNPs aiming at the synthesis of functional magnetic resonance (MR) probes (Yang et al., 2013). With the same goal in sight, polymers, such as polyethylene amine and polyethylene glycol, have also used for the coating of iron oxide nanoparticles (Schweiger et al., 2011). The team led by Somsook described the development of an enhanced catalyst for the selective oxidation of benzyl alcohol by using MNPs with organometallic compounds (Kamonsatikul et al., 2012). Antibodies (Rosen et al., 2012) and enzymes (Kumar et al., 2012) have also been used in the modification of magnetic nanoparticles, showing potential clinical relevance in cancer theranostics. This versatility has opened the window for the application of MNPs in the fields of proteomics and peptidomics (Agrawal et al., 2013; Ray et al., 2011; Tyan et al., 2013). Although MNPs are the most commonly used type of NPs in bioconjugation, other particles have also been explored for this purpose, namely, gold nanoparticles, due to their optical properties and enhanced capability in adsorbing biomolecules (Lee et al., 2014). In this review, we intend to give an updated overview of the different types of bioconjugation at the nanoscale that have been developed, as well as their potential applications in numerous fields of research. Simultaneously, we will attempt to give a comprehensive outline of the limitations of such methodologies, while also looking at the future prospects for this emerging technology. Concepts and early steps Before proceeding, however, it is important to define what constitutes a “nanobioconjugate”. Perhaps one of the first successful uses of such particles in modern days was described by Fawell et al. (1994). In their work, the authors cross-linked Tat peptides to β-galactosidase, horseradish peroxidase, RNase A, and domain III of Pseudomonas exotoxin A (PE). By monitoring the uptake colorimetrically or by cytotoxicity, they showed that Tat chimeras were effective on all cell types tested, with uptake occurring in all cells. This work evidenced that Tat-mediated uptake could allow for the therapeutic delivery of macromolecules previously thought to be impermeable to living cells. However, the first scientifically described nanobioconjugate is assigned to Helcher, who, in 1718, described the use of boiled starch in gold colloids for enhancing their stability (Helcher, 1718). It is possible,

then, to define a nanobioconjugate as a nanomaterial at the submicrometer level that is deliberately interfaced with a biological molecule (or fraction of a biomolecule). Spasford and colleagues further defined a nanobioconjugate as being intentionally produced at the nanoscale, showing discrete functional or structural parts arrayed on its surface or internally (Sapsford et al., 2013) and displaying unique properties or compositions that may not occur in the same material in the bulk scale (Kreyling et al., 2010). Although this definition is consistent with the vast work carried out and summarized in Table 1, what constitutes a NM has been the subject of much debate (Joachim, 2005; Kreyling et al., 2010). Initially, it was considered as a NM any intentionally produced material with at least one dimension b100 nm. Recently, agencies have proposed more generally accepted definitions and terminology, sustaining, however, the upper limit of 100 nm in at least one of the dimensions (ASTM, 2006; ISO, 2008, 2011). Nonetheless, it should be noted that this upper limit is not valid for all NMs (Sapsford et al., 2013). Others consider that novel size-dependent properties alone, rather than particle size, should be the primary criterion in any definition of NPs (Auffan et al., 2009; Skocaj et al., 2011). Such definitions should be carefully considered, as there are regulations and legal restrictions that must be respected (Brayner et al., 2012; EPA, 2010; SCENIHR, 2010). For the purpose of this review, we will consider as nanomaterials any intentionally produced material with at least one dimension inferior or close to 100 nm.

General applications of nanomaterials The innate properties of nanomaterials, and, in particular, nanoparticles, make them especially suited to be used as biomolecular composites. They exhibit unique size-dependent physical, electronic, optical and chemical properties (Sapsford et al., 2013) that can contribute to the resulting conjugate. These include the size-tunable photoluminescence of quantum dots (Zhang et al., 2013c), the Plasmon resonance of gold nanoparticles (Chen et al., 2014), the electrical and mechanical properties of carbon NMs (Zhang et al., 2013a) or the enhanced magnetic moment and catalytic properties of magnetite core–shell NPs (Amarjargal et al., 2013). Nanoparticles also exhibit high surface-to-volume ratios, providing a high reactive surface available for the display of multiple biological components at their surface. These biologicals can potentially be different and, thus, may contribute to enhanced multifunctionality (Sapsford et al., 2013). NPs have also been described as carriers for insoluble materials, including drugs (Guo and Huang, in press) and radioactive isotopes (Di Pasqua et al., 2013), acting as shields and avoiding chemical and/or photodegradation of such compounds. However, the opposite may be also intended: NPs have been designed to undergo gradual degradation in vivo, usually intended for the controlled localized release of drugs (Brannon-Peppas and Blanchette, 2012; Elzoghby et al., 2012; Panyam and Labhasetwar, 2012). As Sapsford et al. (2013) highlight, when considered cumulatively, such properties make nanoparticles an interesting and promising platform for the development of theranostic agents, i. e., designed bionanoconjugates capable of numerous tasks, such as active sensing (Szymanski and Porter, 2013), diagnostics (Sukhanova et al., 2012), tumor-targeting (Conde et al., 2013), and drug (Elzoghby et al., 2012) or image contrast agent (Mi et al., 2014) delivery. As the properties of these materials are better understood and their synthesis methodologies are improved — tackling numerous issues for large scale production, such as the control of particle size and growth (Thorat and Dalvi, 2012) — the vast scope of applications of NMs and their bioconjugates will surely increase. Nanomaterials are particularly interesting for proteomics applications, as they exhibit ideal characteristics affecting, namely, biocatalytic reactions, such as mass transfer resistance, effective enzyme loading and large surface area (Cipolatti et al., 2014).

Please cite this article as: da Costa JP, et al, Bionanoconjugation for Proteomics applications — An overview, Biotechnol Adv (2014), http:// dx.doi.org/10.1016/j.biotechadv.2014.04.013

Particle

Size and morphology

Functionalization or modification

Applications

Observations

Reference

InGaP/ZnS CdSe/ZnS Au CdS Au

25 nm, spheroid 3.8 nm, QD 12 nm, spheroid 4 nm, QD 33 nm, spheroid

Detection of BSA DNA cleavage Detection of BSA Detection of BSA Evaluation of the activity and stability of trypsin

– Texas-red modified nucleic acid was used – QD's capped with thioglycolic acid –

Kumar et al. (2012) Gill et al. (2005) Singh et al. (2013) Singh et al. (2013) Hinterwirth et al. (2012)

Au Au Au Au Galactosylated chitosan oligosaccharide Si Ge ZnO Fe3O4 γFe2O3 Chitosan–ZnS:Mn CdTe Fe3O4–Au (magnetic gold) Fe3O4–Si–PLGAc Methoxyl-PEGf–PCLg Cystamine coated Au PGMAi–CdSe MWCNTj Fe3O4–Si CdSe/ZnS PEGf–PBMk Au Fullerenes (C60) PMLAl Fullerenes (C60)

5.5 nm, spheroid 5 nm, QD 15 nm, spheroid Nanorods ~50 nm, spheroid

Anti-BSA Thiolated nucleic acid Ascorbic acid PEG Mercapto-alkanoic-acid and mercapto-PEGcarboxylic acid Unmodified and thiolated oligonucleotides HIV-1 tat protein transduction domain Quercetin N-terminal cysteine peptide, CLPFFD Adenosine triphosphate (ATP)

Biological and medical assays Nuclear targeting Chemotherapy of visceral leishmaniasis Targeting of β-amyloid aggregates Hepatocellular carcinoma cell targeting

– In vitro Wild and resistant type strains – Potential for drug delivery

Petersen et al. (2009) Berry et al. (2007) Das et al. (2013) Adura et al. (2013) Zhu et al. (2013)

5.5 nm, spheroid 5.5 nm, spheroid 28.5 nm, spheroid 17.5 nm, spheroid 10 nm, spheroid 90 nm, spheroid 2–4 nm, QD ~70 nm, spheroid 150 nm, spheroid 90 nm, spheroid nr 4.6 nm, QD 30–60 nm (diameter) 105 nm, spheroid 3.2 nm, QD ~53 nm, spheroid 10 nm, spheroid 3–4 nm 27 nm, nr 5.5 nm

PEG N,N,N-trimethyl-3(1-propyne) ammonium iodidea β-Galactosidase Urease Decoy oligonucleotide Mannose Denatured BSA-oligonucleotides covalent hybrids Papain Transferrin Angiopeph Organophosphorous hydrolase Biotin Candida antarctica lipase B Multi-enzyme systems L-arginine Biotinylated cyclic RGD peptide EGFR antibody Malonodiserinolamide Antibody-tokine fusion protein Folic acid, L-phenylalanine and L-arginine

Targeting of cancer cells Targeting of cancer cells Lactose hydrolysis Biosensors (analysis of urea in blood and urine) Targeted delivery to cancer cells Fluorescent bioprobes Selective detection of rHuEPO-α protein Enhancement of enzyme stability and efficiency Anti-proliferation of brain glioma cells Anti-proliferation of brain glioma cells Detection of organophosphates Bioprobes Synthesis of pentyl valerate Intensification of molecular interactions FRET-based sensors Drug delivery (DOX3) Thermal damage to targeted cancer cells Drug delivery Immunostimulation and inhibition of tumor growth Photodynamic therapy

– – – PMIDAb used for NP stabilization In vitro – Using anti-rHuEPO-α aptamers linked AuNPs – NPs loaded with DOXd and PTXe; in vivo and in vitro NPs loaded with PTXe; in vivo Paraoxon used as model organophosphates In vitro Synthesis in organic solvents Interactions stimulated under magnetic field Studies carried out using synthetic DNA In vitro Thermal damage induced by radiofrequency – – In vitro

Bhattacharjee et al. (2013) Bhattacharjee et al. (2013) Ansari et al. (2011) Sahoo et al. (2011) Geinguenaud et al. (2012) Jayasree et al. (2011) Sun et al. (2013) Sahoo et al. (2013) Cui et al. (2013) Xin et al. (2012) Kamelipour et al. (2011) Bach et al. (2013) Raghavendra et al. (2013) Zheng et al. (2013) Giri et al. (2012) Loyer et al. (2013) Raoof et al. (2012a) Raoof et al. (2012b) Ding et al. (2013) Hu et al. (2012)

a b c d e f g h i j k l

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Please cite this article as: da Costa JP, et al, Bionanoconjugation for Proteomics applications — An overview, Biotechnol Adv (2014), http:// dx.doi.org/10.1016/j.biotechadv.2014.04.013

Table 1 Nanoparticle conjugates and respective characteristics. Also highlighted are the applications of such compounds.

TMPA — N,N,N-trimethyl-3(1-propyne) ammonium iodide. PMIDA — N-phosphonomethyl iminodiacetic acid. PLGA — poly(D,L-lactic-co-glycolic) acid. DOX — doxorubicin. PTX — paclitaxel. PEG — polyethylene glycol polymer. PCL — polycaprolactone polymer. Angiopep — TFFYGGSRGKRNNFKTEEYC. PGMA — polyglycidyl methacrylate. MWCNT — multi-walled carbon nanotubes. PBM — polybenzyl malate polymer. PMLA — polymalic acid polymer.

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Within the scope of Proteomics General considerations Conjugation and modification intrinsically depend on both the reactive functionalities present on the crosslinking or derivatizing reagents and the functional groups present on molecules that are to be modified (Hermanson, 2010). If these are either unavailable or chemically incompatible, conjugation does not take place. Consequently, this implies that the successful design of a bioconjugation strategy requires the knowledge of the basic mechanisms by which the reactive groups couple to target functionalities (Sleiman, 2006). The most common targets for modification and/or conjugation are proteins and peptides, and, consequently, are the focus of much research (Ansari and Husain, 2012). This is due to the fact the synthesis of labeled proteins/peptides with high specificity for other molecules can greatly affect biological research, clinical diagnostics and therapeutics (Ansari and Husain, 2012; Hermanson, 2010; Sleiman, 2006). Proteomics is one of the most important research areas in the postgenomics era (Li et al., 2013e). The proteome is extremely intricate and versatile, due to splicing and protein modifications. This is further intensified by the different interconnectivity of proteins and highly complex signal pathways, which vary in both time and space (Altelaar et al., 2013). Therefore, the premise of proteomics is the highthroughput separation of proteins and peptides and their subsequent identification (Angel et al., 2012), allowing for a better understanding of fundamental aspects of biology in the processes essential for life. Such studies heavily rely on mass spectrometry (MS)-based methods. In Proteomics, the use of mass spectrometry techniques offers a labelfree and powerful method in high throughput identification and quantification of thousands of proteins (Vaudel et al., 2014). These techniques, however, are expensive and not easily applied in in vivo studies (Coto-García et al., 2011). Moreover, before proceeding to MS analyses it is necessary to perform the controlled degradation of proteins in a short time, which, ideally, results in well-defined and reproducible patterns of peptides (Kim et al., 2010). Conventionally, this is carried out by in-solution digestion, which is a long, laborious and difficult to automate process (Jia et al., 2013; Liu et al., 2013b). Other limitations include chronic autodigestion of enzyme and sample loss, thus severely affecting the comprehensive determination of the proteomic profiles (Li et al., 2013e). Consequentially, many have tried to develop new methods that try to overcome these drawbacks (Lee et al., 2008; Liu et al., 2009, 2010; Liuni et al., 2010; Min et al., 2011). Recent advances in nanobiocatalytic approaches, such as the use of nanoporous materials, magnetic nanoparticles and nanofibers, have improved the performance of protein digestion. This is particularly evident in the unprecedented success of trypsin stabilization (Kalska-Szostko et al., 2012; Kim et al., 2011; Qin et al., 2012). One of the most crucial steps in any Proteomics experiment is sample preparation; whether it is protein solubilization, protease digestion or peptide separation/selection, all methodologies vary greatly among commercially available kits and laboratories and highly depend on the sample to be analyzed (Bell et al., 2009). Also, different mass spectrometers show different mass accuracies of peptide selection for fragmentation and subsequent detection and identification. Typical proteomic strategies can be divided into gel-based and gel-free (Jia et al., 2013). Representative techniques of the former include MALDI-TOF (Vitorino et al., 2012), LC–MS/MS (Wu and French, 2013), 2D gel-MS (Rogowska-Wrzesinska et al., 2013) and NanoMate (Rozen et al., 2013), among others. These techniques, however, require extensive sample preparation and are often quite expensive. As for the latter techniques, commonly used methodologies include 2D gel electrophoresis (Savelonas et al., 2012), protein arrays (Chandra and Srivastava, 2010; Nicolini and LaBaer, 2010), multi-dimensional protein identification (Zhang et al., 2012), isotope tagging (Choi et al., 2012) or hybrid systems (Sugiyama et al., 2013). Though these entail lower

investments, they are sometimes insensitive for low abundance proteins and false negative and positive results are common. Hence, the development of sensitive, robust and high throughput technologies, which allow for the assessment of biological changes in protein expression and regulation, is required (Bell et al., 2009; Jia et al., 2013). Enzyme immobilization and digestion Enzymes can potentially be used in numerous industries, ranging from the low-medium size production of pharmaceuticals (Pollard and Woodley, 2007; Tao and Xu, 2009) and other fine chemical industries to large size food and energy industries (Akoh et al., 2008; Chang et al., 2010). In fact, these molecules allow the performance of rather complex chemical processes in mild experimental conditions, yielding methodologies with low environmental impact (Zhu et al., 2014) Their biological origin, however, makes them unsuitable from an industrial standpoint, as these can be inhibited by substrates, products and/or other components, which are soluble and show no ideal catalytic characteristics when compared to non-physiological substrates (Garcia-Galan et al., 2011). Hence, the necessary improvements can be achieved through a multitude of techniques, ranging from genetic engineering (Liu et al., 2013a) to process (Luo et al., 2013) and enzyme engineering (Bar-Even and Tawfik, 2013). When properly designed, however, immobilization techniques have proven to result in the enhancement of almost all enzymatic properties, namely, activity, stability, reduction of inhibition, specificity and selectivity (Hartmeier, 1985; Mateo et al., 2007). Such process also results in the improvement of reaction control, which contributes to avoid product contamination by the enzyme (which is of particular relevance in food chemistry) and allows the use of different reactor configurations (Garcia-Galan et al., 2011). Many of the other enzyme's limitations may also be thwarted by choosing a successful immobilization technique. One of the most common goals of enzyme immobilization is stabilization (Iyer and Ananthanarayan, 2008). Nonetheless, a controlled immobilization may result not only in the improvement of enzyme stability, but may also reduce enzyme inhibition, improve enzyme selectivity and/or specificity (Sheldon and van Pelt, 2013). Enzymes can be immobilized in magnetic nanoparticles, thus facilitating their separation resorting to magnetic decantation of the reaction mixture or used in magnetically stabilized fluidized bed reactors (Sheldon and van Pelt, 2013; Yiu and Keane, 2012). Owing to their numerous applications, MNPs have become commercially available (Chemicell, 2013; Nano-H., 2013) and are presently used in vast areas of research and commonly used in the immobilization of enzymes. When using MNPs for the immobilization of proteins, two basic methodologies are used. One approach is adsorbing the proteins that will undergo digestion onto the functionalized MNPs and then incubate these in an enzyme-containing solution (Li et al., 2013e). The other approach consists in the reversal of the first: the enzymes are immobilized on the functionalized MNPs and then incubated in the solution containing the proteins to be digested (Monteil et al., 2014). Fig. 1 illustrates these two basic approaches. The first approach has been described to allow for the selective adsorption of phosphorylated proteins (Li et al., 2013a; Zhang et al., 2013b); the latter approach has been demonstrated to result in significant improvements in reaction rates, stability and reusability (Bahrami and Hejazi, 2013; Yu et al., 2013), thus making it more common. MNP-based enzymatic digestion will be further discussed in the following sections. Regarding stabilization of immobilized enzymes, a strategy commonly followed is the rigidification of the enzyme structure, by multipoint covalent immobilization, using short spacer arms (Bolivar et al., 2009; López-Gallego et al., 2005b; Mateo et al., 2000). This technique, nonetheless, requires the immobilization systems to be criteriously chosen, as the relative distances among all residues must be kept unaltered during conformational changes that may be induced by distorting agents, such as the presence of organic solvents

Please cite this article as: da Costa JP, et al, Bionanoconjugation for Proteomics applications — An overview, Biotechnol Adv (2014), http:// dx.doi.org/10.1016/j.biotechadv.2014.04.013

J.P. da Costa et al. / Biotechnology Advances xxx (2014) xxx–xxx

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Fig. 1. Schematics highlighting the two basic routes for protein digestion using functionalized magnetic nanoparticles. Adapted from Li et al. (2013e).

or extreme pH values (Mateo et al., 2007). The immobilization supports should exhibit large internal surface, in order to have good geometrical congruence with the enzyme surface. Additionally, reactive groups should be present in high superficial density and the protein's reactive groups should pose minimal steric hindrance during the reaction (Mateo et al., 2007). Epoxy and glyoxyl groups are usually viewed as adequate (Mateo et al., 2000, 2006). Glutaraldehyde chemistry is also frequently employed (Barbosa et al., 2014; Betancor et al., 2006; Bezbradica et al., 2014), either using glutaraldehyde-activated supports or the treatment of proteins absorbed on aminated supports with glutaraldehyde, aiming at the crosslink of the enzyme with the support (Guisan, 2006). The reaction should take place at moderate temperatures and neutral pH values and the use of buffers, inhibitors or other protein protectors must be carefully evaluated (Mateo et al., 2006). When immobilizing multimeric enzymes, stabilization becomes a specific problem, as these usually undergo inactivation due to the dissociation of their individual subunits (Poltorak et al., 1998). Consequently, the immobilization–stabilization methodology should aim at the multipoint attachment of the protein and its subunits. The principles are quite similar to those described for the multipoint covalent attachment and highly activated supports are generally used for this purpose (Bolivar et al., 2006; López-Gallego et al., 2005a). In considerably complex enzyme structures, it is probable that not all the subunits become attached to the support surface, and, therefore, immobilization may be complemented by chemical crosslinking with polyfunctional polymers of the previously immobilized enzyme (Hidalgo et al., 2003; Lopez-Gallego et al., 2007; Mateo et al., 2007). Whatever the strategy pursued, immobilization–stabilization techniques are becoming increasingly useful in the rational design and development of new biocatalysts with industrial perspectives. After immobilization, digestion follows. Though this is typically carried out in-solution with no additional changes (Hartmann and Jung, 2010; Li et al., 2013e; Zhou and Hartmann, 2012), many efforts have been developed in order to accelerate protein digestion, such as microwave irradiation (Ruan et al., 2013; Wang et al., 2014), ultrasoundassisted (Santos et al., 2010; Shin et al., 2011; Switzar et al., 2013) and pressure-assisted digestion (Lopez-Ferrer et al., 2008; Olszowy et al., 2013), which have been shown to yield equivalent digestion efficiencies in minutes compared to standard overnight in-solution digestions.

Microwave irradiation has been extensively used (Chen et al., 2014; Ruan et al., 2013; Wang and Li, 2012; Wang et al., 2014) and, although there have been concerns that the use of electromagnetic fields could influence the structure and enzymatic digestion of proteins, it has been demonstrated that is not the case (Damm et al., 2012; Reddy et al., 2013). Moreover, some authors have reported that the use of MNPs may contribute to an increase in the efficiency of microwaveassisted digestion (Chen and Chen, 2007), due to the inherent properties of these particles. MNPs can easily absorb the incident radiation, which leads to rapid heating. Secondly, proteins that are present in low amounts in solution can be adsorbed at the MNPs' surface. This is due to the electrostatic interactions that result from the particles' negatively charged functionalities, leading to an increased adsorption of the proteins with opposite charges onto their surfaces. Last, adsorbed proteins are denatured under these conditions and easily digested in the beads (Li et al., 2013e). Perhaps the first report describing an ultrafast method for protein digestion resorting to high pressure was the one published by LopezFerrer et al. (2008), who used pressure cycling technology (PCT). Pressure-assisted digestion was demonstrated to be applicable at room temperature, resulting in the elimination of chemical artifacts ascribed to heating (Li et al., 2013e; Switzar et al., 2013). The use of MNPs in conjugation with PCT has also been proven to result in the fast digestion of proteins (Lee et al., 2011). The coating and crosslinking of enzymes onto MNPs represent a promising strategy for highthroughput platforms, as these functionalized MNPs show high stability, allowing for their prolonged storage and continuous use, with simple magnetic recovery. An alternative approach consists on the use of ultrasound-assisted digestion, which has also been demonstrated to have a positive effect on the enzymatic digestion rate (Santos et al., 2010; Shin et al., 2011; Switzar et al., 2013). Though the exact mechanism of how ultrasoundassisted digestion is not known (Switzar et al., 2013), it has been suggested that the use of ultrasonic energy generates phenomena known as microjetting and microstreaming, leading to a cavitation effect, which creates local regions of high pressure and temperature, thus facilitating the interactions between enzymes and proteins (Kim et al., 2010; Li et al., 2013e). However, to the best of our knowledge, no work has been reported on the use of ultrasound-assisted digestion using MNPs.

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Enrichment of low abundance proteins In peptidomics, studies have demonstrated that some endogenous peptides consist of or contain potential biomarkers for many physiological conditions and pathological states, namely, detection of immunemediated diseases (Rovero, 2010), heart development (Sergeeva and Christoffels, 2013), urinary tract obstruction (Madsen et al., 2013) and Alzheimer's disease (Lehmann et al., 2013). Nonetheless, the use of biomarkers is far more reaching, with applications, among others, in quality control (Sentandreu and Sentandreu, 2011), drug development (Kraus et al., 2011) and pollution monitoring (Viarengo et al., 2007). However, the detection of low abundance peptides and proteins found in biological samples remains a great challenge, not only due to the inherent low quantities available, but also due to the fact that their MS signals are often masked and/or reduced by high abundant proteins/ peptides and contaminants that are commonly used in pre-treatment procedures (Li et al., 2013e). This is especially true for proteins that undergo post-translational modifications (PTMs) — alterations that can determine their activity, interactions with other proteins and localization (Sidoli et al., 2012; Uen et al., 2013) — as, typically, less than 5% of the peptides in a protein digest are either phosphorylated or glycosylated (Li et al., 2013e), two of the most common PTMs (Jensen et al., 2002; Kannicht et al., 2013). Consequently, the corresponding MS signals are greatly suppressed by those matching non-modified peptides. Conventional methods for the enrichment of such peptides, e.g., concentration via evaporation, result in considerable losses of the sample while, simultaneously, concentrating any interferents that may be present, such as salts (Li et al., 2013e). To detect low-abundance proteins and peptides, nanotechnology may offer the required technical platform for the improvement of specificity, biocompatibility and reproducibility of current methods in Proteomics (Jia et al., 2013). Currently, the most used methodologies involving nanoparticles resort to MNPs. In these procedures, the low-abundant peptides/proteins are specifically adsorbed onto the MNPs in the loading buffer. The subsequent washing steps wash away the non-specifically adsorbed substances and the captured peptides/ proteins can then be analyzed, either directly or after elution from the MNPs. These, in turn, are easily separated, due to the high magnetic response (Jia et al., 2013; Kalska-Szostko et al., 2012; Li et al., 2013e; Lin et al., 2011). A wide range of affinity materials has been used for such enrichment, including TiO2 (Yan et al., 2014), antibodies (Whiteaker et al., 2007), C 60 -silica coating (Chen et al., 2009), thermoplastics (poly(methyl methacrylate)) (Chen et al., 2010) or n-alkyl modified column materials (C4, C8 and C18) (Chen et al., 2008). In the case of the latter, reverse-phase chromatographic techniques can enrich peptides/proteins via hydrophobic–hydrophobic interactions (Lu et al., 2010). In theory, the use of smaller particles in the stationary phase in the column can yield higher separation efficiencies; nonetheless, this is not easily achieved, as the required pressure increases with the inverse of the particle diameter squared (Fekete et al., 2012). As a result, making use of the advantages of nanosized particles has not yet been fully achieved in conventional chromatographic methods. On the other hand, the direct use of n-alkyl functionalized MNPs is uncommon, as the modification processes for the modification of the presynthesized MNPs with the n-alkyl chains are complex and the solubility of the nanoparticles in water is very limited, due to their hydrophobic surfaces (Lu et al., 2010). Besides this non-selective enrichment methods based on hydrophobic interactions, other procedures exist, which are based on the selective enrichment of specific peptides/proteins that contain certain amino acids, such as N-blocked peptides (Zhao et al., 2009), tryptophan-containing peptides (Yu et al., 2011), a class of proteins (Suaifan et al., 2013) or specific proteins (Liu et al., 2014). PTM enrichment As previously mentioned, two of the most important PTMs are glycosylation and phosphorylation (Jensen et al., 2002; Kannicht et al., 2013; Silva et al., 2013). A key challenge in both glyco- and

phosphoproteome research is the development of fast and effective enrichment strategies for high-throughput analyses (Černý et al., 2013). Commonly used glyco-specific enrichment methods include hydrazide (Li et al., 2013b) and boric acid (Mitsui et al., 2012) chemistry, hydrophilic-interaction chromatography (HILIC) (Novotny and Alley, 2013) and lectin affinity chromatography (Ferreira et al., 2011; Mortezai et al., 2012). The enrichment of phosphoproteins and peptides generally relies on two methods: immobilized metal ion chromatography (IMAC) and metal oxide affinity chromatography (MOAC) (Li et al., 2013e; Negroni et al., 2012). The first is based on the affinity of positively charged metal ions, such as Fe3 +, Ce4 + or Ti4 +, with the negatively charged proteins/peptides, followed by elution with basic solutions (Janson, 2012). The latter resorts to the same principle, but, rather than using metallic cations, a metal oxide is used. MOAC has the advantage of being more resilient and robust when compared to IMAC, which is greatly affected by some reagents frequently used in biological procedures, such as buffers and detergents (Li et al., 2013e). Moreover, it has been shown that IMAC is not suited for repetitive use (Negroni et al., 2012). A short overview containing examples of MNP-based enrichment methods of both glyco-proteins/ peptides and phospho-proteins/peptides recently developed is shown in Table 2. Magnetic particles have also been used for the selective enrichment of phospo- or glyco-peptides/proteins in suspension. Zirconium-based functionalized magnetic nanoparticles have been described as particularly effective in the enrichment of phosphopeptides (Li et al., 2007a; Lo et al., 2006; Lu et al., 2011; Wei et al., 2008). This enhanced efficiency may be due to the strong interaction between the phosphopeptides and zirconium, which is further enhanced by the high trapping capacity of nanoparticles, attributable to their small size (Li et al., 2007a; Wei et al., 2008). Other particles used in suspension include TiO2-modified, silica-coated magnetic nanoparticles (Li et al., 2013d), coated magnetic carbon nanotubes (Yan et al., 2014) and magnetic polymers (Li et al., 2011). These are facile, reproducible methodologies, which highlight the versatility and advantageous use of magnetic nanoparticles in the selective enrichment of low abundance peptides/proteins, more precisely, phosphorylated and/or glycosylated peptides/proteins. An overview of the MNPs described in the literature aiming at these goals can be found in Table 3. Even though some protocols for the enrichment of samples with PTMs already exist, these are generally based on the interactions with modifications themselves, which allow for the enrichment of all the proteins with the modification under study. To overcome this limitation, the immobilization of biomolecules onto the surface of nanoparticles can be used, as biomolecules specifically interact with the target protein. Previously, our group has developed a lectin-based protocol using MNPs to specific enrich glycosylated proteins. Three different lectins, concanavalin-A (ConA), wheat germ agglutinin (WGA), and Maackia amurensis lectin (MA) were used (Ferreira et al., 2011). In this work, fetuin acted as a reference to characterize the optimal operating conditions of these systems regarding temperature, time and maximum binding capacity. Moreover, we demonstrated the enhanced performance of our lectin-based nanoplatform. Our results showed that, when using the same amount of immobilized lectin, our MNP@ConA exhibited 5 times higher affinity to ovalbumin and fetuin than the commercially available Sepharose@ConA. In order to evaluate the performance of this system in complex samples such as biofluids, incubation in human serum, urine and saliva was carried out. After, the recovered proteins were digested with trypsin and then analyzed by nano-HPLC MALDI-TOF/TOF. This technique allowed the identification of 180 proteins and using bioinformatic tools we estimated that 90% of those proteins were glycosylated, showing the high specificity of our lectin-base nanoplatform. Concluding, the MNP@lectin system proved to be a helpful tool in glycoproteomics, especially when working with low amounts of sample.

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Table 2 Examples of functionalized MNPs for the enrichment of glycosylated proteins/peptides and phosphor-proteins/peptides. Adapted from Li et al. (2013e). Post-translational modification enrichment

Methodology

Type of particle and functionalization

Glycoproteins/glycopeptides

Hydrazide Boronic acid Lectin IMAC MOAC

Fe3O4–PHEMAa–Hydrazide Fe3O4@SiO2–CHO–APBAb Fe3O4–Con Ac Fe3O4@SiO2–IDAd–Mn+ Fe3O4@SiO2@MxOy

Phosphoproteins/phosphopeptides a b c d

Observations

Reference

Size: 15 nm Applied in living cells Size: 15 nm metal ion: Fe3+ Size: 25 nm metal oxide: Ta2O5

Horak et al. (2012) Lin et al. (2011) Yang et al. (2012) Tan et al. (2008) Lin et al. (2009)

PHEMA — poly(2-hydroxyethil methacrylate). APBA — aminophenyl boronic acid. Con A — concanavalin A. IDA — iminodiacetic acid.

Additionally, many authors have extensively reviewed the use of nanoparticles for the immobilization of biomolecules and detection, namely, antibodies and their potential applications, namely, as biosensors (Cipolatti et al., 2014; Gonzalez-Gonzalez et al., 2012; Makaraviciute and Ramanaviciene, 2013; Manjappa et al., 2011). Hence, this will not be covered in the review herein presented. Bridging the gap — linkage The traditional strategies in bioconjugation rule out control of the reactions' regiochemistry, which often leads to the synthesis of heterogeneous products. Moreover, they often result in the loss or decrease of function of the targeted molecule (Kalia and Raines, 2010). Novel methods of bioconjugation are highly specific and the active form of the molecule suffers little, if any, alterations. Additionally, site-specific immobilized molecules can sometimes exhibit higher ligand binding activity (Luk et al., 2004). A wide variety of methods can be used for the immobilization of proteins onto MNPs, although the most commonly used is based in covalent bonding (Li et al., 2013e), in which covalent bonds are formed between active groups at the surface of the particles and the enzymes. This technique presents the advantages of reducing the degree of desorption of enzymes from the matrix, as well as diminishing enzyme deactivation rates, namely, from protease autolysis (Ju et al., 2012; Tural et al., 2013). The use of covalent bonding has, however, the severe drawback of requiring the use of highly thermally stable enzymes, as the strong interaction limits the thermal movement of the protein at elevated temperatures (Aissaoui et al., 2013). Moreover, such strong interaction of the enzyme with the active groups at the surface of the particles may contribute for a reduced activity of the enzyme, as its tertiary structure may be affected (Petkova et al., 2012). Enzymes exhibit many functional groups suitable for covalent bonding, such as the

amino groups of lysine and arginine (Lata et al., 2012) and the carboxyl groups of glutamic and aspartic acids (Fernández-Fernández et al., 2013). Other appropriate groups include the thiol group of cysteine, tyrosine's phenol ring, hydroxyl groups of threonine or serine and the imidazole and indole groups of histidine and tryptophan, respectively (Copeland, 2013; Cowan and Fernandez-Lafuente, 2011; Křenková and Foret, 2004; Li et al., 2013e). Alternative to covalent binding, physical adsorption can also be used for the immobilization of proteases onto MNPs, based on the nonspecific physical adsorption of the first on the latter (Ansari and Husain, 2012). The binding forces include ionic interactions (Khoshnevisan et al., 2011), hydrogen bonding (Datta et al., 2013), van der Waals (Sheldon and van Pelt, 2013) and hydrophobic interactions (Hanefeld et al., 2009). The positive aspect of such immobilization techniques is that, because no reagents are involved, only a small number of steps are required and conformational changes in the protein are not as relevant. However, minor changes in pH or temperature, for example, may result in a significant loss of the adsorbed layer (Zhou and Hartmann, 2012). In Fig. 2, an overview of these immobilization methods is shown, as well as the advantages and drawbacks of each technique. In this section, we review some of the most commonly used linkages in bionanoconjugation. Physical adsorption Though lacking selectivity, the passive adsorption of molecules onto the surface of nanoparticles is still a commonly used strategy. Mostly, such approaches have been used for the direct immobilization of enzymes on the surface of MNPs (Bahar and Çelebi, 2000; Khoshnevisan et al., 2011; Panek et al., 2013), but also of pectins, aiming at the selective binding of microorganisms (Horisberger, 1976). However, as

Table 3 MNP-based procedures for the enrichment of glyco- and/or phospho-peptides peptides/proteins. Type of particle and functionalization a

Fe3O4@PDA C8-amine-Fe3O4 Fe3O4@ZrO2 Boronic acid functionalized Fe3O4 Fe3O4@p(VPAb–EDMAc-x) Fe3O4@Al2O3 Fe3O4@Ta2O5 MB-LAC Con Ad TiO2-coated magnetic carbon nanotubes Fe3O4@TiO2 Fe3O4@ConA Fe3O4@Ti–mSiO2 Fe3O4@ConA a b c d

Peptide or protein enrichment

Protein type (PTM)

Observations

Reference

Human hemoglobin Serum peptides β-Casein peptides Human saliva α- and β-Casein peptides Multiple peptides β-Casein and ovalbumin peptides Multiple proteins β-Casein peptides α-Casein peptides Lactoferrin Serum peptides Membrane glycoproteins

Glycoprotein – Phosphopeptide Glycoproteins Phosphopeptide Phosphopeptides Phosphopeptides Glycoproteins Phosphopeptides Phosphopeptides Glycoprotein Phosphoproteins Glycoproteins

From protein mixture From protein digest From protein digest – From protein digest From protein digest From protein digest – From protein digest From protein digest Protein mixture Mesoporous silica; from protein digest From living HepG-2 cells

Zhou et al. (2010) Yao et al. (2008) Wei et al. (2008) Wang et al. (2013) Li et al. (2011) Li et al. (2007b) Qi et al. (2009) Sparbier et al. (2005) Yan et al. (2014) Li et al. (2013a) Lai et al. (2013) Li et al. (2013c) Yang et al. (2012)

PDA — polydopamine. VPA — vinyl phosphonic acid. EDMA — ethylene glycol dimethacrylate. MB-LAC Con A — magnetic bead–lectin affinity chromatography with Concanavalin A, commercially available kit from Bruker Daltonik, Germany.

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Last, carbonyls, such as ketone or aldehyde groups, can be generated in glycoproteins by oxidizing the post-translational modifications with certain chemical reagents, namely, sodium meta-periodate (NaIO4) (Hayworth, 2013). Table 4 summarizes the different types of crosslinkers described in the following sections. Ideally, any bioconjugation protocol should be such that it can be carried out in mild conditions of pH and temperature, thus preserving the native structure of the peptides/proteins involved (Kapoor). Additionally, some desired features include (Hayworth, 2013; Hermanson, 2013; Solulink, 2013):

Fig. 2. Overview of different strategies for the immobilization of proteins onto MNPs. Advantages (green, upward arrows) and disadvantages (red, downward arrows) of each option are highlighted. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

previously mentioned, minor changes in pH or temperature may result in a significant loss of the adsorbed layer (Zhou and Hartmann, 2012). Consequently, such techniques are of little use within an industrial setting (Cao et al., 2012). These limitations can be overcome resorting to covalent bonding, discussed in the following sections.

Crosslinking In bionanoconjugation, one of the main limitations is the absence of highly reactive chemical groups at the surface of the nanoparticles, making it difficult — and sometimes impossible — to immobilize the biomolecules with significant yields. In order to overcome this limitation, groups are often modified in order to provide functional targets for modification or conjugation of biomolecules. This is achieved resorting to crosslinkers, which allows for the formation of a bond between the nanoparticles and the biological molecules. There are four main types of crosslinkers: a) zero-length, b) homobifunctional, c) heterobifunctional and d) trifunctional crosslinkers (Han and Kim, 2004; Hayworth, 2013; Hermanson, 2010). Among these, the latter is still, to the best of our knowledge, to be explored in the field of bionanoconjugation. Despite the complexity of protein structures, only a limited number of functional groups can be selected for feasible bioconjugation methods (Hayworth, 2013). In fact, the vast majority of crosslinking techniques relies on four chemical targets: primary amines (\NH2), carboxyls (\COOH), sulfhydryls (\SH) and carbonyls (\CHO) (Hayworth, 2013; Hermanson, 2013; Sleiman, 2006). Primary amines exist at the N-terminus of each polypeptide and in the side chain of lysine residues. At physiological conditions, this group exhibits positive charge, and, therefore, are usually facing the outer surface of proteins, meaning that these are targets that, commonly, are accessible for conjugation without denaturing the protein (Subramanian, 2012). Carboxyls, in turn, can be found at the C-terminus of each polypeptide, as well as in the side chains of glutamic and aspartic acids. Similar to primary amines, these groups are usually at the surface of the protein (Wong and Jameson, 2011). Sulfhydryl groups can be found at the side chain of cysteine. However, in secondary or tertiary structure of proteins, cysteine often forms disulfide bonds (\S\S\); hence, they must be reduced to sulfhydryl, in order to make them available for crosslinking reactions (Wong and Jameson, 2011). Although sulfhydryls are the most reactive functional groups, the loss of secondary or tertiary structure, due to the reduction of proteins, often leads to a significant decrease of activity.

a) the conjugation reaction should occur directly and not require the addition of oxidants, reductants or metals; quantification of the incorporated linkers and final conjugate should be readily performed; b) the inherent biological function must be maintained (or enhanced) after modification/conjugation; c) the conjugation reaction should occur directly and not require the addition of oxidants, reductants or metals; d) quantification of the incorporated linkers and final conjugate should be readily performed; e) the modified molecules should be stable over extended periods; f) the reactions should be stoichiometrically efficient; g) fast reaction kinetics should be observed; h) no undesirable covalent side reactions should take place. When considering in vivo crosslinking, protocols should also allow to “freeze” protein interactions in time and place, so that these can be fully characterized spatially and temporarily relative to treatment conditions and should allow for the stabilization of transient complexes and weak interactions for analysis (e.g., by immunoprecipitation and Western blot) (Hayworth, 2013). Zero-length crosslinkers The zero-length crosslinkers are the smallest available reagent systems for bioconjugation, as these compounds lead to the conjugation of two molecules due to the formation of a bond that involves no additional atoms. This means that one atom of the first molecule is directly attached (covalently) to an atom of the second molecule. Usually, the zero-length crosslinking is used to eliminate the potential of crossreactivity. The most commonly used zero-length crosslinkers in bioconjugation are carbodiimides (Carraway and Koshland, 1972; Fuentes et al., 2005; Wragg, 1970), Woodward's reagent K (Llamas et al., 1986), N,N-carbonyldiimidazole (Anderson and Paul, 1958) and reagents leading to Schiff base formation and reductive amination (Gray, 1978; Wolff and Dean, 1987). These reagents can mediate the formation of three types of bonds: the condensation of a primary amine with a carboxylic acid, thus originating an amide linkage; the reaction of an organic phosphate group with a primary amine, leading to a phosphoramidate linkage and the reductive amination of a primary or secondary amine with an aldehyde group, with a resulting secondary or tertiary amine linkage. Such reaction depends on the desired application and on the choice of reagent, which can be performed in aqueous or non-aqueous environments in a very efficient way. Although these reactions are widely employed in bioconjugation, conjugation of biomolecules onto nanoparticles using carbodiimides is the most frequently followed methodology. For this reason, we will only cover the reaction between nanoparticles and biomolecules mediated by carbodiimides as zero crosslinkers. Carbodiimides can catalyze the formation of amide bonds between amines and carboxylates, as well as of phophoramidate linkages between phosphate groups and amines. Due to the nature of the reaction and their inherent efficiency, carbodiimides are highly explored for the conjugation of biomolecules, leading to the formation of protein– protein, protein–peptide or even oligonucleotide–protein complexes. They also can be used to intermediate the linkage between a biomolecule

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Table 4 Crosslinkers at a glance. Examples of the different types of crosslinkers commonly used are succinctly described, as well as some works reporting their use. The chemical structure is also shown.

Type of crosslinker

Target

Reactive groups

Examples

Structure

Ref.

Carboxyl to amine

NHS ester

EDC + NHS

Handké et al., (2013)

Carboxyl to amine

NHS ester

DCC + NHS

Joullié and Lassen, (2010)

Amine to amine

NHS ester

Gluteraldehyde

Mateo et al., (2007)

Sulfhydryl to sulfhydryl

Maleimides

BMOEa

Fink, (2013)

Amine to sulfhydryl

NHS ester/maleimide

SPDPb

Manjappa et al., (2011)

Sulfhydryl to carbohydrate

Maleimide/Hydrazide

MPBHc

Hermanson, (2010)

Amine to non-selective

NHS ester/Aryl azide

ANB-NOSd

Hayworth, (2013)

Zero-length

Homobifunctional

Heterobifunctional

a

BMOE — bismaleimidoethane; bSPDP — (N-succinimidyl-3-(2-pyridyldithio)propionate; cMPBH — 4-(4-N-Maleimidophenylbutyric acid hydrazide)·HCl; dANB-NOS — N-5-azido-2nitrobenzoyloxysuccinimide.

and the surface of a particle (Hermanson, 2010). Carbodiimides can be divided in two basic types: water-soluble and waterinsoluble. Since most macromolecules of biological origin are in aqueous buffer solutions, the water-soluble carbodiimides are the best suited for bioconjugation. Moreover, the use of water-soluble carbodiimides yields a by-product, an isourea, which is also a water-soluble compound, thus facilitating the subsequent purification steps. On the other hand, water-insoluble carbodiimides seem to be an alternative to conjugate water-insoluble biomolecules. Due to their nature, peptides have low solubility in water and are sometimes insoluble. For this reason, these kinds of carbodiimides are very popular in peptide synthesis (Han and Kim, 2004). It is important to note that not only these carbodiimides are organic-soluble, but also their isourea by-products are hydrophobic and, consequently, water insoluble. The most popular water-soluble and waterinsoluble carbodiimides are the EDC (N-(3-dimethylaminopropyl)-N ′-ethylcarbodiimide hydrochloride) plus NHS (N-hydroxysuccinimide) and the DCC (N,N′-dicyclohexylcarbodiimide) systems, respectively. Water-soluble carbodiimides. Commonly, EDC is a water-soluble carbodiimide used for the conjugation of carboxylates and amines from biological samples, such as proteins (Hermanson, 2010). Nowadays, the system resorting to the use of EDC together with NHS is the most commonly used bioconjugation method (Handké et al., 2013;

Shan et al., 2008). Furthermore, its application in coupling biomolecules into nanoparticles is very well documented and characterized. Because EDC and NHS are water-soluble, their addition to the reaction medium is facilitated and the purification steps are improved using techniques such as centrifugation, filtration, dialysis or even magnetic separation (Hermanson, 2013). Despite the advantages of using EDC as a crosslinker, some precautions should be taken into consideration. Due to the fact that EDC is very unstable in the presence of water, it should be stored at − 20 °C in desiccated environment, and warmed up to room temperature just prior to its use, in order to avoid condensation and consequent decomposition of the reagent over time. Using EDC as a crosslinker facilitates the formation of an amide bond, which can be used to create a wide variety of chemical conjugates, by reacting an amine group with a carboxylate group. The reaction between Nsubstituted carbodiimides with carboxylic acids leads to the formation of a highly reactive intermediate, o-acylisourea. In the presence of a nucleophile in the medium, such as primary amines, o-acylisourea will be attacked and form an amide bond (Williams and Ibrahim, 1981). One of the main disadvantages in using this approach is the lack of selectivity of the active species, and, thus, it can react with other nucleophiles, such as oxygen atoms and sulfhydryl groups. For this reason, as highlighted in Fig. 3, in aqueous solution, the water molecules can hydrolyze o-acylisourea, leading to the formation of an isourea and regeneration of the original carboxylate group (Gilles et al., 1990).

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Fig. 3. Amide bond formation by a carbodiimide-catalyzed reaction using the EDC plus NHS system.

Due to relevance of carbodiimides in bioconjugation, but also in other areas, various authors have tried to evaluate the mechanism of amide formation mediated by carbodiimides in aqueous solution. In 1995, Nakajima and Ikada (1995) developed a study where different types of carboxylic- and amine-containing molecules were evaluated using 13C NMR and IR to assess the reaction products. They concluded that EDC activity rapidly decreases in aqueous media at low pH, leading almost exclusively to the production of the corresponding urea derivative. Under the used conditions, EDC activity was very stable in neutral and higher pH conditions. In a low pH range, between 3.5 and 4.5, they suggested that EDC could effectively react with carboxylic groups, while the highest yields for the amide bond formation occurred between pHs 4–6. Despite the fact that hydrolysis of EDC is higher at acidic pH values, the carbodiimide stability in solution increases with higher pH values, and the best results are achieved with pH near or above 6.5.

Although EDC-mediated amide bond formation between peptides and proteins can occur almost at any pH, high bioconjugation yields of these biomolecules can be achieved using a pH range between 4.5 and 7.5. A clear disadvantage of using EDC to conjugate these kinds of biomolecules is that both peptides and proteins are constituted by aminoacids and, therefore, both amine and carboxylic groups exist in the same substance. This may result in self-polymerization, instead of reacting with the desired target (Han and Kim, 2004). Such restrictions may be circumvented by performing the conjugations in mild alkaline conditions, which contributes to restrict the degree of polymerization between the peptides/proteins and still allows for the facile coupling of proteins and particles (Han and Kim, 2004; Hermanson, 2010). Consequently, various buffers can be used for this purpose, though it should be noted that buffers containing amine or carboxylate salts should be avoided, as these components can also react with carbodiimides leading

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to undesired cross reaction products. Usually, a MES (2-(N-morpholino) ethanesulfonic acid) buffer is a good option, as it exhibits a pKa of approximately 6.15 at 25 °C, and it can be used to buffer the solution between pH values in the range of 5.5–6.7 (Hayworth, 2013). As previously mentioned, the highly reactive intermediate o-acylisourea can react with nucleophiles. For this reason, when conjugating proteins onto the surface of particles, side reactions between the active intermediate and the sulfhydryl groups of the protein can take place (Carraway and Triplett, 1970; Hermanson, 2013). Moreover, EDC or NHS can react with the side chains of the aminoacids present in the peptide/protein, leading to the formation of various undesired side products (Carraway and Koshland, 1968; Cuatrecasas and Parikh, 1972) or, ultimately, to the loss of activity if the conjugation occurs in the active site of the protein. One way to overcome this disadvantage is to protect the active site of the protein by adding reversible inhibitors that do not contain primary amines or carboxylate groups into the reaction medium (e.g., benzamidine) (Hermanson, 2010). The rationale behind the use of a water soluble carbodiimide, such as EDC, is to mediate the formation of a highly reactive ester functionality with the carboxylate of the targeted molecule. In the presence of NHS there is the formation of the isourea by-product and the NHS ester intermediate is formed (Fig. 3). This formed ester is hydrophilic in nature, and, consequently, it rapidly reacts with the amines on the targeted molecule, forming stable amide bonds (Beth et al., 1986; Denny and Blobel, 1984; Kotite et al., 1984; Staros, 1982). Because o-acylisourea is relatively unstable in water, the addition of NHS also has the advantage of leading to the formation of an intermediate (NHS-ester intermediate) which shows a higher stability and that will react with the nucleophile amine. Although NHS is a water-soluble compound, its solubility is comparatively low to that of sulfonated-NHS, sometimes used to enhance solubility. Hence, when using sulfonated-NHS, the intermediate ester formed also shows higher solubility in water and less hydrolysis in aqueous media. In the presence of nucleophiles, such as amines, the carbonyl group of the intermediate ester will be attacked and the sulfo-NHS group will rapidly leave and a stable amide bond between the carboxylic group and amine group will be formed. This is also the case for the o-acylisourea intermediate: the NHS-ester intermediate reacts with sulfhydryl and hydroxyl groups, leading to the formation of thioesters and esters as by-products, although these are less stable then the amide bond. Another disadvantage of using this system is that the reaction between amines and NHS-ester intermediate is slow and this intermediate can hydrolyze in aqueous medium (Hoare and Koshland, 1967). Thus, the active carboxylate should react with the targeted amine before undergoing hydrolysis. This limitation can be overcome by increasing the concentration of NHS, leading to the formation of the NHS ester intermediate, thus greatly increasing the resulting amide bond formed (Hermanson, 2010). Water-insoluble carbodiimides. Since 1955, when Sheehan and Hess first reported its use (Sheehan and Hess, 1955), DCC has been widely used as a zero-length crosslinker in organic medium, in particular in peptide synthesis. Contrary to the reaction involving EDC and NHS, in which both reagents and by-products are water-soluble, in a DCC-catalyzed reaction both reagents and by-products are water-insoluble. For this reason, the use of DCC is a valid solution for overcoming the disadvantage of hydrolysis when using EDC as a coupling agent. DCC is a highly effective crosslinker that, initially, reacts with the carboxylic acid forming an o-acylisourea. This compound then mediates the reaction to form different end-products. The main goal is the direct coupling with an amine group, in order to form an amide bond, as shown in Fig. 4. In other cases, especially when working in anhydrous environment and in the absence of the amine group, if a high amount of carboxylic acid is added to the medium, the intermediate oacylisourea can react with a second carboxylic group and create an symmetrical anhydride that will react with an amine group, resulting in the formation of an amide bond and one of the original carboxylic

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group (Selinger and Lapidot, 1966). In addition, this o-acylisourea can undergo rearrangements and form an inactive N-acyl urea as an undesired by-product (Stewart and Young, 1984). Finally, it can also react with aminoacids and form an azlactone (Joullié and Lassen, 2010), which can also react with amine groups, forming an amide bond different than the desired zero-length crosslink (Hermanson, 2010). As in the EDC crosslinking system, some additives have been suggested in order to boost the reactivity of the intermediate. König and Geiger suggested the use of HOBt (hydroxybenzotriazole) (König and Geiger, 1970), as this compound readily reacts with the intermediary o-acylisourea to result in an oxybenzotriazole (OBT) active ester. This intermediate has increased reactivity, since it establishes hydrogen bonds with the amine groups and leads to the amide bond formation (Valeur and Bradley, 2009). Other related benzotriazole derivatives have also been proposed as additives for this reaction, such as HOAt (1-hydroxy-7azabenzotriazole) (Carpino, 1993) and PyBOP (benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate) (Valeur and Bradley, 2009). Despite the advantages of the use of zero-length crosslinkers, the surfaces of the nanoparticles sometimes do not possess the reactive groups suited for bioconjugation. Moreover, it may be necessary to add a chain (spacer) between the surface of the nanoparticle and the targeted biomolecule in order to avoid steric hindrance between the conjugates. Hence, the use of different kinds of crosslinkers, described below, is sometimes required. Homobifunctional crosslinkers Homobifunctional crosslinkers are characterized by the fact that they exhibit identical reactive groups at either end of a spacer arm (Hermanson, 2010), and, generally, they are used in one-step reactions to randomly fix or polymerize molecules that show identical functional groups (Hermanson, 2013). These reagents are used like a “molecular rope” that bridges two biomolecules covalently or even a surface/particle to a biomolecule, reacting with common groups in both ends. Homobifunctional crosslinkers have been widely used in order to add a spacer and to change the reactive group existing at the particle surface to other groups with higher reactivity or higher specificity for the target molecules (Steck, 1972; Trester-Zedlitz et al., 2003). One example is the addition of an amine-to-amine crosslinker to a cell lysate. This will result in the random conjugation of protein subunits, interacting proteins and any additional peptides that happen to show lysine side chains in proximity of one another in the solution (Hayworth, 2013). Some studies have also been carried out in order to assess how different groups present at the surface of nanoparticles could influence the resulting system and its activity. One example is the work developed by Li et al. (2010), in which they evaluated the influence of different reactive groups at the surface of magnetic nanoparticles in the immobilization of trypsin and the influence on the resulting proteolytic activity. Two homobifunctional crosslinkers were used, aiming at the derivatization of the amine groups exhibited at the surface of the MNPs with aldehyde and carboxylic groups, using gluteraldehyde and succinic acid, respectively. They concluded that the derivatization was successful and that the carboxylic-functionalized nanoparticles showed both higher immobilization efficiency and proteolytic activity. Due to the enormous variety of applications using homobifunctional crosslinkers, nowadays there are multiple commercially available crosslinkers possessing a wide variety in both length and desired reactivity (ThermoScientific, 2013a, 2013b). Although these crosslinkers have been extensively used, they show some inherent disadvantages. The use of homobifunctional crosslinkers in proteomics may be used to gather a broad understanding of all protein interactions (Singh et al., 2012), but it does not offer the specificity and precision required in other crosslinking applications, such as the preparation of an antibody–enzyme conjugate without the formation of antibody–antibody conjugates (Hayworth, 2013; Hermanson, 2010). Moreover, the possibility of cross reaction at multiple sites within the protein often leads

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Fig. 4. Amide bond formation by a carbodiimide-catalyzed reaction using the DCC system.

to a significant loss of activity (Hayworth, 2013; Hermanson, 2010). In some particular cases, strategies can be used in order to minimize these limitations. For example, in our work developed by Ferreira et al. (2011) were lectins were immobilized into the MNP surface, suberic acid bis(N-hydroxysuccinimide ester) (DSS) was used in a first step to modify the particle surface with carboxylic groups. Since the activation of the carboxylic groups of the DSS is prior activated, activation of the carboxylic groups of biomolecules does not occur, which avoids biomolecule polymerization. Heterobifunctional crosslinkers In heterobifunctional conjugation, two different reactive groups can couple to two distinct functional targets on a protein (or other macromolecule). This means that, for example, one part of the crosslinker may contain a sulfhydryl-reactive group, while another exhibits an amine-reactive group, thus resulting in the ability to direct the crosslinking reaction to specific parts of the targeted molecule, offering a better control over the bioconjugation process (Hermanson, 2010). Typically, one protein undergoes modification with a heterobifunctional compound with the most reactive (or most labile) end of the crosslinker. After a quick purification step, such as gel filtration or rapid dialysis, the other end of the crosslinker can be used to conjugate another molecule (Hermanson, 2013). Because of the multi-step nature of these methodologies, there is a great control over the size and the

molar ratios of the components with the crosslinked product. Consequently, low- or high-weight molecule conjugates can be obtained, depending on the intended use (Johnson, 2010) and these sequential steps minimize undesired polymerization or self-conjugation (Hayworth, 2013). Heterobifunctional crosslinkers can also be used to site-direct a conjugation reaction, i.e., toward a specific part or section of the targeted molecule. This means that amines may be coupled on one molecule, while carbohydrates or sulfhydryls target another (Hermanson, 2010). This is of special relevance, as effective conjugation of certain molecules — such as antibodies — requires them to preserve critical epitopes or active sites available. For all heterobifunctional crosslinkers, there is a third component, which is the spacer, also referred to as cross-bridge, that connects the two reactive ends (Hermanson, 2010, 2013). Thus, selecting a crosslinker may not only depend on its reactivity, but also on the length and nature of spacer. One example is polyethylene glycol-(PEG) based cross-bridges, which endow the bioconjugates with increased water solubility, due to the creation of hydrophilic reagents (Rao et al., 2013), offering great flexibility in the experimental design. Interestingly, when used as a spacer arm in vivo, PEG-based systems have demonstrated to increase immunogenicity (Huang et al., 2013). Other crosslinkers may alter the reactivity of their functional groups. For example, a maleimide group that is adjacent to an aromatic ring is less stable to ring opening and loss of activity than a maleimide group

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neighboring an aliphatic ring (Hermanson, 2010; Myers et al., 1989). We will briefly review some of the most frequently used heterobifunctional reagents reported in the literature. The most popular heterobifunctional crosslinkers are those containing both amine-reactive and sulfhydryl reactive ends (Hermanson, 2010). While the latter portion may be one of several functional groups (mostly, alkylating agents (Wong and Jameson, 2011)), the amine-reactive group is, commonly, an active ester, often, a N-hydroxysuccinimide (NHS) ester (Hermanson, 2013). The alkylating agent is capable of creating thioether or disulfide linkages with the molecules containing the sulfhydryl ends. Sulfosuccinimidyl 4(N-maleimidomethyl)cyclohexane-1-carboxylate (or Sulfo-SMCC, Fig. 5) is a popular crosslinker that exhibits such reactive ends (Hayworth, 2013), although others are also commonly used (Fig. 5) (Hermanson, 2010). Due to the fact that the NHS ester is less stable in aqueous solution, it is usually reacted to one protein first, and, if the second protein does not possess native sulfhydryl groups, they can be added in a prior step using sulfhydryl-addition reagents (Hayworth, 2013; Wong and Jameson, 2011), such as those commercially available (PierceNet, 2013). An important class of heterobifunctional reagents is the photoreactive crosslinkers. These are chemically inert compounds that exhibit one end that can undergo photolysis to initiate coupling, when exposed to ultraviolet or visible radiation (Hermanson, 2010). The most popular are aryl azides (Mehenni and Bakr, 2011), commonly used in labeling reagents and crosslinking (Hayworth, 2013). When such compounds are exposed to UV radiation, a nitrene group is formed (Khan et al., 2013). These groups can then initiate not only addition

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reactions with double bonds, but also insertions into C\H and N\H sites. They can also undergo ring expansion to react with nucleophiles, such as primary amines (Hayworth, 2013). Photoreactive crosslinking reagents are used to capture binding partner interactions, by using a protein which is labeled with the crosslinker using either the sulfhydryl or amine reactive end. This labeled protein can then be subsequently added to a lysate sample and allowed to bind with the interactor (Hayworth, 2013; Mehenni and Bakr, 2011). When subjected to UV light, conjugation is initiated via the phenyl azide group (Hermanson, 2013). Although some successful experimental set-ups have been reported in the literature (Khan et al., 2013; Kim et al., 2012; Mehenni and Bakr, 2011; Pham et al., 2013), the use of such reagents presents obvious drawbacks. Such experiments must be performed in attenuated light and in either opaque or foil-covered vessels until photoactivation is initiated. This heavily limits the conditions in which such reagents can be used, and, therefore, only a few reports on their applications are available. Nonetheless, some interesting approaches have been reported on the use of photoreactive crosslinkers for the bioconjugation of cDNA, aiming at the development of self-assembly protein microarrays (Ramachandran et al., 2004, 2008) In bionanoconjugation a new family of heterofunctional groups is becoming increasingly popular: the organosilane precursors. Typically, these compounds have a functional organic group (amine, thiol, among others) in one end and at least one alkoxysilane group on the other. The hydrolysis of the alkoxy groups yields silanol-containing species that can condensate with hydroxyl groups at the nanoparticles'

Fig. 5. In A, sulfo-SMCC, a popular crosslinker showing an amine reactive sulfo-NHS ester group (on the left) and a sulfhydryl reactive maleimide group (on the right), with a cyclohexane spacer arm. In B, SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate. The NHS ester reacts with amine-containing molecules and the pyridyl dissulfide group reacts with sulfhydryl groups. In C, LC–SPDP, the long-chain version of SPDP. Adapted from Hayworth (2013) and Hermanson (2010).

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Challenges

Fig. 6. Functionalization steps using APTES. In A), the hydrolysis of the APTES molecule. In B), the condensation of the hydrolyzed APTES molecule onto the surface of the silicacoated magnetic nanoparticle.

surface, leading to the introduction of novel functional groups onto the surface of the nanoparticles. This strategy allows for the covalent immobilization of biomolecules onto the surface of inorganic nanomaterials such as magnetic iron oxides and silica coated nanoparticles. Perhaps the most commonly used organosilane is APTES ((3aminopropyl)triethoxy silane). The hydrolytic condensation of APTES on nanoparticles' silica shell confers reactive amine groups to the surface of the particle, allowing for the immobilization of biomolecules. Fig. 6 highlights this process. Besides APTES, other organosilanes have been also investigated, such as MPTES (3-mercaptopropyltriethoxysilane) (Vinoba et al., 2011) or THPMP (3(trihydroxysiliyl)propylmethylphosphonate) (Palani et al., 2008) for the surface modification with sulfhydryl and phosphonate groups respectively. Last, hetero-functional chemistries based on the use of 3(2-amineethylene) propyl-metyl dimethoxylane (MANAE) have been successfully used for the manufacture of antibody arrays (González-González et al., 2014) It is important to notice that, often, the bionanoconjugation is carried out in a multiple-step process, and hetero and homobifunctional crosslinkers can be both used for the immobilization of biomolecules onto the nanoparticle' surface. The importance of crosslinking reagents in bionanoconjugation is irrefutable, as, without these, conjugation yields would be insipid. However, when working with biomolecules, such as amino acid chains, self-polymerization can occur, due to the wide range of available functional groups, which is an obvious drawback in bionanoconjugation.

Protecting groups during crosslinking In order to prevent the formation of unexpected crosslinks, one possible approach is the use of protecting groups at different stages of the crosslinking reaction. One example is the strategy followed by Fan et al. (2003) which have synthesized a pentapeptide (Phe-AlaAla-Ala-Ala) onto the surface of gold nanoparticles following a peptide elongation approach. The surface of the nanoparticles was first functionalized with alkanethiolate compounds. Then the pentapeptide was produced onto the surface of the particles, step-by-step, using N-BocL-amino acids to avoid the polymerization of the amino acids. This assembly was successfully achieved using PyBOP (benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate) and HOBt as crosslinking agents and DIPEA (N,N-diisopropylethylamine) as a catalyst. This strategy was highly efficient (95% each step) and the resulting polypeptide showed no racemization.

The advent of nanoproteomics has renewed the attention in enzyme immobilization and enzymatic reactors, though at the micro and nanoscales (Kim et al., 2010). The implementation of enzymatic digestion at this level allows for a decrease of the amount of consumables and for enhanced sensitivity. Moreover, assays can be conducted faster and better reproducibility can be achieved. Finally, enzymatic reactions taking place in microchannels are suitable for long-term automation (Li et al., 2013e; Switzar et al., 2013). Nevertheless, many challenges need to be overcome. Difficulties arise in the packing of the enzymeimmobilized nanoparticles in the microchannels, which require intricate capillary chemistry and reproducible filling (Li et al., 2013e). Although the use of MNPs may contribute to the eradication of such limitations, as the packing step is particularly straightforward and reproducible (Switzar et al., 2013), most of the work published resorting to such tactics is carried out in off-line mode (Li et al., 2013e; Liu et al., 2013b; Switzar et al., 2013). This may be because the on-line enzymatic digestion (Min et al., 2011) based on magnetic nanoparticles may be limited due to the increase of the backpressure of the packed bed with the number of MNPs. If sufficiently high, the forces that result from the backpressure would overpower the axial field gradients, and the packed bed would simply flush away. If, on the contrary, a low amount of MNPs is used in order to circumvent the high backpressure, insufficient or inefficient digestion can occur, thus not meeting the requirements for the on-line coupling with the subsequent separation step, such as liquid chromatography or capillary electrophoresis. There is also the inherent danger of contaminating the mass spectrometer in which the analyses are to be performed, as some beads may leak from the packed bed (Li et al., 2013e). An outlook for the next decade The development of simplified synthesis methods for the preparation of magnetic nanoparticles with higher increased biocompatibility and magnetic responsivity, as well as more active sites and multifunctionality will continue as bustling research topic. Moreover, the magnetic core of the MNPs may open the doors to automation, resulting in minimal operation errors. Although only a few reports exist describing the automated manipulation and handling of MNPs, this is certainly a possibility. Recently, the introduction of additional functionalities in MNPs through surface modification and functionalization has gained increasing interest. Multifunctional MNPs showing tailored active sites, ranging from enzymes to ligands and metal oxides, have been demonstrated not only to improve the efficiency of protein digestion, but also the enrichment of low-abundance proteins and peptides. Most publications dealing on this issue, nonetheless, focus on the process itself, resorting to standardized activity assays to determine and compare the activity of the immobilized enzyme. Only limited conclusions can be drawn from the commonly used activity assays, such as UV–vis measurements, with respect to the stability and activity of the enzyme in continuously operated reactors. This lack of correlation is often neglected. Moreover, different immobilization techniques are difficult to compare amongst each other, as, usually, the comparison is done toward the free enzyme. Whatever the method of protein immobilization in MNPs, the vast array of research in this area shows that it is one of the most promising techniques in numerous applications, such as environmental monitoring, biotransformation, theranostics, pharmaceutical and food industries. Enzyme-based methodologies are steadily replacing conventional chemical methods in academia, but also in industry, owing to their better performance and increased efficiency. Commercially, however, only a handful of products is available, and the associated costs and storage issues still need to be overcome. It is also clear that every protein is different, and to this date, no “onesize-fits-all” method has been described for enzyme immobilization.

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