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Magnetic metal-organic frameworks containing abundant carboxylic groups for highly effective enrichment of glycopeptides in breast cancer serum
Talanta 204 (2019) 446–454
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Magnetic metal-organic frameworks containing abundant carboxylic groups for highly effective enrichment of glycopeptides in breast cancer serum
T
Xufang Hua, Qianjing Liua, Yonglei Wua, Zhiqiang Dengc, Jian Longc,∗∗, Chunhui Denga,b,∗ a
Department of Chemistry, And Department of Gastroenterology and Hepatology, Zhongshan Hospital, Fudan University, Shanghai, 200433, China Institutes of Biomedical Sciences, And Collaborative Innovation Center of Genetics and Development, Fudan University, Shanghai, 200433, China c First People's Hospital of Fuzhou, Jiangxi, 344000, China b
ARTICLE INFO
ABSTRACT
Keywords: Hydrophilic interaction liquid chromatography Magnetic metal-organic frameworks Glycopeptides enrichment Breast cancer serum Mass spectrometry
A mercaptosuccinic acid functionalized hydrophilic magnetic metal-organic framework nanocomposite (denoted as mMOF@Au-MSA) was proposed and synthesized to provide an excellent platform for glycopeptide analysis. The novel nanomaterial integrated favorable advantages such as robust magnetic response from Fe3O4 magnetic nanoparticles, large surface area contributed by MOF, abundant ultra-high hydrophilic carboxylic groups from mercaptosuccinic acid, as well as unbiased affinity toward different types of glycopeptides. This nanocomposite was successfully utilized to capture glycopeptides from standard protein digests with the high selectivity and great sensitivity of 0.5 fmol μL−1. Notably, 307 glycopeptides assigned to 96 glycoproteins were identified from only 2 μL serum of breast cancer patient. The satisfying achievement indicated that the as-prepared nanopartical had promising potential in exploring the knowledge of glycoproteins in breast cancer.
1. Introduction Glycosylation, recognized as one of the most significant posttranslational modifications (PTMs) of proteins and peptides, is cohesively associated with the process of the cellular metabolism and regulation of multiple biological course [1]. Immense amount of researches have verified that the abnormal variation of glycosylation frequently lead to dysfunction of protein and then causing a series of serious diseases, including cancer especially [2,3]. It is therefore undisputed that multiple clinical biomarkers and therapeutic targets are glycosylated proteins [4]. Additionally, from a pharmacological point of view, these glycosylated proteins generally exist in body fluids easily accessible such as blood serum [5], urine [6], saliva [7] and so forth. Hence, it is of great value to have better understanding of glycosylation information. Especially, breast cancer has inspired arising concern attributed to its high incidence and low survival rates in women worldwide [8]. Many reports have proved that glycoproteins can act as biomarkers of breast cancer [9]. As a result, to explore the knowledge of glycoproteins in patient means a lot to early diagnosis of breast cancer. Shotgun proteomics based on mass spectrometric (MS) detection has developed into a sophisticated and powerful platform for the in-depth glycoproteomes profiling [10]. However, challenges are existent for direct
identification of target analytes by MS on account of some intrinsic properties of glycopeptides, including the low abundance of glycoproteins, the complexity of organism, as well as the interference of non-glycoproteins, etc [11]. In addition, proteolysis of biological protein sample with trypsin, a widely utilized protease for MS analysis, consequently produce more peptides per protein and lead to increased complexity of sample [12]. Therefore, pre-concentration is a vital step prior to MS analysis to improve the detection sensitivity of low abundance glycopeptides. In order to overcome these obstacles, considerable researches have been devoted to exploiting diverse techniques to enrich glycopeptides before MS analysis [13,14]. In particular, hydrophilic interaction liquid chromatography (HILIC) has attracted increasing attention owing to its remarkable enrichment performance, excellent reproducibility and unbiased affinity towards glycopeptides [15,16]. Glycopeptides were isolated from non-glycopeptides with the fact that they generally possessed more hydrophilic capacity. It was mentionable that the isolation was achieved with no damage to the glycans structure of glycopeptides [17]. So far, large amount of biocompatible nanomaterials, on whose surfaces were modified with hydrophilic functional groups, have been employed to selectively separate and capture glycopeptides [18–21]. Materials utilized in HILIC are of great importance since the versatile stationary phase can enhance the binding capability and enrichment performance.
Corresponding author. Department of Chemistry, and Department of Gastroenterology and Hepatology, Zhongshan Hospital, Fudan University, Shanghai, 200433, China. ∗∗ Corresponding author. E-mail addresses: [email protected] (J. Long), [email protected] (C. Deng). ∗
https://doi.org/10.1016/j.talanta.2019.06.037 Received 11 April 2019; Received in revised form 3 June 2019; Accepted 9 June 2019 Available online 11 June 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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And it also has been demonstrated that the more functional groups were grafted on the surface of HILIC substrates, the better enrichment efficiency would be achieved [22]. Thus, it is crucial to explore efficient functionalized materials for sample preparation in proteomics. Metal-organic frameworks (MOFs), a unique class of hybrid inorganic-organic microporous coordination polymers, display various merits including large surface areas, ultra-high porosity, adjustable pore size, flexible functionalization, various modifiable organic linkers as well as admirable chemical durability and thermostability [23]. On account of these advantages, MOFs have been extensively explored in many significant fields [24,25]. Currently, a large number of MOFs can be synthesized via easy-operation and low-cost methods. And the richness and diversity of the MOFs facilitated the selection of an appropriate MOF as the candidate substrate for HILIC-based methods [26]. Additionally, MOFs can not only act as the carriers for subsequent functionalization but also has been demonstrated as function moieties being grafted onto other carriers such as magnetic nanoparticles and graphene [27]. As a result, extensive researches focusing on hydrophilic MOFs for glycopeptide enrichment have sprung up. In particular, the UiO‐66 series of MOFs aroused great passion involved in various fields because of its better stability against moisture, hyperthermia and acidic chemical situations compared with many others [28]. UiO-66-NH2, of which structure is consist of Zr6O4(OH)4 clusters bridged together by twelve 2-amino-1,4-benzenedicarboxylate (NH2-BDC) linkers, is certified to possess higher biocompatibility [29]. Moreover, the amino groups have broadened the possibility of postmodification of UiO-66-NH2 with multiple properties, which greatly expanded its applications in drug delivery, tumor monitoring and many other fields [30–32]. It is notable that our groups’ previous works have successfully proven the brilliant promise of the performance of UiO-66NH2 in glycopeptide enrichment [15,33]. Molecules with acid groups are recognized as talented functional candidates of HILIC materials due to the high hydrophilicity originating from their polar acid. The ability to capture glycopeptides can be improved through the affinity to the glycan moieties by hydrogen bonding interaction. Diverse HILIC-based enrichment methods have been carried out with the assistance of acid functional molecules involving in mercaptoacetic acid [34], phytic acid [35], iminodiacetic acid [36] and the like. Mercaptosuccinic acid (MSA), a typical acid compound possessing high content of hydrophilic free carboxyl groups, exhibits advantages of distinguished biocompatibility and accessibility with low cost. The extraordinary achievements of MSA in DNA sensing [37] and neurotoxin inhibiting [38] have hinted its fantastic potential application in other biological fields including glycopeptide enrichment especially. Herein, mercaptosuccinic acid-modified magnetic MOF (denoted as mMOF@Au-MSA) was synthesized to selectively capture low-abundance glycopeptides based on hydrophilic interaction. Briefly, UiO-66NH2 was coated on the surface of magnetic Fe3O4 via hydrophilic polydopamine as a linker (mMOF). Then, Au nanoparticles were fabricated on the surface of MOF (mMOF@Au) to establish the bridge connection between UiO-66-NH2 and MSA attribute to the formation of Au–S bond (mMOF@Au-MSA). By virtue of the unique characterizations of UiO-66NH2, ultra-hydrophilicity of mercaptosuccinic acid, as well as formidable magnetic property to simplify the enrichment process, the biocompatible HILIC materials exhibited outstanding enrichment performance towards the HRP and IgG digestion. Especially, 307 glycopeptides corresponding to 96 glycoproteins were successfully captured by mMOF@Au-MSA from breast cancer serum. The synthetic method of mMOF@Au-MSA offers a new idea for the exploration of HILIC-based MOF materials and paves the way for in-depth glycoproteome studies.
dopamine chloride, zirconiun tetrachloride (ZrCl4), 2-aminoterephthalic acid (H2BDC-NH2), chloroauric acid (HAuCl4·4H2O), trisodium citrate, mercaptosuccinic acid (MSA), phosphoric acid (H3PO4), ammonium bicarbonate (NH4HCO3), ethylene glycol(EG), dimethylformamide (DMF) and ethanol were purchased from Shanghai Chemical Corp (Shanghai, China). Tris(hydroxymethyl) aminomethane (Tris) and 2,5-dihydroxybenzoic acid (DHB) were bought from J&K Scientific. Horseradish peroxidase (HRP), Immunoglobulin G (IgG), bovine serum albumin (BSA), trypsin from bovine pancreas, dithiothreitol (DTT) and indoacetamide (IAA) were bought from SigmaAldrich. ACN (HPLC grade), trifluoroacetic acid (TFA) and formic acid (FA) were purchased from Merck (Darmstadt, Germany). PNGase F was purchased from Genetimes Technology (Shanghai, China). Serum of breast cancer was provided by First people's hospital of Fuzhou (Jiangxi province, China). All aqueous solutions were prepared by ultrapure water via a Milli-Q system (Millipore, Bedford, MA, USA). 2.2. Synthesis of Fe3O4@PDA@ UiO-66-NH2 (mMOF) Firstly, Fe3O4 nanoparticles were synthesized solvothermally in accordance with the previous report [39]. Secondly, Dopamine is selfpolymerized on the surface of Fe3O4 at room temperature. A 0.12 g portion of obtained Fe3O4 nanoparticles was dispersed in the 80 mL Tris buffer (pH 8.5) containing 21.09 mmol/l dopamine chloride. The mixture was consistently stirred at room temperature for 8 h. The Fe3O4@ PDA was obtained and then washed by deionized water as well as ethanol each for three times and dried in vacuum at 50 °C. Next, UiO66-NH2 was coated on the Fe3O4@PDA shell by one-pot synthesis. In brief, 0.1 g Fe3O4@PDA was dispersed into 60 mL DMF which containing 11.04 mmol/L 2-aminoterephthalic acid and 11.44 mmol/L ZrCl4. The mixture was ultrasonicated for 5 min, followed by stirring at 120 °C for 1 h. The obtained Fe3O4@PDA@UiO-66-NH2 (denoted as mMOF) was washed with DMF for three times and then dried in vacuum at 50 °C. 2.3. Synthesis of mMOF@Au-MSA Initially, Au nanoparticles were grown in-situ on the surface of mMOF. 30 mg mMOF was dispersed into 60 mL deionized water at 85 °C with stirring. HAuCl4 aqueous solution was added into the reaction solution dropwise with the final concentration of 0.24 mmol/L. After continuous stirred 0.5 h, 1.6 mL trisodium citrate (1 mol/L) was poured into the system followed by stirring for another 1 h. The as synthesized mMOF@Au was washed by deionized water three times and dried in vacuum at 50 °C. Subsequently, 10 mg MOF@Au was added into 30 mL ethanol with 10 mmol/L mercaptosuccinic acid and stirred at 60 °C for 3 h. The prepared mMOF@Au-MSA was collected by magnet and washed with ethanol for three times then dried in vacuum at 50 °C for the following experiments. 2.4. Sample preparation Standard glycoprotein (HRP or IgG, 2 mg) was dissolved in 25 mmol/L NH4HCO3 aqueous solution and then denatured in boiling water for 10 min. Afterwards, appropriate amount of trypsin (trypsin/ protein = 1/40, w/w) was added into the standard glycoprotein solution and digested at 37 °C for 16 h. The obtained tryptic digests were diluted with loading buffer for enrichment experiments. For BSA, preparation steps were the same as the previous description except the adding amount was 10 mg. As for the preparation of breast cancer serum digestion, 2 μL of serum was diluted to 20 μL with NH4HCO3 aqueous solution (25 mmol/ L, pH 7.9) and denatured in boiling water for 10 min. After that, the obtained serum solution was reduced using DTT for 0.5 h at 60 °C and then alkylated by IAA in dark for 1 h at 37 °C. The mixture was treated with a certain amount of trypsin (trypsin/protein = 1/40, w/w) and
2. Experimental 2.1. Materials and chemicals Iron chloride hexahydrate (FeCl3·6H2O), sodium citrate(NaAc), 447
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incubated for 16 h at 37 °C. Tryptic digestion of breast cancer serum was obtained and lyophilized for further experiments.
was further investigated with Fourier transform infrared (FTIR) spectroscopy in Fig. S3. Compared with the spectrogram of Fe3O4@PDA, after coating with the UiO-66-NH2, the peaks at 766.99 cm−1 and 629.33 cm−1 were detected attributed to N–H stretching vibration. The adsorption band at 1435.16 cm−1 originated from the O–H bending vibration. Although the peaks that corresponding to MOF decreased after modified with MSA since the surface of the materials was covered intensively with Au nanoparticles, the newly generated peak at 1695.31 was provided by the C–O stretching vibration from the carboxyl groups of MSA [40]. Wide-angle X-ray diffraction (XRD) was also conducted to confirm the crystalline structure of mMOF. As shown in Fig. S4, diffraction peaks at 7.25°, 8.4°, 25.7° and 43.2° were distinctively caused by UiO-66-NH2. And those at 30.2° and 35.8°, 57.5° as well as 63.3° were from the magnetic core of Fe3O4 [33]. It directly demonstrated that the MOF layer was successfully fabricated. Finally, zeta potential analysis was operated in aqueous solution to monitor the surface potential change of the product on each step (Fig. S5). In the synthetic process of mMOF, the values firstly dropped and then went up, which was ascribed to the negative acidic catechol hydroxyl groups of PDA and positive amino groups from MOFs. As to the functionalization of MSA, the Zeta potential decreased thanks to the carboxyl groups of MSA. All these characterization investigations have given a satisfactory result that the mMOF@Au-MSA was completely constructed. Meanwhile, large specific area is a significant property leading to the highly efficient enrichment performance. The nitrogen adsorption−desorption experiment revealed that the mMOF@Au-MSA processed a favorable BET surface area of 229.23 m2 g−1 (Fig. S6). Besides, hydrophilicity is absolutely another key point for HILIC-based glycopeptide enrichment. The mMOF@ Au-MSA was tested to be 28.04° of water contact angle (Fig. S7 b), exhibiting greatly improved hydrophilicity compared with that of mMOF (Fig. S7 a). Moreover, magnetic responsiveness was also a crucial factor which can greatly facilitate the separating procedures and minimize the waste of samples. The satisfying magnetic responsiveness of mMOF@Au-MSA was illustrated in Fig. S8, while being equably dispersed in water (Fig. S8a), the nanomaterials can be separated from solution in a few seconds (Fig. S8b) with the help of a magnetic block.
2.5. Enrichment protocol For enrichment of glycopetides from standard protein digestion, mMOF@Au-MSA (200 μg) and a specific amount of digests were dispersed in 100 μL loading buffer (ACN/H2O/TFA, 90/9.9/0.1, v/v/v). The obtained mixture solution was incubated at 37 °C for 45 min. Subsequently, the materials were separated from solution by a magnet field and then washed by loading buffer for three times. After that, the enriched glycopetides were released from the materials by 10 μL eluting buffer (ACN/H2O/FA, 30/70/0.1, v/v/v) under continuous vibration for 30 min at 37 °C. Finally, the collected eluent was mixed with DHB matrix (20 mg/mL) and analyzed by MALDI-TOF MS. Breast cancer serum was donated by two patients. The same enrichment and eluting treatments were carried out. After that, the elution was lyophilized and redissolved in 25 mmol/L NH4HCO3 aqueous solution. Then, N-linked glycan moieties were removed by adding 1 μL PNGaseF into the solution and incubating at 37 °C for 16 h. The obtained glycopetides solution was lyophilized again for Nano-HPLC–MS/ MS analysis. 2.6. MALDI-TOF MS analysis Generally, 1 μL of eluent was dropped upon the steel plate and dried naturally. Then, 1 μL of DHB (20 mg/mL) was added. Slightly change was made only when taking the sensitivity experiment. A total of 1 μL eluent was dropped upon the steel plate in two batches, allowing the former one to get dried naturally before another droplet was added, followed by 0.5 μL of DHB (20 mg/mL). MALDI-TOF MS analysis were accomplished on a 5800 Proteomics Analyzer (Applied Biosystems, U.S.A.) in positive ion mode at a frequency of 200 Hz and the acceleration voltage of 20 kV. The Nd:YAG laser was 355 nm. 2.7. Characterization, nano-LC-ESI-MS/MS analysis and database searching Detailed information about characterization, Nano-LC-ESI-MS/MS analysis and database searching was illustrated in the Supporting Information.
3.2. Investigation of the performance of mMOF@Au-MSA nanocomposities for glycopeptides enrichment The glycopetides enrichment workflow was displayed in Scheme 2 and involved four steps: incubating, washing, eluting as well as MS detecting. According to the hydrophilic affinity interaction between nanomaterials and glycosylated peptides, glycopeptides can be adsorbed on the surface of the materials, on the contrary, non-glycopeptides can be washed away. To achieve the best enrichment efficiency, the tryptic digest of horseradish peroxidase (HRP) was utilized as standard glycopeptides to optimize the capture conditions. Firstly, incubation conditions with appropriate hydrophilicity as well as suitable pH condition were crucial factors severely affecting the enrichment efficiency. Thus, the loading buffer with different amount of acetonitrile (ACN) and trifluoroactic acid (TFA) was studied using of 250 fmol μL−1 HRP digest. The MS peak intensities of glycopeptides were systematically compared. As seen in Fig. S9a and Fig. S9b, best result was obviously achieved with the loading buffer composition of ACN/H2O/TFA (90/9.9/0.1, v/v/v) and the subsequent experiments were operated in this condition. Besides, the incubation duration was also investigated and demonstrated in Fig. S 9c. The incubation period of 45 min is sufficient to get a relatively satisfying result since longer incubation time can hardly improve the peak intensities of glycopeptides. With the optimized incubation conditions, outstanding performance was achieved using the as-synthesized nanoparticles to enrich glycopeptides in the standard protein digests of HRP as well as immunoglobulin G (IgG). As illustrated in Fig. 1, the mass spectra of HRP
3. Results and discussion 3.1. Synthesis and characterization of mMOF@Au-MSA The fabrication process of mMOF@Au-MSA was depicted in Scheme 1. First, A layer of polydopamine was coated on the magnetic Fe3O4 core via a self-assembly procedure. Then, a one-pot synthesis protocol was applied to grafted UiO-66-NH2 shell onto the surface of Fe3O4@ PDA (mMOF). Next, Au nanoparticles were intensively grown in-situ on the surface of mMOF. Finally, mercaptosuccinic acid was covalently combined to the Au and the mMOF@Au-MSA was synthesized successfully. The morphology and construction of mMOF@Au-MSA were systematically certified through diverse characterizations. At first, the TEM image of Fe3O4@PDA@ UiO-66-NH2 (Fig. S1a) shows an undulating layer of MOF outside a magnetic core. Au nanoparticles with the diameters of approximately 15 nm were apparently immobilized on magnetic MOFs from the observation of TEM image (Fig. S1b) as well as SEM image (Fig. S2a) of mMOF@Au-MSA. Besides, the components of mMOF@Au-MSA were investigated in the energy dispersive X-ray (EDX) analysis. The results were depicted in Fig. S2b and detailed in Table S1. They demonstrated the existence of Zr, S and Au elements, proving that the MOF was successfully synthesized and the MSA was attached to Au as desired. Moreover, the structure of mMOF@Au-MSA 448
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Scheme 1. The synthetic procedure for mMOF@Au-MSA.
and IgG tryptic digests without enrichment were occupied by plenty of non-glycopeptides and only six and seven glycopeptide peaks were observed from 250 fmol μL−1 HRP and 300 fmolμL−1 IgG tryptic digest respectively. Nevertheless, after extracted with mMOF@Au-MSA, both 29 glycopeptide peaks from HRP and IgG were clearly detected, and the intensities of glycopeptide peaks were extensively improved. The detail information of the captured glycopeptides from HRP and IgG digests were provided in Table S2 and Table S3 respectively. Besides, the enrichment experiments of Fe3O4@PDA@UiO-66-NH2 (mMOF) were also estimated under the same conditions which were performed in Fig. 2 for comparison. As showed in Fig. S10, fewer and less intensive glycopeptides peaks were detected. It indirectly confirmed that the MSA was successfully grafted on mMOF@Au and greatly improved the enrich efficiency towards glycopeptides. Furthermore, to investigate the enrichment sensitivity of mMOF@Au-MSA nanocomposites for glycopeptides, a series of diluted HRP tryptic digests were employed in the enrich process. As shown in Fig. 2, three glycopeptide peaks still can be detected even though the concentration of the HRP digest was decreased to 0.5 fmol μL−1, which means mMOF@Au-MSA nanocomposites possess competitive sensitivity towards glycopeptides compared with the previous reports [16,35]. This good result could mostly be credited to the high hydrophilicity origin from MSA with abundant carboxylic groups and large surface area of the MOF-based composites. Moreover, digestion of protein-bovine serum albumin (BSA),a
typical non-glycoprotein, was employed to monitor the enrichment selectivity of mMOF@Au-MSA towards glycopeptides. As shown in Fig. 3, when the mass ratio was 1 : 50 (HRP/BSA), only two glycopeptide peak can be detected due to the strong signal suppression from BSA digests. Whereas, after enrichment with mMOF@Au-MSA, the glycopeptide peaks of HRP digests were detected distinctly and the peaks of BSA digests were barely observed in the meanwhile. Remarkably, the mMOF@Au-MSA still exhibited a good ability of selective enrichment towards glycopeptides even if the mass ratio increased to 1:100. As a consequence, the excellent selective performance compared to those reported studies [22,41] indicated that the mMOF@Au-MSA possessed great potential in capturing glycopeptide from more complicated bio-samples containing multiple coexistent non-glycopeptides interferences. In addition, the binding capacity of mMOF@Au-MSA nanocomposites was tested as well. In brief, 2 μg HRP digests was extracted by different amounts of mMOF@Au-MSA nanocomposites. After incubation, each supernatant was collected and analyzed by MS. Through monitoring the S/N ratio of residual glycopeptides in the supernatants, the enrichment capacity of mMOF@Au-MSA was calculated to be 100 mg g−1 (Fig. S11). Meanwhile, the reusability of mMOF@Au-MSA for glycopeptides enrichment was further evaluated. After each enrichment experiment, the nanomaterials were recollected and washed with eluent buffer repeatedly to remove the potential residues. As revealed in Fig. S12, the number of glycopeptides in HRP digest presented
Scheme 2. Flowchart of glycopetides enrichment. 449
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Fig. 1. MALDI-TOF mass spectra for the glycopeptide enrichment from 250 fmol μL−1 HRP tryptic digest: (a) before enrichment, and (b) after treatment; from 300 fmol μL−1 IgG: (c) before enrichment, and (d) after treatment. Peaks of glycopeptides are marked with red diamonds . The detail information of the marked glycopeptides from HRP and IgG digests were provided in Table S2 and Table S3 respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 450
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Fig. 2. MALDI-TOF mass spectra of tryptic digested HRP after enrichment by mMOF@Au-MSA nanomaterials with the concentration of: (a) 50 fmol μL−1, (b) 10 fmol μL−1, (c) 1 fmol μL−1 and (d) 0.5 fmol μL−1. Peaks of glycopeptides are marked with red diamonds . The detail information of the marked glycopeptides was provided in Table S2. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
almost the same with the prior ones except a little decrease in peak intensities at the fifth reutilization, suggesting the great stability and reusability of mMOF@Au-MSA nanomaterials in glycopeptides enrichment. According to all the above excellent results including great selectivity, large binding capacity, high sensitivity, good stability and reusability, the mMOF@Au-MSA were further applied to enrich
glycopeptides in biological sample. Breast cancer serum, a universal and promising clinical specimen, possessed high potential to discovery glycopeptides of biomarkers for early diagnosis of this disease. In this work, three isolated replicates of 2 μL serums of breast involved two cancer patients were respectively enriched by the as prepared nanoparticles. As listed in Table S4, a total of 307 unique glycopeptides assigned to 96 glycoproteins were enriched and identified eventually 451
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Fig. 3. MALDI-TOF mass spectra for the glycopeptide enrichment from a mixture of tryptic digests of HRP and BSA at a mass ratio of 1 : 50 (a) before and (b) after treatment with mMOF@Au-MSA nanoparticles; HRP and BSA at a mass ratio of 1: 100 before (c) and after (d) treatment with mMOF@Au-MSA nanoparticles. Glycopeptides are marked with red diamonds . The detail information of the marked glycopeptides was provided in Table S2. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 452
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with the nano-LC-ESI-MS/MS analysis. It is doubtless that the mMOF@Au-MSA possessed overwhelming enrichment advantage toward glycopeptide in biological samples.
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4. Conclusions Magnetic metal–organic frameworks containing abundant carboxylic groups (mMOF@Au-MSA) nanocomposites were designed and synthesized to enrich N-glycopeptides from breast cancer serum. The as-synthesized nanomaterials integrated the advantages of robust magnetic responsiveness of Fe3O4, large surface area of the MOF, as well as extraordinary hydrophilicity and biocompatibility of mercaptosuccinic acid. As a consequence, the mMOF@Au-MSA demonstrated brilliant performance in glycopeptide enrichment including remarkable selectivity, high sensitivity, good repeatability and sustainability. Especially, mMOF@Au-MSA was applied to capture glycopeptides from serum of breast cancer. The satisfying results not only confirmed the promising potential of mMOF@Au-MSA in glycopeptidome profiling based on MS strategies, but also extended our knowledge of glcopeptides in breast cancer. Moreover, this work provided a guide for hydrophilic post-modification of MOFs and its application in glycoproteome research. Conflicts of interest The authors declare no competing financial interest. Acknowledgments This work was financially supported by National Key R&D Program of China (2018YFA0507501) and the National Natural Science Foundation of China (21425518). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.06.037. References [1] Y. Yang, V. Franc, A.J.R. Heck, Glycoproteomics, A balance between highthroughput and in-depth analysis, Trends Biotechnol. 35 (7) (2017) 598–609. [2] L. Cao, J.K. Diedrich, D.W. Kulp, M. Pauthner, L. He, S.R. Park, D. Sok, C.Y. Su, C.M. Delahunty, S. Menis, R. Andrabi, J. Guenaga, E. Georgeson, M. Kubitz, Y. Adachi, D.R. Burton, W.R. Schief, J.R. Yates III, J.C. Paulson, Global site-specific N-glycosylation analysis of HIV envelope glycoprotein, Nat. Commun. 8 (2017) 14954. [3] M. Murakami, T. Kiuchi, M. Nishihara, K. Tezuka, R. Okamoto, M. Izumi, Y. Kajihara, Chemical synthesis of erythropoietin glycoforms for insights into the relationship between glycosylation pattern and bioactivity, Sci. Adv. 2 (1) (2016) e1500678. [4] H. Zhang, X.-j. Li, D.B. Martin, R. Aebersold, Identification and quantification of Nlinked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry, Nat. Biotechnol. 21 (2003) 660. [5] N. Sun, C. Deng, Y. Li, X. Zhang, Highly selective enrichment of N-linked glycan by carbon-functionalized ordered graphene/mesoporous silica composites, Anal. Chem. 86 (4) (2014) 2246–2250. [6] G.S.M. Kammeijer, J. Nouta, J. de la Rosette, T.M. de Reijke, M. Wuhrer, An indepth glycosylation assay for urinary prostate-specific antigen, Anal. Chem. 90 (7) (2018) 4414–4421. [7] Y. Li, J. Wang, N. Sun, C.-h. Deng, Glucose-6-Phosphate-Functionalized magnetic microsphere as novel hydrophilic probe for specific capture of N-linked glycopeptides, Anal. Chem. 89 (20) (2017) 11151–11158. [8] P. Minicozzi, L. Van Eycken, F. Molinie, K. Innos, M. Guevara, R. Marcos-Gragera, C. Castro, E. Rapiti, A. Katalinic, A. Torrella, T. Žagar, M. Bielska-Lasota, P. Giorgi Rossi, N. Larrañaga, J. Bastos, M.J. Sánchez, M. Santthe European HR Working Group on breast cancer, Comorbidities, age and period of diagnosis influence treatment and outcomes in early breast cancer, Int. J. Cancer 144 (9) (2019) 2118–2127. [9] S.S. Pinho, C.A. Reis, Glycosylation in cancer: mechanisms and clinical implications, Nat. Rev. Canc. 15 (2015) 540. [10] J. Cox, M.Y. Hein, C.A. Luber, I. Paron, N. Nagaraj, M. Mann, Accurate proteomewide label-free quantification by delayed normalization and maximal peptide ratio
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novel affinity probe for highly selective capture of endogenous phosphopeptides, ACS Sustain. Chem. Eng. 6 (3) (2018) 4382–4389. [40] J. Zhou, Y. Liang, X. He, L. Chen, Y. Zhang, Dual-functionalized magnetic metal–organic framework for highly specific enrichment of phosphopeptides, ACS Sustain. Chem. Eng. 5 (12) (2017) 11413–11421. [41] Q. Liu, Y. Xie, C. Deng, Y. Li, One-step synthesis of carboxyl-functionalized metalorganic framework with binary ligands for highly selective enrichment of N-linked glycopeptides, Talanta 175 (2017) 477–482.
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Magnetic metal-organic frameworks containing abundant carboxylic groups for highly effective enrichment of glycopeptides in breast cancer serum
T
Xufang Hua, Qianjing Liua, Yonglei Wua, Zhiqiang Dengc, Jian Longc,∗∗, Chunhui Denga,b,∗ a
Department of Chemistry, And Department of Gastroenterology and Hepatology, Zhongshan Hospital, Fudan University, Shanghai, 200433, China Institutes of Biomedical Sciences, And Collaborative Innovation Center of Genetics and Development, Fudan University, Shanghai, 200433, China c First People's Hospital of Fuzhou, Jiangxi, 344000, China b
ARTICLE INFO
ABSTRACT
Keywords: Hydrophilic interaction liquid chromatography Magnetic metal-organic frameworks Glycopeptides enrichment Breast cancer serum Mass spectrometry
A mercaptosuccinic acid functionalized hydrophilic magnetic metal-organic framework nanocomposite (denoted as mMOF@Au-MSA) was proposed and synthesized to provide an excellent platform for glycopeptide analysis. The novel nanomaterial integrated favorable advantages such as robust magnetic response from Fe3O4 magnetic nanoparticles, large surface area contributed by MOF, abundant ultra-high hydrophilic carboxylic groups from mercaptosuccinic acid, as well as unbiased affinity toward different types of glycopeptides. This nanocomposite was successfully utilized to capture glycopeptides from standard protein digests with the high selectivity and great sensitivity of 0.5 fmol μL−1. Notably, 307 glycopeptides assigned to 96 glycoproteins were identified from only 2 μL serum of breast cancer patient. The satisfying achievement indicated that the as-prepared nanopartical had promising potential in exploring the knowledge of glycoproteins in breast cancer.
1. Introduction Glycosylation, recognized as one of the most significant posttranslational modifications (PTMs) of proteins and peptides, is cohesively associated with the process of the cellular metabolism and regulation of multiple biological course [1]. Immense amount of researches have verified that the abnormal variation of glycosylation frequently lead to dysfunction of protein and then causing a series of serious diseases, including cancer especially [2,3]. It is therefore undisputed that multiple clinical biomarkers and therapeutic targets are glycosylated proteins [4]. Additionally, from a pharmacological point of view, these glycosylated proteins generally exist in body fluids easily accessible such as blood serum [5], urine [6], saliva [7] and so forth. Hence, it is of great value to have better understanding of glycosylation information. Especially, breast cancer has inspired arising concern attributed to its high incidence and low survival rates in women worldwide [8]. Many reports have proved that glycoproteins can act as biomarkers of breast cancer [9]. As a result, to explore the knowledge of glycoproteins in patient means a lot to early diagnosis of breast cancer. Shotgun proteomics based on mass spectrometric (MS) detection has developed into a sophisticated and powerful platform for the in-depth glycoproteomes profiling [10]. However, challenges are existent for direct
identification of target analytes by MS on account of some intrinsic properties of glycopeptides, including the low abundance of glycoproteins, the complexity of organism, as well as the interference of non-glycoproteins, etc [11]. In addition, proteolysis of biological protein sample with trypsin, a widely utilized protease for MS analysis, consequently produce more peptides per protein and lead to increased complexity of sample [12]. Therefore, pre-concentration is a vital step prior to MS analysis to improve the detection sensitivity of low abundance glycopeptides. In order to overcome these obstacles, considerable researches have been devoted to exploiting diverse techniques to enrich glycopeptides before MS analysis [13,14]. In particular, hydrophilic interaction liquid chromatography (HILIC) has attracted increasing attention owing to its remarkable enrichment performance, excellent reproducibility and unbiased affinity towards glycopeptides [15,16]. Glycopeptides were isolated from non-glycopeptides with the fact that they generally possessed more hydrophilic capacity. It was mentionable that the isolation was achieved with no damage to the glycans structure of glycopeptides [17]. So far, large amount of biocompatible nanomaterials, on whose surfaces were modified with hydrophilic functional groups, have been employed to selectively separate and capture glycopeptides [18–21]. Materials utilized in HILIC are of great importance since the versatile stationary phase can enhance the binding capability and enrichment performance.
Corresponding author. Department of Chemistry, and Department of Gastroenterology and Hepatology, Zhongshan Hospital, Fudan University, Shanghai, 200433, China. ∗∗ Corresponding author. E-mail addresses: [email protected] (J. Long), [email protected] (C. Deng). ∗
https://doi.org/10.1016/j.talanta.2019.06.037 Received 11 April 2019; Received in revised form 3 June 2019; Accepted 9 June 2019 Available online 11 June 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
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And it also has been demonstrated that the more functional groups were grafted on the surface of HILIC substrates, the better enrichment efficiency would be achieved [22]. Thus, it is crucial to explore efficient functionalized materials for sample preparation in proteomics. Metal-organic frameworks (MOFs), a unique class of hybrid inorganic-organic microporous coordination polymers, display various merits including large surface areas, ultra-high porosity, adjustable pore size, flexible functionalization, various modifiable organic linkers as well as admirable chemical durability and thermostability [23]. On account of these advantages, MOFs have been extensively explored in many significant fields [24,25]. Currently, a large number of MOFs can be synthesized via easy-operation and low-cost methods. And the richness and diversity of the MOFs facilitated the selection of an appropriate MOF as the candidate substrate for HILIC-based methods [26]. Additionally, MOFs can not only act as the carriers for subsequent functionalization but also has been demonstrated as function moieties being grafted onto other carriers such as magnetic nanoparticles and graphene [27]. As a result, extensive researches focusing on hydrophilic MOFs for glycopeptide enrichment have sprung up. In particular, the UiO‐66 series of MOFs aroused great passion involved in various fields because of its better stability against moisture, hyperthermia and acidic chemical situations compared with many others [28]. UiO-66-NH2, of which structure is consist of Zr6O4(OH)4 clusters bridged together by twelve 2-amino-1,4-benzenedicarboxylate (NH2-BDC) linkers, is certified to possess higher biocompatibility [29]. Moreover, the amino groups have broadened the possibility of postmodification of UiO-66-NH2 with multiple properties, which greatly expanded its applications in drug delivery, tumor monitoring and many other fields [30–32]. It is notable that our groups’ previous works have successfully proven the brilliant promise of the performance of UiO-66NH2 in glycopeptide enrichment [15,33]. Molecules with acid groups are recognized as talented functional candidates of HILIC materials due to the high hydrophilicity originating from their polar acid. The ability to capture glycopeptides can be improved through the affinity to the glycan moieties by hydrogen bonding interaction. Diverse HILIC-based enrichment methods have been carried out with the assistance of acid functional molecules involving in mercaptoacetic acid [34], phytic acid [35], iminodiacetic acid [36] and the like. Mercaptosuccinic acid (MSA), a typical acid compound possessing high content of hydrophilic free carboxyl groups, exhibits advantages of distinguished biocompatibility and accessibility with low cost. The extraordinary achievements of MSA in DNA sensing [37] and neurotoxin inhibiting [38] have hinted its fantastic potential application in other biological fields including glycopeptide enrichment especially. Herein, mercaptosuccinic acid-modified magnetic MOF (denoted as mMOF@Au-MSA) was synthesized to selectively capture low-abundance glycopeptides based on hydrophilic interaction. Briefly, UiO-66NH2 was coated on the surface of magnetic Fe3O4 via hydrophilic polydopamine as a linker (mMOF). Then, Au nanoparticles were fabricated on the surface of MOF (mMOF@Au) to establish the bridge connection between UiO-66-NH2 and MSA attribute to the formation of Au–S bond (mMOF@Au-MSA). By virtue of the unique characterizations of UiO-66NH2, ultra-hydrophilicity of mercaptosuccinic acid, as well as formidable magnetic property to simplify the enrichment process, the biocompatible HILIC materials exhibited outstanding enrichment performance towards the HRP and IgG digestion. Especially, 307 glycopeptides corresponding to 96 glycoproteins were successfully captured by mMOF@Au-MSA from breast cancer serum. The synthetic method of mMOF@Au-MSA offers a new idea for the exploration of HILIC-based MOF materials and paves the way for in-depth glycoproteome studies.
dopamine chloride, zirconiun tetrachloride (ZrCl4), 2-aminoterephthalic acid (H2BDC-NH2), chloroauric acid (HAuCl4·4H2O), trisodium citrate, mercaptosuccinic acid (MSA), phosphoric acid (H3PO4), ammonium bicarbonate (NH4HCO3), ethylene glycol(EG), dimethylformamide (DMF) and ethanol were purchased from Shanghai Chemical Corp (Shanghai, China). Tris(hydroxymethyl) aminomethane (Tris) and 2,5-dihydroxybenzoic acid (DHB) were bought from J&K Scientific. Horseradish peroxidase (HRP), Immunoglobulin G (IgG), bovine serum albumin (BSA), trypsin from bovine pancreas, dithiothreitol (DTT) and indoacetamide (IAA) were bought from SigmaAldrich. ACN (HPLC grade), trifluoroacetic acid (TFA) and formic acid (FA) were purchased from Merck (Darmstadt, Germany). PNGase F was purchased from Genetimes Technology (Shanghai, China). Serum of breast cancer was provided by First people's hospital of Fuzhou (Jiangxi province, China). All aqueous solutions were prepared by ultrapure water via a Milli-Q system (Millipore, Bedford, MA, USA). 2.2. Synthesis of Fe3O4@PDA@ UiO-66-NH2 (mMOF) Firstly, Fe3O4 nanoparticles were synthesized solvothermally in accordance with the previous report [39]. Secondly, Dopamine is selfpolymerized on the surface of Fe3O4 at room temperature. A 0.12 g portion of obtained Fe3O4 nanoparticles was dispersed in the 80 mL Tris buffer (pH 8.5) containing 21.09 mmol/l dopamine chloride. The mixture was consistently stirred at room temperature for 8 h. The Fe3O4@ PDA was obtained and then washed by deionized water as well as ethanol each for three times and dried in vacuum at 50 °C. Next, UiO66-NH2 was coated on the Fe3O4@PDA shell by one-pot synthesis. In brief, 0.1 g Fe3O4@PDA was dispersed into 60 mL DMF which containing 11.04 mmol/L 2-aminoterephthalic acid and 11.44 mmol/L ZrCl4. The mixture was ultrasonicated for 5 min, followed by stirring at 120 °C for 1 h. The obtained Fe3O4@PDA@UiO-66-NH2 (denoted as mMOF) was washed with DMF for three times and then dried in vacuum at 50 °C. 2.3. Synthesis of mMOF@Au-MSA Initially, Au nanoparticles were grown in-situ on the surface of mMOF. 30 mg mMOF was dispersed into 60 mL deionized water at 85 °C with stirring. HAuCl4 aqueous solution was added into the reaction solution dropwise with the final concentration of 0.24 mmol/L. After continuous stirred 0.5 h, 1.6 mL trisodium citrate (1 mol/L) was poured into the system followed by stirring for another 1 h. The as synthesized mMOF@Au was washed by deionized water three times and dried in vacuum at 50 °C. Subsequently, 10 mg MOF@Au was added into 30 mL ethanol with 10 mmol/L mercaptosuccinic acid and stirred at 60 °C for 3 h. The prepared mMOF@Au-MSA was collected by magnet and washed with ethanol for three times then dried in vacuum at 50 °C for the following experiments. 2.4. Sample preparation Standard glycoprotein (HRP or IgG, 2 mg) was dissolved in 25 mmol/L NH4HCO3 aqueous solution and then denatured in boiling water for 10 min. Afterwards, appropriate amount of trypsin (trypsin/ protein = 1/40, w/w) was added into the standard glycoprotein solution and digested at 37 °C for 16 h. The obtained tryptic digests were diluted with loading buffer for enrichment experiments. For BSA, preparation steps were the same as the previous description except the adding amount was 10 mg. As for the preparation of breast cancer serum digestion, 2 μL of serum was diluted to 20 μL with NH4HCO3 aqueous solution (25 mmol/ L, pH 7.9) and denatured in boiling water for 10 min. After that, the obtained serum solution was reduced using DTT for 0.5 h at 60 °C and then alkylated by IAA in dark for 1 h at 37 °C. The mixture was treated with a certain amount of trypsin (trypsin/protein = 1/40, w/w) and
2. Experimental 2.1. Materials and chemicals Iron chloride hexahydrate (FeCl3·6H2O), sodium citrate(NaAc), 447
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incubated for 16 h at 37 °C. Tryptic digestion of breast cancer serum was obtained and lyophilized for further experiments.
was further investigated with Fourier transform infrared (FTIR) spectroscopy in Fig. S3. Compared with the spectrogram of Fe3O4@PDA, after coating with the UiO-66-NH2, the peaks at 766.99 cm−1 and 629.33 cm−1 were detected attributed to N–H stretching vibration. The adsorption band at 1435.16 cm−1 originated from the O–H bending vibration. Although the peaks that corresponding to MOF decreased after modified with MSA since the surface of the materials was covered intensively with Au nanoparticles, the newly generated peak at 1695.31 was provided by the C–O stretching vibration from the carboxyl groups of MSA [40]. Wide-angle X-ray diffraction (XRD) was also conducted to confirm the crystalline structure of mMOF. As shown in Fig. S4, diffraction peaks at 7.25°, 8.4°, 25.7° and 43.2° were distinctively caused by UiO-66-NH2. And those at 30.2° and 35.8°, 57.5° as well as 63.3° were from the magnetic core of Fe3O4 [33]. It directly demonstrated that the MOF layer was successfully fabricated. Finally, zeta potential analysis was operated in aqueous solution to monitor the surface potential change of the product on each step (Fig. S5). In the synthetic process of mMOF, the values firstly dropped and then went up, which was ascribed to the negative acidic catechol hydroxyl groups of PDA and positive amino groups from MOFs. As to the functionalization of MSA, the Zeta potential decreased thanks to the carboxyl groups of MSA. All these characterization investigations have given a satisfactory result that the mMOF@Au-MSA was completely constructed. Meanwhile, large specific area is a significant property leading to the highly efficient enrichment performance. The nitrogen adsorption−desorption experiment revealed that the mMOF@Au-MSA processed a favorable BET surface area of 229.23 m2 g−1 (Fig. S6). Besides, hydrophilicity is absolutely another key point for HILIC-based glycopeptide enrichment. The mMOF@ Au-MSA was tested to be 28.04° of water contact angle (Fig. S7 b), exhibiting greatly improved hydrophilicity compared with that of mMOF (Fig. S7 a). Moreover, magnetic responsiveness was also a crucial factor which can greatly facilitate the separating procedures and minimize the waste of samples. The satisfying magnetic responsiveness of mMOF@Au-MSA was illustrated in Fig. S8, while being equably dispersed in water (Fig. S8a), the nanomaterials can be separated from solution in a few seconds (Fig. S8b) with the help of a magnetic block.
2.5. Enrichment protocol For enrichment of glycopetides from standard protein digestion, mMOF@Au-MSA (200 μg) and a specific amount of digests were dispersed in 100 μL loading buffer (ACN/H2O/TFA, 90/9.9/0.1, v/v/v). The obtained mixture solution was incubated at 37 °C for 45 min. Subsequently, the materials were separated from solution by a magnet field and then washed by loading buffer for three times. After that, the enriched glycopetides were released from the materials by 10 μL eluting buffer (ACN/H2O/FA, 30/70/0.1, v/v/v) under continuous vibration for 30 min at 37 °C. Finally, the collected eluent was mixed with DHB matrix (20 mg/mL) and analyzed by MALDI-TOF MS. Breast cancer serum was donated by two patients. The same enrichment and eluting treatments were carried out. After that, the elution was lyophilized and redissolved in 25 mmol/L NH4HCO3 aqueous solution. Then, N-linked glycan moieties were removed by adding 1 μL PNGaseF into the solution and incubating at 37 °C for 16 h. The obtained glycopetides solution was lyophilized again for Nano-HPLC–MS/ MS analysis. 2.6. MALDI-TOF MS analysis Generally, 1 μL of eluent was dropped upon the steel plate and dried naturally. Then, 1 μL of DHB (20 mg/mL) was added. Slightly change was made only when taking the sensitivity experiment. A total of 1 μL eluent was dropped upon the steel plate in two batches, allowing the former one to get dried naturally before another droplet was added, followed by 0.5 μL of DHB (20 mg/mL). MALDI-TOF MS analysis were accomplished on a 5800 Proteomics Analyzer (Applied Biosystems, U.S.A.) in positive ion mode at a frequency of 200 Hz and the acceleration voltage of 20 kV. The Nd:YAG laser was 355 nm. 2.7. Characterization, nano-LC-ESI-MS/MS analysis and database searching Detailed information about characterization, Nano-LC-ESI-MS/MS analysis and database searching was illustrated in the Supporting Information.
3.2. Investigation of the performance of mMOF@Au-MSA nanocomposities for glycopeptides enrichment The glycopetides enrichment workflow was displayed in Scheme 2 and involved four steps: incubating, washing, eluting as well as MS detecting. According to the hydrophilic affinity interaction between nanomaterials and glycosylated peptides, glycopeptides can be adsorbed on the surface of the materials, on the contrary, non-glycopeptides can be washed away. To achieve the best enrichment efficiency, the tryptic digest of horseradish peroxidase (HRP) was utilized as standard glycopeptides to optimize the capture conditions. Firstly, incubation conditions with appropriate hydrophilicity as well as suitable pH condition were crucial factors severely affecting the enrichment efficiency. Thus, the loading buffer with different amount of acetonitrile (ACN) and trifluoroactic acid (TFA) was studied using of 250 fmol μL−1 HRP digest. The MS peak intensities of glycopeptides were systematically compared. As seen in Fig. S9a and Fig. S9b, best result was obviously achieved with the loading buffer composition of ACN/H2O/TFA (90/9.9/0.1, v/v/v) and the subsequent experiments were operated in this condition. Besides, the incubation duration was also investigated and demonstrated in Fig. S 9c. The incubation period of 45 min is sufficient to get a relatively satisfying result since longer incubation time can hardly improve the peak intensities of glycopeptides. With the optimized incubation conditions, outstanding performance was achieved using the as-synthesized nanoparticles to enrich glycopeptides in the standard protein digests of HRP as well as immunoglobulin G (IgG). As illustrated in Fig. 1, the mass spectra of HRP
3. Results and discussion 3.1. Synthesis and characterization of mMOF@Au-MSA The fabrication process of mMOF@Au-MSA was depicted in Scheme 1. First, A layer of polydopamine was coated on the magnetic Fe3O4 core via a self-assembly procedure. Then, a one-pot synthesis protocol was applied to grafted UiO-66-NH2 shell onto the surface of Fe3O4@ PDA (mMOF). Next, Au nanoparticles were intensively grown in-situ on the surface of mMOF. Finally, mercaptosuccinic acid was covalently combined to the Au and the mMOF@Au-MSA was synthesized successfully. The morphology and construction of mMOF@Au-MSA were systematically certified through diverse characterizations. At first, the TEM image of Fe3O4@PDA@ UiO-66-NH2 (Fig. S1a) shows an undulating layer of MOF outside a magnetic core. Au nanoparticles with the diameters of approximately 15 nm were apparently immobilized on magnetic MOFs from the observation of TEM image (Fig. S1b) as well as SEM image (Fig. S2a) of mMOF@Au-MSA. Besides, the components of mMOF@Au-MSA were investigated in the energy dispersive X-ray (EDX) analysis. The results were depicted in Fig. S2b and detailed in Table S1. They demonstrated the existence of Zr, S and Au elements, proving that the MOF was successfully synthesized and the MSA was attached to Au as desired. Moreover, the structure of mMOF@Au-MSA 448
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Scheme 1. The synthetic procedure for mMOF@Au-MSA.
and IgG tryptic digests without enrichment were occupied by plenty of non-glycopeptides and only six and seven glycopeptide peaks were observed from 250 fmol μL−1 HRP and 300 fmolμL−1 IgG tryptic digest respectively. Nevertheless, after extracted with mMOF@Au-MSA, both 29 glycopeptide peaks from HRP and IgG were clearly detected, and the intensities of glycopeptide peaks were extensively improved. The detail information of the captured glycopeptides from HRP and IgG digests were provided in Table S2 and Table S3 respectively. Besides, the enrichment experiments of Fe3O4@PDA@UiO-66-NH2 (mMOF) were also estimated under the same conditions which were performed in Fig. 2 for comparison. As showed in Fig. S10, fewer and less intensive glycopeptides peaks were detected. It indirectly confirmed that the MSA was successfully grafted on mMOF@Au and greatly improved the enrich efficiency towards glycopeptides. Furthermore, to investigate the enrichment sensitivity of mMOF@Au-MSA nanocomposites for glycopeptides, a series of diluted HRP tryptic digests were employed in the enrich process. As shown in Fig. 2, three glycopeptide peaks still can be detected even though the concentration of the HRP digest was decreased to 0.5 fmol μL−1, which means mMOF@Au-MSA nanocomposites possess competitive sensitivity towards glycopeptides compared with the previous reports [16,35]. This good result could mostly be credited to the high hydrophilicity origin from MSA with abundant carboxylic groups and large surface area of the MOF-based composites. Moreover, digestion of protein-bovine serum albumin (BSA),a
typical non-glycoprotein, was employed to monitor the enrichment selectivity of mMOF@Au-MSA towards glycopeptides. As shown in Fig. 3, when the mass ratio was 1 : 50 (HRP/BSA), only two glycopeptide peak can be detected due to the strong signal suppression from BSA digests. Whereas, after enrichment with mMOF@Au-MSA, the glycopeptide peaks of HRP digests were detected distinctly and the peaks of BSA digests were barely observed in the meanwhile. Remarkably, the mMOF@Au-MSA still exhibited a good ability of selective enrichment towards glycopeptides even if the mass ratio increased to 1:100. As a consequence, the excellent selective performance compared to those reported studies [22,41] indicated that the mMOF@Au-MSA possessed great potential in capturing glycopeptide from more complicated bio-samples containing multiple coexistent non-glycopeptides interferences. In addition, the binding capacity of mMOF@Au-MSA nanocomposites was tested as well. In brief, 2 μg HRP digests was extracted by different amounts of mMOF@Au-MSA nanocomposites. After incubation, each supernatant was collected and analyzed by MS. Through monitoring the S/N ratio of residual glycopeptides in the supernatants, the enrichment capacity of mMOF@Au-MSA was calculated to be 100 mg g−1 (Fig. S11). Meanwhile, the reusability of mMOF@Au-MSA for glycopeptides enrichment was further evaluated. After each enrichment experiment, the nanomaterials were recollected and washed with eluent buffer repeatedly to remove the potential residues. As revealed in Fig. S12, the number of glycopeptides in HRP digest presented
Scheme 2. Flowchart of glycopetides enrichment. 449
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Fig. 1. MALDI-TOF mass spectra for the glycopeptide enrichment from 250 fmol μL−1 HRP tryptic digest: (a) before enrichment, and (b) after treatment; from 300 fmol μL−1 IgG: (c) before enrichment, and (d) after treatment. Peaks of glycopeptides are marked with red diamonds . The detail information of the marked glycopeptides from HRP and IgG digests were provided in Table S2 and Table S3 respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 450
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Fig. 2. MALDI-TOF mass spectra of tryptic digested HRP after enrichment by mMOF@Au-MSA nanomaterials with the concentration of: (a) 50 fmol μL−1, (b) 10 fmol μL−1, (c) 1 fmol μL−1 and (d) 0.5 fmol μL−1. Peaks of glycopeptides are marked with red diamonds . The detail information of the marked glycopeptides was provided in Table S2. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
almost the same with the prior ones except a little decrease in peak intensities at the fifth reutilization, suggesting the great stability and reusability of mMOF@Au-MSA nanomaterials in glycopeptides enrichment. According to all the above excellent results including great selectivity, large binding capacity, high sensitivity, good stability and reusability, the mMOF@Au-MSA were further applied to enrich
glycopeptides in biological sample. Breast cancer serum, a universal and promising clinical specimen, possessed high potential to discovery glycopeptides of biomarkers for early diagnosis of this disease. In this work, three isolated replicates of 2 μL serums of breast involved two cancer patients were respectively enriched by the as prepared nanoparticles. As listed in Table S4, a total of 307 unique glycopeptides assigned to 96 glycoproteins were enriched and identified eventually 451
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Fig. 3. MALDI-TOF mass spectra for the glycopeptide enrichment from a mixture of tryptic digests of HRP and BSA at a mass ratio of 1 : 50 (a) before and (b) after treatment with mMOF@Au-MSA nanoparticles; HRP and BSA at a mass ratio of 1: 100 before (c) and after (d) treatment with mMOF@Au-MSA nanoparticles. Glycopeptides are marked with red diamonds . The detail information of the marked glycopeptides was provided in Table S2. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 452
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with the nano-LC-ESI-MS/MS analysis. It is doubtless that the mMOF@Au-MSA possessed overwhelming enrichment advantage toward glycopeptide in biological samples.
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4. Conclusions Magnetic metal–organic frameworks containing abundant carboxylic groups (mMOF@Au-MSA) nanocomposites were designed and synthesized to enrich N-glycopeptides from breast cancer serum. The as-synthesized nanomaterials integrated the advantages of robust magnetic responsiveness of Fe3O4, large surface area of the MOF, as well as extraordinary hydrophilicity and biocompatibility of mercaptosuccinic acid. As a consequence, the mMOF@Au-MSA demonstrated brilliant performance in glycopeptide enrichment including remarkable selectivity, high sensitivity, good repeatability and sustainability. Especially, mMOF@Au-MSA was applied to capture glycopeptides from serum of breast cancer. The satisfying results not only confirmed the promising potential of mMOF@Au-MSA in glycopeptidome profiling based on MS strategies, but also extended our knowledge of glcopeptides in breast cancer. Moreover, this work provided a guide for hydrophilic post-modification of MOFs and its application in glycoproteome research. Conflicts of interest The authors declare no competing financial interest. Acknowledgments This work was financially supported by National Key R&D Program of China (2018YFA0507501) and the National Natural Science Foundation of China (21425518). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.06.037. References [1] Y. Yang, V. Franc, A.J.R. Heck, Glycoproteomics, A balance between highthroughput and in-depth analysis, Trends Biotechnol. 35 (7) (2017) 598–609. [2] L. Cao, J.K. Diedrich, D.W. Kulp, M. Pauthner, L. He, S.R. Park, D. Sok, C.Y. Su, C.M. Delahunty, S. Menis, R. Andrabi, J. Guenaga, E. Georgeson, M. Kubitz, Y. Adachi, D.R. Burton, W.R. Schief, J.R. Yates III, J.C. Paulson, Global site-specific N-glycosylation analysis of HIV envelope glycoprotein, Nat. Commun. 8 (2017) 14954. [3] M. Murakami, T. Kiuchi, M. Nishihara, K. Tezuka, R. Okamoto, M. Izumi, Y. Kajihara, Chemical synthesis of erythropoietin glycoforms for insights into the relationship between glycosylation pattern and bioactivity, Sci. Adv. 2 (1) (2016) e1500678. [4] H. Zhang, X.-j. Li, D.B. Martin, R. Aebersold, Identification and quantification of Nlinked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry, Nat. Biotechnol. 21 (2003) 660. [5] N. Sun, C. Deng, Y. Li, X. Zhang, Highly selective enrichment of N-linked glycan by carbon-functionalized ordered graphene/mesoporous silica composites, Anal. Chem. 86 (4) (2014) 2246–2250. [6] G.S.M. Kammeijer, J. Nouta, J. de la Rosette, T.M. de Reijke, M. Wuhrer, An indepth glycosylation assay for urinary prostate-specific antigen, Anal. Chem. 90 (7) (2018) 4414–4421. [7] Y. Li, J. Wang, N. Sun, C.-h. Deng, Glucose-6-Phosphate-Functionalized magnetic microsphere as novel hydrophilic probe for specific capture of N-linked glycopeptides, Anal. Chem. 89 (20) (2017) 11151–11158. [8] P. Minicozzi, L. Van Eycken, F. Molinie, K. Innos, M. Guevara, R. Marcos-Gragera, C. Castro, E. Rapiti, A. Katalinic, A. Torrella, T. Žagar, M. Bielska-Lasota, P. Giorgi Rossi, N. Larrañaga, J. Bastos, M.J. Sánchez, M. Santthe European HR Working Group on breast cancer, Comorbidities, age and period of diagnosis influence treatment and outcomes in early breast cancer, Int. J. Cancer 144 (9) (2019) 2118–2127. [9] S.S. Pinho, C.A. Reis, Glycosylation in cancer: mechanisms and clinical implications, Nat. Rev. Canc. 15 (2015) 540. [10] J. Cox, M.Y. Hein, C.A. Luber, I. Paron, N. Nagaraj, M. Mann, Accurate proteomewide label-free quantification by delayed normalization and maximal peptide ratio
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