聚酰基氨基脲类多孔有机聚合物的制备及其在糖肽富集中的应用

通过均苯三甲酰肼(BTZ)和对苯二异氰酸酯(PDI)的聚合反应制备了一种新型的聚酰基氨基脲类多孔有机聚合物(POP)。采用傅里叶变换红外光谱(FT-IR)、交叉极化魔角自旋碳13核磁共振(CP-MAS13C NMR)、氮气物理吸附/脱附实验和水接触角等手段对POP-1的物理性质进行表征。其中比表面积为166 m~2/g,水接触角为46.4°,表明这种多孔材料具有良好的亲水性。因此,尝试将POP-1应用于标准蛋白质和复杂生物样品中糖肽的亲水色谱富集。通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF/MS)分析,从500 fmol人血清免疫球蛋白G(IgG)酶解液中共鉴定到19条典型的N-连接糖肽;通过毛细管液相色谱-串联质谱(c LC-MS/MS)分析,从100μg鼠肝蛋白酶解液中共鉴定到属于605个糖蛋白的1 919个糖肽的1 350个N-糖基化位点。这些结果表明POP-1在糖蛋白质组学研究中具有较强的应用潜力。     

The determination of glycopeptides by liquid chromatography/mass spectrometry with collision-induced dissociation

Glycopeptides derived from ribonuclease B and ovomucoid have been subjected to collision-induced dissociation (CID) in the second quadrupole of a triple quadrupole mass spectrometer. Doubly charged parent ions gave predictable fragmentation that yielded partial sequence information of the attached oligosaccharide as Hex and HexNAc units. Common oxonium ions are observed in the product ion mass spectra of the glycopeptides that correspond to HexNAc⁺ (m/z 204) and HexHexNAc⁺ (m/z 366). A strategy for locating the glycopeptides in the proteolytic digest mixtures of glycoproteins by ions spray liquid chromatography mass spectrometry (LC/MS) is described by utilizing CID in the declustering region of the atmospheric pressure ionization mass spectrometer to produce these characteristic oxonium ions. This LC/CID/MS approach is used to identify glycopeptides in proteolytic digest mixtures of ovomucoid, asialofetuin, and fetuin. LC/CID/MS in the selected ion monitoring mode may be used to identify putative glycopeptides from the proteolytic digest of fetuin.     

半胱氨酸基麦芽糖修饰亲水硅胶的制备及其对糖肽的富集

糖蛋白与糖肽在复杂生物体系中丰度一般较低,为了在糖蛋白质组学研究中深入和全面分析鉴定糖基化位点与糖链,通常需要进行富集前处理操作。该文设计并合成了一种半胱氨酸基麦芽糖修饰亲水硅胶分离介质(Cys-Mal@SiO_2),将其装填入固相萃取柱中制备新型亲水固相萃取柱。在以人免疫球蛋白G酶解液为样品进行富集鉴定时,Cys-Mal@SiO_2鉴定糖肽的质谱信号强度和信噪比比半胱氨酸修饰硅胶(Cys@SiO_2)、麦芽糖修饰硅胶(Mal@SiO_2)和商品化ZIC-HILIC更高。在复杂的鼠肝蛋白质提取物的糖蛋白质组学分析中,Cys-Mal@SiO_2共鉴定出1 551条糖肽,属于466个糖蛋白的906个N-糖基化位点,比Cys@SiO_2和Mal@SiO_2鉴定糖肽数、蛋白质数和N-糖基化位点数分别多211、67、127个和289、76、193个。将Cys-Mal@SiO_2成功应用于低丰度糖肽的选择性富集与鉴定,在糖蛋白质组学研究中体现出良好的应用潜力。     

Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry

We present herein an efficient mass spectrometric method for the localization of the glycation sites of a model neoglycoconjugate vaccine formed by a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases, and the digests were then analyzed by MALDI-TOF/TOF-CID-MS/MS and nano-LC-ESI-QqTOF-CID-MS/MS. The sequences of the unknown peptides analyzed by MALDI-TOF/TOF-CID-MS/MS, following digestion with the GluC V8 endoproteinase, allowed us to recognize three glycopeptides whose glycation occupancies were, respectively, on Lys 235, Lys 420, and Lys 498. Similarly, the same analysis was performed on the tryptic digests, which permitted us to recognize two glycation sites on Lys 100 and Lys 374. In addition, we have also used LC-ESI-QqTOF-CID-MS/MS analysis for the identification of the tryptic digests. However, this analysis identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate-protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during the synthetic carbohydrate-spacer-carrier protein conjugation. Likewise, the LC-ESI-QqTOF-MS/MS analysis of the GluC V8 digest also identified 17 different glycation sites on the synthetic glycoconjugate.     

Structural elucidation of O-linked glycopeptides by high energy collision-induced dissociation

O-linked glycopeptides that bear a GalNAc core with and without the presence of sialic acid have been analyzed by high energy collision-induced dissociation (CID). We show that the CID spectra from the glycosylated precursor ions contain sufficient information to identify the peptide sequence and to determine the glycosylated site(s). Asialo O-linked glycopeptides, previously prepared from a tryptic digest of bovine fetuin were studied. One of the glycopeptides contained only a single Hex (hexose)-HexNAc (N-acetylhexosamine) substitution at Thr²⁶², whereas the other exhibited Hex-HexNAc moieties at both Thr²⁶² and Ser²⁶⁴. In addition, sialo and asialo fetuin glycopeptides from a pronase digest were derivatized with t-butoxycarbonyl-tyrosine, and characterized by high energy CID analysis. The presence of a Galβ(1,3)GalNAc core structure at Ser²⁶⁴ was confirmed by using the substrate specificity of endo-α-N-acetylgalactosaminidase. These studies revealed the presence of a β-galactosidase specific for β(1,4) linkages in the endo-α-N-acetylgalactosaminidase preparation employed. Finally, the relative stability of N-and O-glycosyl bonds to high energy CID is addressed based upon comparison of the behavior of a synthetic N-linked glycopeptide with analogous O-linked structures.     

基于Endo-M N175Q糖链转移反应与丙酮富集糖肽的LC-MS分析糖蛋白N-糖链

基于化学酶标记和丙酮富集糖肽方法,建立了一种可靠、有效、简单的糖蛋白N-糖链分析方法。以唾液酸糖肽(SGP)为模型糖肽,比较了样品中丙酮加入量对SGP的富集效果,最终选择加入样品体积5倍量的丙酮。用丙酮富集经胰蛋白处理的核糖核酸酶B(RNase B)酶解液中的糖肽,以富集分离得到的糖肽(糖基供体)和PDPZ-Boc-Asn-GlcNAc(糖基受体)作为酶反应底物,进行Endo-M N175Q的转糖基反应,得到PDPZBoc-Asn-GlcNAc-N-糖链标记物。采用YMC C18色谱柱为分析柱,10 mmol/L甲酸铵-乙腈为流动相梯度洗脱,经液相色谱-串联质谱(LC-MS)检测得到5种高甘露糖型糖链。结果表明,丙酮可有效地富集大量肽和少量糖肽混合溶液中的糖肽,Endo-M N175Q可将天然糖肽的糖链转移到PDPZ-Boc-Asn-GlcNAc受体上。将该方法应用于胎球蛋白N-糖链分析,检测到5种复杂型N-糖链。该研究为各种糖蛋白N-糖链检测提供了新的分析方法。     

Improving confidence in detection and characterization of protein N-glycosylation sites and microheterogeneity

Protein glycosylation is one of the most common post-translational modifications, estimated to occur in over 50% of human proteins. Mass spectrometry (MS)-based approaches involving different fragmentation mechanisms have been frequently used to detect and characterize protein N-linked glycosylations. In addition to the popular Collision-Induced Dissociation (CID), high-energy C-trap dissociation (HCD) fragmentation, which is a feature of a linear ion trap orbitrap hybrid mass spectrometer (LTQ Orbitrap), has been recently used for the fragmentation of tryptic N-linked glycopeptides in glycoprotein analysis. The oxonium ions observed with high mass accuracy in the HCD spectrum of glycopeptides can be combined with characteristic fragmentation patterns in the CID spectrum resulting from consecutive glycosidic bond cleavages, to improve the detection and characterization of N-linked glycopeptides. As a means of automating this process, we describe here GlypID 2.0, a software tool that implements several algorithmic approaches to utilize MS information including accurate precursor mass and spectral patterns from both HCD and CID spectra, thus allowing for an unequivocal and accurate characterization of N-linked glycosylation sites of proteins.     

Reliable Determination of Site-Specific In Vivo Protein N-Glycosylation Based on Collision-Induced MS/MS and Chromatographic Retention Time

Site-specific glycopeptide mapping for simultaneous glycan and peptide characterization by MS is difficult because of the heterogeneity and diversity of glycosylation in proteins and the lack of complete fragmentation information for either peptides or glycans with current fragmentation technologies. Indeed, multiple peptide and glycan combinations can readily match the same mass of glycopeptides even with mass errors less than 5 ppm providing considerably ambiguity and analysis of complex mixtures of glycopeptides becomes quite challenging in the case of large proteins. Here we report a novel strategy to reliably determine site-specific N-glycosylation mapping by combining collision-induced dissociation (CID)-only fragmentation with chromatographic retention times of glycopeptides. This approach leverages an experimental pipeline with parallel analysis of glyco- and deglycopeptides. As the test case we chose ABCA4, a large integral membrane protein with 16 predicted sites for N-glycosylation. Taking advantage of CID features such as high scan speed and high intensity of fragment ions together combined with the retention times of glycopeptides to conclusively identify the non-glycolytic peptide from which the glycopeptide was derived, we obtained virtually complete information about glycan compositions and peptide sequences, as well as the N-glycosylation site occupancy and relative abundances of each glycoform at specific sites for ABCA4. The challenges provided by this example provide guidance in analyzing complex relatively pure glycoproteins and potentially even more complex glycoprotein mixtures.

Targeted glycoproteomics: serial lectin affinity chromatography in the selection of O-glycosylation sites on proteins from the human blood proteome

Although lectin selection is gaining increasing acceptance as a tool for targeting glycosylation in glycoproteomics, most of the work has been directed at N-glycosylation. The work reported here focuses on the use of lectins in the study of O-glycosylation. The problem with using lectins for studying O-glycosylation is that they are not sufficiently specific. This paper reports that through the use of serial lectin affinity chromatography (SLAC) it is possible to select predominantly O-glycosylated peptides from tryptic digests of human serum. Jacalin is relatively specific for O-glycosylation but has the problem that it also selects high mannose N-type glycans. This problem was addressed by using a concanavalin A affinity column to first remove high mannose, hybrid-type and biantennary complex-type N-type glycans before application of the Jacalin columns. When used in a serial format, concanavalin A and Jacalin together provide essentially O-glycosylated peptides. The glycoprotein parents of glycopeptides were identified by deglycosylating the selected O-glycopeptides by oxidative elimination. These peptides were then separated by RPC and further analyzed using ESI-MS/MS and MALDI-MS/MS. Using this approach all the O-glycosylated sites in a model protein (fetuin) and over thirty glycoprotein parents from human serum were identified. It is concluded that a serial combination of Con A and Jacalin can be of utility in the study of O-glycosylation in glycoproteomics.     

Quantitative mapping of glycoprotein micro‐heterogeneity and macro‐heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro‐heterogeneity) and evaluate the molar site occupancy (macro‐heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N‐glycans was chemically synthesised by solid‐phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N‐acetylglucosamine‐linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well‐defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI‐IT, ESI‐Q‐TOF, MALDI‐TOF, ESI/MALDI‐FT‐ICR‐MS). Depending on the ion source/mass analyser, glycopeptides carrying complex‐type N‐glycans exhibited clearly lower signal strengths (10–50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano‐ESI and medium‐pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro‐heterogeneity and macro‐heterogeneity by label‐free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics. Copyright © 2013 John Wiley & Sons, Ltd.     

Glycosylation analysis of interleukin-23 receptor: elucidation of glycosylation sites and characterization of attached glycan structures

Interleukin-23 (IL-23) is a heterodimeric cytokine, a central factor in chronic/autoimmune inflammation. It signals through a heterodimeric receptor consisting of IL-23r, which is heavily glycosylated. The structural characterization of IL-23r has not been reported. In this work, glycosylation profiles of soluble recombinant human IL-23r (rhIL-23r) were established using mass spectrometry (MS), which included defining glycosylation sites, degree of glycosylation occupancy of each site and structure of attached oligosaccharides. Specifically, precursor ion scan of oxonium ion protonated N-acetylglucosamine (GlcNAc(+)) (m/z 204) was performed using a triple quadrupole MS instrument to locate the retention time of glycopeptides. Both the glycopeptides and their corresponding deglycosylated forms in each collected HPLC fraction were studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (LTQ-Orbitrap) for glycosylation site profiling. The attached glycan structures were elucidated by collision-induced dissociation (CID) fragmentation of target glycopeptides in combination with accurate mass measurement. Eight glycosylation sites were identified on IL-23r (Asn24, Asn209, Asn239, Asn157, Asn118, Asn250, Asn58 and Asn6). Most of the glycosylation sites were > 95% occupied except Asn250 and Asn6. Those two sites were 88% and 45% occupied by estimation from trypsin digestion and were 55% and 42% occupied from LysC digestion. Multiple glycoforms were observed in IL-23r. Most of them were bi-, tri- or tetra-antennary complex type structures with fucose and sialic acid. High mannose and hybrid type glycans were only observed on Asn157. The structural characterization on IL-23r glycosylation provides useful information for better understanding of the biological function of IL-23r.     

Combining gel and capillary electrophoresis, nano-LC and mass spectrometry for the elucidation of post-translational modifications of Trichoderma reesei cellobiohydrolase I

N-Glycosylation of cellobiohydrolase I from the fungus Trichoderma reesei (strain Rut-C30) is studied using a combination of electrophoretic, chromatographic and mass spectrometric techniques. As four potential N-glycosylation sites and several uncharged and phosphorylated high-mannose glycans are present, a large number of glycoforms and phospho-isoforms can be expected. Isoelectric focusing both in gel and in capillary format was successfully applied for the separation of the phospho-isoforms. They were extracted in their intact form from the gel and subsequently analysed by nanospray-Q-TOF-MS, thereby making use of a powerful two-dimensional technique. Nano-LC/MS/MS on a Q-Trap MS further allowed the determination of the glycosylation sites. As a novel approach, an oxonium ion was used in precursor ion scanning for selective detection of glycopeptides containing phosphorylated high-mannose glycans.     

GlycoMiner: a new software tool to elucidate glycopeptide composition

New computer software, GlycoMiner, has been developed to automatically identify tandem (MS/MS) spectra obtained in liquid chromatography/mass spectrometry (LC/MS) runs which correspond to N-glycopeptides. The program complements conventional proteomics analysis, and can be used in a high-throughput environment. The program interprets the spectra and determines the structure of the corresponding glycopeptides. GlycoMiner runs under Windows, can process spectra obtained on various instruments, and can be downloaded from our website (w3.chemres.hu/ms/glycominer). The algorithm works similarly to a human expert; evaluates the low mass oxonium ions; deduces oligosaccharide losses from the protonated molecule; and identifies the mass of the peptide residue. The program has been tested on tryptic digests of two glycopeptides: AGP (which has five different N-glycosylation sites) and transferrin (with two N-glycosylation sites). Results have been evaluated both manually and by GlycoMiner. Out of 3132 MS/MS spectra 338 were found to correspond to glycopeptides; identification by GlycoMiner showed a 0.1% false positive and 0.1% false negative rate. From these it was possible to identify 196 glycan structures manually; GlycoMiner correctly identified all of these, with no false positives. The rest were low quality spectra, not suitable for structure assignment.     

Unusual Fragmentation Pathways in Collagen Glycopeptides

Collagens are the most abundant glycoproteins in the body. One characteristic of this protein family is that the amino acid sequence consists of repeats of three amino acids –(X—Y—Gly)_n. Within this motif, the Y residue is often 4-hydroxyproline (HyP) or 5-hydroxylysine (HyK). Glycosylation in collagen occurs at the 5-OH group in HyK in the form of two glycosides, galactosylhydroxylysine (Gal-HyK) and glucosyl galactosylhydroxylysine (GlcGal-HyK). In collision induced dissociation (CID), collagen tryptic glycopeptides exhibit unexpected gas-phase dissociation behavior compared to typical N- and O-linked glycopeptides (i.e., in addition to glycosidic bond cleavages, extensive cleavages of the amide bonds are observed). The Gal- or GlcGal- glycan modifications are largely retained on the fragment ions. These features enable unambiguous determination of the amino acid sequence of collagen glycopeptides and the location of the glycosylation site. This dissociation pattern was consistent for all analyzed collagen glycopeptides, regardless of their length or amino acid composition, collagen type or tissue. The two fragmentation pathways—amide bond and glycosidic bond cleavage—are highly competitive in collagen tryptic glycopeptides. The number of ionizing protons relative to the number of basic sites (i.e., Arg, Lys, HyK, and N-terminus) is a major driving force of the fragmentation. We present here our experimental results and employ quantum mechanics calculations to understand the factors enhancing the labile character of the amide bonds and the stability of hydroxylysine glycosides in gas phase dissociation of collagen glycopeptides.

Matrix-assisted laser desorption/ionization on-target method for the investigation of oligosaccharides and glycosylation sites in glycopeptides and glycoproteins

The significance of glycoproteins in living systems instigates the ceaseless expansion of new techniques and procedures for the analysis of biological samples. Many of these applications are focused on improving the detection limit of analyzed material. In a previous study, we described a procedure for the detection of oligosaccharides cleaved from tryptic glycopeptides. Treatment of deglycosylated fractions with phenylhydrazine gave rise to peaks consistent with labeled glycans, and both types of compounds--deglycosylated peptides and oligosaccharides--were recorded from one spot and observed in one matrix-assisted laser desorption/ionization (MALDI) mass spectrum for the first time. Here, we added an additional step to this simple procedure of deglycosylating glycopeptides directly from the target spot of the first analyzed glycosylated peptides. For the purpose of this new study, a mixture of 2-aza-2-thiothymine and phenylhydrazine hydrochloride showed to be an excellent matrix for glycopeptides, oligosaccharides, deglycosylated peptides and moreover it allowed PNGaseF to be active enough to cleave oligosaccharides from peptides. The efficiency of this procedure is demonstrated on a series of intact glycoproteins and on the analysis of tryptic peptides obtained from IgG and total mouse serum. This one-step on-target deglycosylation method with subsequent derivatization on the same spot makes MALDI-MS analyses of glycopeptides fast, simple and accessible for biological samples, where classical procedures cannot produce useful results.     

Characterization of glycosylation sites for a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein by liquid chromatography-mass spectrometry peptide mapping

Liquid chromatography mass spectrometry (LC-MS) peptide mapping can be a versatile technique for characterizing protein glycosylation sites without the need to remove the attached glycans as in conventional oligosaccharide mapping methods. In this way, both N-linked and O-linked sites of glycosylation can each be directly identified, characterized, and quantified by LC-MS as intact glycopeptides in a single experiment. LC-MS peptide mapping of the individual glycosylation sites avoids many of the limitations of preparing and analyzing an entire pool of released N-linked oligosaccharides from all sites mixed together. In this study, LC interfaced to a linear ion trap mass spectrometer (ESI-LIT-MS) were used to characterize the glycosylation of a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein with multiple sites of N-and O-glycosylation. Samples were reduced, S-carboxyamidomethylated, and cleaved with either trypsin or endoproteinase Asp-N. Enhanced detection for minor IgG1 glycoforms (～0.1 to 1.0 mol% level) was obtained by LC-MS of the longer 32-residue Asp-N glycopeptide (4+ protonated ion) compared to the 9-residue tryptic glycopeptide (2+ ion). LC-MS peptide mapping was run according to a general procedure: (1) Locate N-linked and/or O-linked sites of glycosylation by selected-ion-monitoring of carbohydrate oxonium fragment ions generated by ESI in-source collision-induced dissociation (CID), i.e. 204, 366, and 292 Da marker ions for HexNAc, HexNAc-Hex, and NeuAc, respectively; (2) Characterize oligosaccharides at each site via MS and MSMS. Use selected ion currents (SIC) to estimate relative amounts of each glycoform; and (3) Measure the percentage of site-occupancy by searching for any corresponding nonglycosylated peptide.     

Characterization of oligosaccharide moieties of glycopeptides by microwave-assisted partial acid hydrolysis and mass spectrometry

Microwave-assisted partial acid hydrolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used to study oligosaccharide structures of glycopeptides. Tryptic N-glycosylated peptides of horseradish peroxidase, with MH+ ions at m/z 2533, 2612, 3355, 3673, and 5647, were used as test cases. Within a microwave exposure with trifluoroacetic acid of 2 min, partial cleavages of the oligosaccharides of these tryptic N-glycosylated peptides were observed. The data showed that the most labile group within the oligosaccharides is the fucose (Fuc) residue, and that a majority of the end cleavage products are peptides with one N-acetylglucosamine (GlcNAc) residue linked to asparagine (Asn). In addition, the glycopeptides with m/z 3355 and 3673 carry an oligosaccharide (Xyl)Man3(Fuc)GlcNAc2, the glycopeptide at m/z 5647 carries two oligosaccharides (Xyl)Man3(Fuc)GlcNAc2, and the glycopeptides at m/z 2612 and 2533 carry (Xyl)Man3GlcNAc2 and (Fuc)GlcNAc, respectively. However, the glycosylation site of the m/z 2612 peptide at Asn286 is partially occupied. This simple and rapid method is particularly useful in identifying glycopeptides and obtaining monosaccharide compositions of glycopeptides.     

Structural analysis of a glycoprotein by liquid chromatography-mass spectrometry and liquid chromatography with tandem mass spectrometry. Application to recombinant human thrombomodulin

Using recombinant human thrombomodulin (rhTM) expressed in Chinese hamster ovary (CHO) cells, we studied the structural analysis of a glycoprotein by liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS-MS). First, we analyzed the structure of both the O- and N-linked glycans in rhTM by oligosaccharide mapping using LC-MS equipped with a graphitized carbon column (GCC-LC-MS). Major O- and N-linked glycans were determined to be core 1 structure and fucosyl biantennary containing NeuAc(0-2) respectively. Next, the post-translational modifications and their heterogeneities, including the site-specific glycosylation, were analyzed by mass spectrometric peptide/glycopeptide mapping of trypsin-digested rhTM and precursor-ion scanning. Precursor-ion scanning was successful in the detection of five glycopeptides. Four N-glycosylation sites and their site-specific carbohydrate heterogeneity were determined by their mass spectra. O-Glycosylation could be estimated on the basis of its mass spectrum. We were able to identify partial beta-hydroxylation on Asn324 and Asn439, and O-linked glucose on Ser287 from the peptide/glycopeptide map and their mass spectra. We demonstrated that a sequential analysis of LC-MS and LC-MS-MS are very useful for the structural analysis of O- and N-linked glycans, polypeptides, and post-translational modifications and their heterogeneities, including site-specific glycosylation in a glycoprotein. Our method can be applied to a glycoprotein in biological samples.     

Rapid glycopeptide enrichment and N-glycosylation site mapping strategies based on amine-functionalized magnetic nanoparticles

Glycoproteins secreted or expressed on the cell surface at specific pathophysiological stages are well-recognized disease biomarkers and therapeutic targets. While mapping of specific glycan structures can be performed at the level of released glycans, site-specific glycosylation and identification of specific protein carriers can only be determined by analysis of glycopeptides. A key enabling step in mass spectrometry (MS)-based glycoproteomics is the ability to selectively or non-selectively enrich for the glycopeptides from a total pool of a digested proteome for MS analysis since the highly heterogeneous glycopeptides are usually present at low abundance and ionize poorly compared with non-glycosylated peptides. Among the most common approaches for non-destructive and non-glycan-selective glycopeptide enrichment are strategies based on various forms of hydrophilic interaction liquid chromatography (HILIC). We present here a variation of this method using amine-derivatized Fe₃O₄ nanoparticles, in concert with in situ peptide N-glycosidase F digestion for direct matrix-assisted laser desorption/ionization–mass spectrometry analysis of N-glycosylation sites and the released glycans. Conditions were also optimized for efficient elution of the enriched glycopeptides from the nanoparticles for on-line nanoflow liquid chromatography–MS/MS analysis. Successful applications to single glycoproteins as well as total proteomic mixtures derived from biological fluids established the unrivaled practical versatility of this method, with enrichment efficiency comparable to other HILIC-based methods.     

Off-line hyphenation of boronate affinity monolith-based extraction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for efficient analysis of glycoproteins/glycopeptides

Boronate affinity materials have attracted increasing attentions as sample enrichment platforms for glycoproteomic analysis in recent years. However, most of the boronate affinity materials that have already employed for proteomic analysis are suffering from apparent disadvantages, such as alkaline pH for binding, weak affinity, and relatively poor selectivity. Benzoboroxoles are a unique class of boronic acids which have showed excellent binding properties for the recognition of cis-diol-containing compounds. Recently, a 3-carboxy-benzoboroxole-functionalized monolithic column had been reported and it had exhibited the best selectivity and affinity as well as the lowest binding pH among all reported boronate affinity monolithic columns. In this study, an off-line hyphenation of this boronate affinity monolithic column-based extraction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was developed and the powerfulness of this hyphenated approach in the analysis of glycoproteins and glycopeptides in complex samples was investigated. The approach was first applied to the analysis of glycopeptides in the tryptic digest of horseradish peroxidase (HRP). Totally 22 glycopeptides were identified. To the best of our knowledge, this is the best performance among all the boronic acid-functionalized materials. We further employed this approach to the analysis of intact proteins in human saliva. Totally 6 intact glycoproteins were successfully identified. As comparison, when the samples were analyzed without extraction, only a few glycopeptides were identified from the tryptic digest of HRP while no glycoproteins were found from the saliva samples.