Tools for glycoproteomic analysis: size exclusion chromatography facilitates identification of tryptic glycopeptides with N-linked glycosylation sites

Proteomic techniques, such as HPLC coupled to tandem mass spectrometry (LC-MS/MS), have proved useful for the identification of specific glycosylation sites on glycoproteins (glycoproteomics). Glycosylation sites on glycopeptides produced by trypsinization of complex glycoprotein mixtures, however, are particularly difficult to identify both because a repertoire of glycans may be expressed at a particular glycosylation site, and because glycopeptides are usually present in relatively low abundance (2% to 5%) in peptide mixtures compared to nonglycosylated peptides. Previously reported methods to facilitate glycopeptide identification require either several pre-enrichment steps, involve complex derivatization procedures, or are restricted to a subset of all the glycan structures that are present in a glycoprotein mixture. Because the N-linked glycans expressed on tryptic glycopeptides contribute substantially to their mass, we demonstrate that size exclusion chromatography (SEC) provided a significant enrichment of N-linked glycopeptides relative to nonglycosylated peptides. The glycosylated peptides were then identified by LC-MS/MS after treatment with PNGase-F by the monoisotopic mass increase of 0.984 Da caused by the deglycosylation of the peptide. Analyses performed on human serum showed that this SEC glycopeptide isolation procedure results in at least a 3-fold increase in the total number of glycopeptides identified by LC-MS/MS, demonstrating that this simple, nonselective, rapid method is an effective tool to facilitate the identification of peptides with N-linked glycosylation sites.     

Approaches to the study of N-linked glycoproteins in human plasma using lectin affinity chromatography and nano-HPLC coupled to electrospray linear ion trap--Fourier transform mass spectrometry

In this publication, we will describe the combination of lectin affinity chromatography with nano high performance liquid chromatography (HPLC) coupled to a linear ion trap Fourier transform mass spectrometer (capillary LC-LTQ/FTMS) to characterize N-linked glycosylation structures in human plasma proteins. We used a well-characterized glycoprotein, tissue plasminogen activator (rt-PA), which is present at low levels in blood, as a standard to determine the dynamic range of this approach. N-linked glycopeptides derived from rt-PA could be characterized at a ratio of 1:200 in human plasma (rtPA: total plasma protein, w/w) by accurate mass measurement in the FTMS and fragmentation (MS(n)) in the linear ion trap. We demonstrated that this platform has the potential to characterize the general N-linked glycosylation structures of abundant glycoproteins present in human plasma without the requirement for antibody-based purification, or additional carbohydrate analytical protocols. This conclusion was supported by the determination of carbohydrate structures for three glycoproteins, IgG, haptoglobin, and alpha-1-acid glycoprotein, at their natural levels in a human plasma sample, but only after the lectin enrichment step.     

Protein glycosylation regulates the externalization of two distinct classes of glucocorticoid-induced glycoproteins in rat hepatoma cells

The relationship of protein glycosylation to the externalization of glucocorticoid inducible alpha1-acid glycoprotein and mouse mammary tumor virus glycoproteins was examined in M1.54, a clonal population of mouse mammary tumor virus-infected rat hepatoma cells. Multiple freeze-thaw of isolated microsomes revealed that while alpha 1-acid glycoprotein is carried through the cell as a soluble component of vesicles, extracellular viral glycoproteins are initially membrane-associated. At concentrations of tunicamycin that specifically inhibited N-linked protein glycosylation, alpha 1-acid glycoprotein fractionated between the cellular and extracellular compartments. Thus, approximately one half of the newly synthesized, nonglycosylated (22,000 Mr) alpha 1-acid glycoprotein was rapidly secreted with kinetics similar to its glycosylated counterpart (release half-time of 60 min), while the remaining species first localized in an undefined intracellular compartment prior to its slow secretion (release half-time of 24 h). The same distribution of nonglycosylated alpha 1-acid glycoprotein was observed at various absolute levels of polypeptide, suggesting that this was not due simply to the saturation of an efficient secretory pathway at high polypeptide levels. In contrast to alpha 1-acid glycoprotein, no labeled viral antigens were released by tunicamycin-treated M1.54, while a nonglycosylated viral precursor glycopolyprotein was expressed intracellularly. Taken together, these results suggest that carbohydrate attachment strongly regulates the externalization of both alpha 1-acid glycoprotein and mouse mammary tumor virus species, which represent two distinct classes of extracellular glycoproteins.     

N-Glycan structure annotation of glycopeptides using a linearized glycan structure database (GlyDB)

While glycoproteins are abundant in nature, and changes in glycosylation occur in cancer and other diseases, glycoprotein characterization remains a challenge due to the structural complexity of the biopolymers. This paper presents a general strategy, termed GlyDB, for glycan structure annotation of N-linked glycopeptides from tandem mass spectra in the LC-MS analysis of proteolytic digests of glycoproteins. The GlyDB approach takes advantage of low-energy collision-induced dissociation of N-linked glycopeptides that preferentially cleaves the glycosidic bonds while the peptide backbone remains intact. A theoretical glycan structure database derived from biosynthetic rules for N-linked glycans was constructed employing a novel representation of branched glycan structures consisting of multiple linear sequences. The commonly used peptide identification program, Sequest, could then be utilized to assign experimental tandem mass spectra to individual glycoforms. Analysis of synthetic glycopeptides and well-characterized glycoproteins demonstrate that the GlyDB approach can be a useful tool for annotation of glycan structures and for selection of a limited number of potential glycan structure candidates for targeted validation.     

The distribution of glycan structures in individual N-glycosylation sites in animal and plant glycoproteins

Glycopeptides representing each individual N-glycosylation site in six animal and plant glycoproteins (ovoinhibitor and ovotransferrin, orosomucoid, antitrypsin, phaseolin, and phytohemagglutinin) have been isolated and compared by mass spectrometric analysis. Since the isolation step separates each individual peptide regardless of the nature of the glycan attached to it, it is possible to observe the entire spectrum of glycans associated with each site from the mass spectrum of the corresponding glycopeptide. The three glycosylation sites in ovoinhibitor have very similar but not identical glycans; they are significantly different from those observed in the single site of ovotransferrin. The three sites in serum antitrypsin also have quite similar glycans, whereas the five sites in orosomucoid show considerable variation in both the nature and the relative amount of glycans. The two plant glycoproteins each have two sites with very different glycan structures. Except for the first and third glycosylation sites of antitrypsin which were found to have remarkably homogeneous glycans (97 and 90% of a biantennary complex structure), all the individual glycosylation sites contained heterogeneous mixtures of glycan structures. The results support the proposition that each N-linked glycan in a glycoprotein is affected by its unique protein environment to such an extent that each one may be displayed to the processing enzymes as a unique structural entity. On the basis of a limited number of observations of the glycan interfering with chymotryptic but not tryptic cleavage in the proximity of the glycan attachment site, it is proposed that hydrophobic interactions between the protein and the glycan may be involved in the conformational modulation of the glycans.     

Characterization of glycoprotein digests with hydrophilic interaction chromatography and mass spectrometry

A new hydrophilic interaction chromatography (HILIC) column packed with amide 1.7 μm sorbent was applied to the characterization of glycoprotein digests. Due to the impact of the hydrophilic carbohydrate moiety, glycopeptides were more strongly retained on the column and separated from the remaining nonglycosylated peptides present in the digest. The glycoforms of the same parent peptide were also chromatographically resolved and analyzed using ultraviolet and mass spectrometry detectors. The HILIC method was applied to glyco-profiling of a therapeutic monoclonal antibody and proteins with several N-linked and O-linked glycosylation sites. For characterization of complex proteins with multiple glycosylation sites we utilized 2D LC, where RP separation dimension was used for isolation of glycopeptides and HILIC for resolution of peptide glycoforms. The analysis of site-specific glycan microheterogeneity was illustrated for the CD44 fusion protein.     

Development of quantitative plasma N-glycoproteomics using label-free 2-D LC-MALDI MS and its applicability for biomarker discovery in hepatocellular carcinoma

There has been rapid progress in the development of clinical proteomic methodologies with improvements in mass spectrometric technologies and bioinformatics, leading to many new methodologies for biomarker discovery from human plasma. However, it is not easy to find new biomarkers because of the wide dynamic range of plasma proteins and the need for their quantification. Here, we report a new methodology for relative quantitative proteomic analysis combining large-scale glycoproteomics with label-free 2-D LC-MALDI MS. In this method, enrichment of glycopeptides using hydrazide resin enables focusing on plasma proteins with lower abundance corresponding to the tissue leakage region. On quantitative analysis, signal intensities by 2-D LC-MALDI MS were normalized using a peptide internal control, and the values linked to LC data were treated with DeView? software. Our proteomic method revealed that the quantitative dynamic ranged from 102 to 10? pg/mL of plasma proteins with good reproducibility, and the limit of detection was of the order of a few ng/mL of proteins in biological samples. To evaluate the applicability of our method for biomarker discovery, we performed a feasibility study using plasma samples from patients with hepatocellular carcinoma, and identified biomarker candidates, including ceruloplasmin, alpha-1 antichymotrypsin, and multimerin-1.     

Identification and quantification of N-linked oligosaccharides released from glycoproteins: an inter-laboratory study

As characterization of glycosylation is required for the licensing of recombinant glycoprotein therapeutics, technique comparability must be assessed. Eleven UK laboratories (seven industrial, two regulatory or government, two academic) participated in an inter-laboratory study to analyze N-glycans present in four mixtures prepared by PNGase F cleavage of commercial glycoproteins: human alpha1-acid glycoprotein (H alpha1), bovine alpha1-acid glycoprotein (B alpha1), bovine pancreatic ribonuclease B (RNaseB), and human serum immunoglobulin G (hIgG). Participants applied their routine glycan mapping methodology using predominantly chromatography and mass spectrometry to identify and quantify components. Data interpretation focused on the relative amounts of different glycan structures present, the degree of sialylation, antennary and the galactosylation profiles, fucosylation and bisecting GlcNAc content, and the number of glycan components identified. All laboratories found high levels of sialylation for H alpha1 and B alpha1 (Z-numbers 271 +/- 24 and 224 +/- 18, respectively), but varying ratios of di-, tri-, and tetra-antennary chains. The Z-score for hIgG glycans had high variability as values obtained from mass spectrometric and chromatographic methods clustered separately. The proportion of the major penta-mannosyl chain from RNaseB was between 29 and 62%. Proportions of fucosylated and bisected GlcNAc chains from hIgG were between 58 and 96% and 9 and 23%, respectively. Mass spectrometric approaches consistently identified more glycan species, especially when both N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac) were present. These data highlight the need for well-characterized reference standards to support method validation and regulatory guidance on selection of approaches. Pharmacopoeial specifications must acknowledge method variability.     

Identification of glycan structure and glycosylation sites in cellobiohydrolase II and endoglucanases I and II from Trichoderma reesei

Mass spectrometric techniques combined with enzymatic digestions were applied to determine the glycosylation profiles of cellobiohydrolase (CBH II) and endoglucanases (EG I, II) purified from filamentous fungus Trichoderma reesei. Electrospray mass spectrometry (ESMS) analyses of the intact cellulases revealed the microheterogeneity in glycosylation where glycoforms were spaced by hexose units. These analyses indicated that glycosylation accounted for 12-24% of the molecular mass and that microheterogeneity in both N- and O-linked glycans was observed for each glycoprotein. The identification of N-linked attachment sites was carried out by MALDI-TOF and capillary liquid chromatography-ESMS analyses of tryptic digests from each purified cellulase component with and without PNGase F incubation. Potential tryptic glycopeptide candidates were first detected by stepped orifice-voltage scanning and the glycan structure and attachment site were confirmed by tandem mass spectrometry. For purified CBH II, 74% of glycans found on Asn310 were high mannose, predominantly Hex(7-9)GlcNAc(2), whereas the remaining amount was single GlcNAc; Asn289 had 18% single GlcNAc occupancy, and Asn14 remained unoccupied. EG I presented N-linked glycans at two out of the six potential sites. The Asn56 contained a single GlcNAc residue, and Asn182 showed primarily a high-mannose glycan Hex(8)GlcNAc(2) with only 8% being occupied with a single GlcNAc. Finally, EG II presented a single GlcNAc residue at Asn103. It is noteworthy that the presence of a single GlcNAc in all cellulase enzymes investigated and the variability in site occupancy suggest the secretion of an endogenous endo H enzyme in cultures of T. reesei.     

N-linked glycans of the human insulin receptor and their distribution over the crystal structure

The human insulin receptor (IR) homodimer is heavily glycosylated and contains a total of 19 predicted N-linked glycosylation sites in each monomer. The recent crystal structure of the IR ectodomain shows electron density consistent with N-linked glycosylation at the majority of sites present in the construct. Here, we describe a refined structure of the IR ectodomain that incorporates all of the N-linked glycans and reveals the extent to which the attached glycans mask the surface of the IR dimer from interaction with antibodies or other potential therapeutic binding proteins. The usefulness of Fab complexation in the crystallization of heavily glycosylated proteins is also discussed. The compositions of the glycans on IR expressed in CHO-K1 cells and the glycosylation deficient Lec8 cell line were determined by protease digestion, glycopeptide purification, amino acid sequence analysis, and mass spectrometry. Collectively the data reveal: multiple species of complex glycan at residues 25, 255, 295, 418, 606, 624, 742, 755, and 893 (IR-B numbering); multiple species of high-mannose glycan at residues 111 and 514; a single species of complex glycan at residue 671; and a single species of high-mannose glycan at residue 215. Residue 16 exhibited a mixture of complex, hybrid, and high-mannose glycan species. Of the remaining five predicted N-linked sites, those at residues 397 and 906 were confirmed by amino acid sequencing to be glycosylated, while that at residue 78 and the atypical (NKC) site at residue 282 were not glycosylated. The peptide containing the final site at residue 337 was not recovered but is seen to be glycosylated in the electron density maps of the IR ectodomain. The model of the fully glycosylated IR reveals that the sites carrying high-mannose glycans lie at positions of relatively low steric accessibility.     

Human plasma protein <Emphasis Type="Italic">N</Emphasis>-glycosylation

Glycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be involved in multiple biological processes, and to affect proteins physical properties, and has consequentially been labeled a critical quality attribute of biopharmaceuticals. Additionally, due to recent advances in analytical methods and analysis software, glycosylation is targeted in the search for disease biomarkers for early diagnosis and patient stratification. Biofluids such as saliva, serum or plasma are of great use in this regard, as they are easily accessible and can provide relevant glycosylation information. Thus, as the assessment of protein glycosylation is becoming a major element in clinical and biopharmaceutical research, this review aims to convey the current state of knowledge on the N-glycosylation of the major plasma glycoproteins alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, vitronectin, and zinc-alpha-2-glycoprotein. In addition, the less abundant immunoglobulins D and E are included because of their major relevance in immunology and biopharmaceutical research. Where available, the glycosylation is described in a site-specific manner. In the discussion, we put the glycosylation of individual proteins into perspective and speculate how the individual proteins may contribute to a total plasma N-glycosylation profile determined at the released glycan level.     

BlotGlycoABCTM, an integrated glycoblotting technique for rapid and large scale clinical glycomics

Recent progress in mass spectrometry has led to new challenges in glycomics, including the development of rapid glycan enrichment techniques. A facile technique for exploration of a carbohydrate-related biomarker is important because proteomics research targets glycosylation, a posttranslational modification. Here we report an "all-in-one" protocol for high throughput clinical glycomics. This new technique integrates glycoblotting-based glycan enrichment onto the BlotGlycoABC bead, on-bead stabilization of sialic acids, and fluorescent labeling of oligosaccharides in a single workflow on a multiwell filter plate. The advantage of this protocol and MALDI-TOF MS was demonstrated through differentiation of serum N-glycan profiles of subjects with congenital disorders of glycosylation and hepatocellular carcinoma and healthy donors. The method also permitted total cellular glycomics analysis of human prostate cancer cells and normal human prostate epithelial cells. These results demonstrate the potentials of glycan enrichment/processing for biomarker discovery.     

18O Labeling for a Quantitative Proteomic Analysis of Glycoproteins in Hepatocellular Carcinoma

Introduction

Quantitative proteomics using tandem mass spectrometry is an attractive approach for identification of potential cancer biomarkers. Fractionation of complex tissue samples into subproteomes prior to mass spectrometric analyses increases the likelihood of identifying cancer-specific proteins that might be present in low abundance. In this regard, glycosylated proteins are an interesting class of proteins that are already established as biomarkers for several cancers.

Materials and Methods

In this study, we carried out proteomic profiling of tumor and adjacent non-cancer liver tissues from hepatocellular carcinoma (HCC) patients. Glycoprotein enrichment from liver samples using lectin affinity chromatography and subsequent ¹⁸O/¹⁶O labeling of peptides allowed us to obtain relative abundance levels of lectin-bound proteins. As a complementary approach, we also examined the relative expression of proteins in HCC without glycoprotein enrichment. Lectin affinity enrichment was found to be advantageous to quantitate several interesting proteins, which were not detected in the whole proteome screening approach. We identified and quantitated over 200 proteins from the lectin-based approach. Interesting among these were fetuin, cysteine-rich protein 1, serpin peptidase inhibitor, leucine-rich alpha-2-glycoprotein 1, melanoma cell adhesion molecule, and heparan sulfate proteoglycan-2. Using lectin affinity followed by PNGase F digestion coupled to ¹⁸O labeling, we identified 34 glycosylation sites with consensus sequence N-X-T/S. Western blotting and immunohistochemical staining were carried out for several proteins to confirm mass spectrometry results.

Conclusion

This study indicates that quantitative proteomic profiling of tumor tissue versus non-cancerous tissue is a promising approach for the identification of potential biomarkers for HCC.     

A strategy to reveal potential glycan markers from serum glycoproteins associated with breast cancer progression

Aberrant glycosylation on glycoproteins that are either presented on the surface or secreted by cancer cells is a potential source of disease biomarkers and provides insights into disease pathogenesis. N-Glycans of the total serum glycoproteins from advanced breast cancer patients and healthy individuals were sequenced by HPLC with fluorescence detection coupled with exoglycosidase digestions and mass spectrometry. We observed a significant increase in a trisialylated triantennary glycan containing alpha1,3-linked fucose which forms part of the sialyl Lewis x epitope. Following digestion of the total glycan pool with a combination of sialidase and beta-galactosidase, we segregated and quantified a digestion product, a monogalactosylated triantennary structure containing alpha1,3-linked fucose. We compared breast cancer patients and controls and detected a 2-fold increase in this glycan marker in patients. In 10 patients monitored longitudinally, we showed a positive correlation between this glycan marker and disease progression and also demonstrated its potential as a better indicator of metastasis compared to the currently used biomarkers, CA 15-3 and carcinoembryonic antigen (CEA). A pilot glycoproteomic study of advanced breast cancer serum highlighted acute-phase proteins alpha1-acid glycoprotein, alpha1-antichymotrypsin, and haptoglobin beta-chain as contributors to the increase in the glycan marker which, when quantified from each of these proteins, marked the onset of metastasis in advance of the CA 15-3 marker. These preliminary findings suggest that specific glycans and glycoforms of proteins may be candidates for improved markers in the monitoring of breast cancer progression.     

Recent progress in quantitative glycoproteomics

Protein glycosylation is acknowledged as one of the major posttranslational modifications that elicit significant effects on protein folding, conformation, distribution, stability, and activity. The changes in glycoprotein abundance, glycosylation degree, and glycan structure are associated with a variety of diseases. Therefore, the quantitative study of glycoproteomics has become a new and popular research topic, and is quickly emerging as an important technique for biomarker discovery. Mass spectrometry-based protein quantification technologies provide a powerful tool for the systematic and quantitative assessment of the quantitative differences in the protein profiles of different samples. Combined with various glycoprotein/glycopeptide enrichment strategies and other glycoprotein analysis methods, these techniques have been further developed for application in quantitative glycoproteomics. A comprehensive quantitative analysis of the glycoproteome in a complex biological sample remains challenging because of the enormous complexity of biological samples, intrinsic characteristics of glycoproteins, and lack of universal quantitative technology. In this review, recently developed technologies in quantitative glycoproteome, especially those focused on two of the most common types of glycosylation (N-linked and O-linked glycoproteome), were summarized. The strengths and weaknesses of the various approaches were also discussed.     

Characterization of the structurally diverse N-linked glycans of Campylobacter species

The Gram-negative bacterium Campylobacter jejuni encodes an extensively characterized N-linked protein glycosylation system that modifies many surface proteins with a heptasaccharide glycan. In C. jejuni, the genes that encode the enzymes required for glycan biosynthesis and transfer to protein are located at a single pgl gene locus. Similar loci are also present in the genome sequences of all other Campylobacter species, although variations in gene content and organization are evident. In this study, we have demonstrated that only Campylobacter species closely related to C. jejuni produce glycoproteins that interact with both a C. jejuni N-linked-glycan-specific antiserum and a lectin known to bind to the C. jejuni N-linked glycan. In order to further investigate the structure of Campylobacter N-linked glycans, we employed an in vitro peptide glycosylation assay combined with mass spectrometry to demonstrate that Campylobacter species produce a range of structurally distinct N-linked glycans with variations in the number of sugar residues (penta-, hexa-, and heptasaccharides), the presence of branching sugars, and monosaccharide content. These data considerably expand our knowledge of bacterial N-linked glycan structure and provide a framework for investigating the role of glycosyltransferases and sugar biosynthesis enzymes in glycoprotein biosynthesis with practical implications for synthetic biology and glycoengineering.     

Focused differential glycan analysis with the platform antibody-assisted lectin profiling for glycan-related biomarker verification

Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and high-throughput manner. Thus, the established system should provide a powerful pipeline in support of on-going efforts in glyco-biomarker discovery.     

Monitoring of glycoprotein products in cell culture lysates using lectin affinity chromatography and capillary HPLC coupled to electrospray linear ion trap-Fourier transform mass spectrometry (LTQ/FTMS)

In this paper, we describe the combination of lectin chromatography with capillary LC coupled to a linear ion trap-Fourier transform mass spectrometer (LTQ/FTMS) to enrich and characterize overexpressed glycoproteins from a cell culture lysate. A well-characterized glycoprotein, recombinant tissue plasminogen activator (rt-PA), was used as a standard, and we demonstrated that the three N-linked glycopeptides (including glycan structures) present in a tryptic digest of the rt-PA standard could be characterized in the new hybrid MS platform. A feature of this approach is that a significant amount of information can be obtained about the carbohydrate structures by direct analysis of the tryptic digest without the need for additional time-consuming sample preparation protocols. A combination of lectins was then studied for improved recovery of captured glycopeptides and was related to the selectivity of different lectins for specific glycosylation motifs. This approach was then extended to the lysate of a cell line routinely used in biotechnology manufacture (Chinese hamster ovary, CHO). This study showed that the combinations of lectins could enrich glycoproteins significantly from a CHO cell lysate. We also demonstrated that with this level of enrichment and with the new hybrid mass spectrometer, we could study the structures of N-linked glycopeptides of rt-PA present in a crude CHO cell lysate, at a ratio of 1:200 (rtPA:total cell lysate protein, w/w) by accurate mass measurement in the FTMS and tandem MSn in the linear ion trap. The generic and high throughput nature of the lectin approach combined with the ability to directly analyze the glycan structures in the tryptic digest suggest that this platform has the potential to routinely monitor glycoprotein products at early stage manufacturing in the biotech industry.     

A novel N-linked flagellar glycan from Methanococcus maripaludis

The archaea Methanococcus maripaludis strain Mm900 produces flagella that are glycosylated with an N-linked tetrasaccharide. Mass spectrometric analysis of flagellar tryptic peptides identified a number of tryptic glycopeptides carrying a glycan of mass 1036.4 Da, and fragmentation of the glycan oxonium ion indicated that the glycan was a tetrasaccharide. The glycan was purified, following extensive pronase digestion of flagellar filaments, by size-exclusion and anion-exchange chromatography. NMR spectroscopy revealed that the glycan had the following structure:Sug-4-β-ManNAc3NAmA6Thr-4-β-GlcNAc3NAcA-3-β-GalNAc-Asnwhere Sug is a novel monosaccharide unit, (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. This oligosaccharide has significant similarity to the oligosaccharide that was found previously in Methanococcus voltae.     

二维电泳和质谱技术鉴定肝癌血清差异表达的N-连接糖蛋白

缺乏有效的早期诊断方法是导致肝细胞癌(hepatocellular carcinoma, HCC)预后极差的主要原因之一.蛋白质异常糖基化与恶性肿瘤细胞侵袭、转移等生物学过程关系密切,人体内至少有50%的蛋白质发生了糖基化修饰.本实验采用IgY12去除血清高丰度蛋白、多植物凝集素亲和层析技术分别从20例肝癌和年龄、性别匹配的20例非癌慢性肝病患者血清中纯化N 连接糖蛋白、二维电泳分析差异表达的蛋白质斑点,质谱检测、生物信息学等技术鉴定了18个差异表达的糖蛋白和/或其异质体（12种高表达和6种低表达）.ExPASy数据库比对结果表明,本实验鉴定的糖蛋白质分子含有至少1个已报道的N 糖基化位点.这些差异表达的糖蛋白属于急性期反应蛋白,分别具有蛋白酶抑制、生物转运、凝血和纤溶等功能,表明肝癌的发生发展过程中机体产生的急性期反应物可能是潜在的肝癌血清标志物.