Human urinary glycoproteomics; attachment site specific analysis of N- and O-linked glycosylations by CID and ECD

Urine is a complex mixture of proteins and waste products and a challenging biological fluid for biomarker discovery. Previous proteomic studies have identified more than 2800 urinary proteins but analyses aimed at unraveling glycan structures and glycosylation sites of urinary glycoproteins are lacking. Glycoproteomic characterization remains difficult because of the complexity of glycan structures found mainly on asparagine (N-linked) or serine/threonine (O-linked) residues. We have developed a glycoproteomic approach that combines efficient purification of urinary glycoproteins/glycopeptides with complementary MS-fragmentation techniques for glycopeptide analysis. Starting from clinical sample size, we eliminated interfering urinary compounds by dialysis and concentrated the purified urinary proteins by lyophilization. Sialylated urinary glycoproteins were conjugated to a solid support by hydrazide chemistry and trypsin digested. Desialylated glycopeptides, released through mild acid hydrolysis, were characterized by tandem MS experiments utilizing collision induced dissociation (CID) and electron capture dissociation fragmentation techniques. In CID-MS(2), Hex(5)HexNAc(4)-N-Asn and HexHexNAc-O-Ser/Thr were typically observed, in agreement with known N-linked biantennary complex-type and O-linked core 1-like structures, respectively. Additional glycoforms for specific N- and O-linked glycopeptides were also identified, e.g. tetra-antennary N-glycans and fucosylated core 2-like O-glycans. Subsequent CID-MS(3), of selected fragment-ions from the CID-MS(2) analysis, generated peptide specific b- and y-ions that were used for peptide identification. In total, 58 N- and 63 O-linked glycopeptides from 53 glycoproteins were characterized with respect to glycan- and peptide sequences. The combination of CID and electron capture dissociation techniques allowed for the exact identification of Ser/Thr attachment site(s) for 40 of 57 putative O-glycosylation sites. We defined 29 O-glycosylation sites which have, to our knowledge, not been previously reported. This is the first study of human urinary glycoproteins where "intact" glycopeptides were studied, i.e. the presence of glycans and their attachment sites were proven without doubt.     

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.     

Multiattribute Glycan Identification and FDR Control for Glycoproteomics

Rapidly improving methods for glycoproteomics have enabled increasingly large-scale analyses of complex glycopeptide samples, but annotating the resulting mass spectrometry data with high confidence remains a major bottleneck. We recently introduced a fast and sensitive glycoproteomics search method in our MSFragger search engine, which reports glycopeptides as a combination of a peptide sequence and the mass of the attached glycan. In samples with complex glycosylation patterns, converting this mass to a specific glycan composition is not straightforward; however, as many glycans have similar or identical masses. Here, we have developed a new method for determining the glycan composition of N-linked glycopeptides fragmented by collisional or hybrid activation that uses multiple sources of information from the spectrum, including observed glycan B-type (oxonium) and Y-type ions and mass and precursor monoisotopic selection errors to discriminate between possible glycan candidates. Combined with false discovery rate estimation for the glycan assignment, we show that this method is capable of specifically and sensitively identifying glycans in complex glycopeptide analyses and effectively controls the rate of false glycan assignments. The new method has been incorporated into the PTM-Shepherd modification analysis tool to work directly with the MSFragger glyco search in the FragPipe graphical user interface, providing a complete computational pipeline for annotation of N-glycopeptide spectra with false discovery rate control of both peptide and glycan components that is both sensitive and robust against false identifications.     

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.     

Higher Energy Collision Dissociation (HCD) Product Ion-Triggered Electron Transfer Dissociation (ETD) Mass Spectrometry for the Analysis of N-Linked Glycoproteins

Large scale mass spectrometry analysis of N-linked glycopeptides is complicated by the inherent complexity of the glycan structures. Here, we evaluate a mass spectrometry approach for the targeted analysis of N-linked glycopeptides in complex mixtures that does not require prior knowledge of the glycan structures or pre-enrichment of the glycopeptides. Despite the complexity of N-glycans, the core of the glycan remains constant, comprising two N-acetylglucosamine and three mannose units. Collision-induced dissociation (CID) mass spectrometry of N-glycopeptides results in the formation of the N-acetylglucosamine (GlcNAc) oxonium ion and a [mannose+GlcNAc] fragment (in addition to other fragments resulting from cleavage within the glycan). In ion-trap CID, those ions are not detected due to the low m/z cutoff; however, they are detected following the beam-type CID known as higher energy collision dissociation (HCD) on the orbitrap mass spectrometer. The presence of these product ions following HCD can be used as triggers for subsequent electron transfer dissociation (ETD) mass spectrometry analysis of the precursor ion. The ETD mass spectrum provides peptide sequence information, which is unobtainable from HCD. A Lys-C digest of ribonuclease B and trypsin digest of immunoglobulin G were separated by ZIC-HILIC liquid chromatography and analyzed by HCD product ion-triggered ETD. The data were analyzed both manually and by search against protein databases by commonly used algorithms. The results show that the product ion-triggered approach shows promise for the field of glycoproteomics and highlight the requirement for more sophisticated data mining tools.     

Solid-phase extraction of N-linked glycopeptides

Protein glycosylation is a common post-translational modification and has been increasingly recognized as one of the most prominent biochemical alterations associated with malignant transformation and tumorigenesis. N-linked glycosylation is prevalent in proteins on the extracellular membrane, and many clinical biomarkers and therapeutic targets are glycoproteins. Here, we describe a protocol for solid-phase extraction of N-linked glycopeptides and subsequent identification of N-linked glycosylation sites (N-glycosites) by tandem mass spectrometry. The method oxidizes the carbohydrates in glycopeptides into aldehydes, which can be immobilized on a solid support. The N-linked glycopeptides are then optionally labeled with a stable isotope using deuterium-labeled succinic anhydride and the peptide moieties are released by peptide-N-glycosidase. In a single analysis, the method identifies hundreds of N-linked glycoproteins, the site(s) of N-linked glycosylation and the relative quantity of the identified glycopeptides.     

Glycoproteomics based on tandem mass spectrometry of glycopeptides

Next to the identification of proteins and the determination of their expression levels, the analysis of post-translational modifications (PTM) is becoming an increasingly important aspect in proteomics. Here, we review mass spectrometric (MS) techniques for the study of protein glycosylation at the glycopeptide level. Enrichment and separation techniques for glycoproteins and glycopeptides from complex (glyco-)protein mixtures and digests are summarized. Various tandem MS (MS/MS) techniques for the analysis of glycopeptides are described and compared with respect to the information they provide on peptide sequence, glycan attachment site and glycan structure. Approaches using electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) of glycopeptides are presented and the following fragmentation techniques in glycopeptide analysis are compared: collision-induced fragmentation on different types of instruments, metastable fragmentation after MALDI ionization, infrared multi-photon dissociation, electron-capture dissociation and electron-transfer dissociation. This review discusses the potential and limitations of tandem mass spectrometry of glycopeptides as a tool in structural glycoproteomics.     

Complexities and algorithms for glycan sequencing using tandem mass spectrometry

Determining glycan structures is vital to comprehend cell-matrix, cell-cell, and even intracellular biological events. Glycan sequencing, which determines the primary structure of a glycan using tandem mass spectrometry (MS/MS), remains one of the most important tasks in proteomics. Analogous to peptide de novo sequencing, glycan de novo sequencing determines the structure without the aid of a known glycan database. We show in this paper that glycan de novo sequencing is NP-hard. We then provide a heuristic algorithm and develop a software program to solve the problem in practical cases. Experiments on real MS/MS data of glycopeptides demonstrate that our heuristic algorithm gives satisfactory results on practical data.     

Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures

Extensive site-specific glycosylation analysis of individual glycoproteins is difficult due to the nature and complexity of glycosylation in proteins. In protein mixtures, these analyses are even more difficult. We present an approach combining nonspecific protease digestion, nanoflow liquid chromatography, and tandem mass spectrometry (MS/MS) aimed at comprehensive site-specific glycosylation analysis in protein mixtures. The strategy described herein involves the analysis of a complex mixture of glycopeptides generated from immobilized-Pronase digestion of a cocktail of glycoproteins consisting of bovine lactoferrin, kappa casein, and bovine fetuin using nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC-Q-TOF MS). The resulting glycopeptides were chromatographically separated on a micro fluidic chip packed with porous graphitized carbon and analyzed via MS and MS/MS analyses. In all, 233 glycopeptides (identified based on composition and including isomers) corresponding to 18 glycosites were observed and determined in a single mixture. The glycopeptides were a mixture of N-linked glycopeptides (containing high mannose, complex and hybrid glycans) and O-linked glycopeptides (mostly sialylated). Results from this study were comprehensive as detailed glycan microheterogeneity information was obtained. This approach presents a platform to simultaneously characterize N- and O-glycosites in the same mixture with extensive site heterogeneity.     

Identification of N-glycosylated proteins from the central nervous system of Drosophila melanogaster

Although the function of many glycoproteins in the nervous system of fruit flies is well understood, information about the glycosylation profile and glycan attachment sites for such proteins is scarce. In order to fill this gap and to facilitate the analysis of N-linked glycosylation in the nervous system, we have performed an extensive survey of membrane-associated glycoproteins and their N-glycosylation sites isolated from the adult Drosophila brain. Following subcellular fractionation and trypsin digestion, we used different lectin affinity chromatography steps to isolate N-glycosylated glycopeptides. We identified a total of 205 glycoproteins carrying N-linked glycans and revealed their 307 N-glycan attachment sites. The size of the resulting dataset furthermore allowed the statistical characterization of amino acid distribution around the N-linked glycosylation sites. Glycan profiles were analyzed separately for glycopeptides that were strongly and weakly bound to Concanavalin A (Con A), or that failed to bind Concanavalin A, but did bind to wheat germ agglutinin (WGA). High- or paucimannosidic glycans dominated each of the profiles, although the wheat germ agglutinin-bound glycan population was enriched in more extensively processed structures. A sialylated glycan structure was unambiguously detected in the wheat germ agglutinin-bound fraction. Despite the large amount of starting material, insufficient amount of glycopeptides was retained by the Wisteria floribunda (WFA) and Sambucus nigra columns to allow glycan or glycoprotein identification, providing further evidence that the vast majority of glycoproteins in the adult Drosophila brain carry primarily high-mannose, paucimannose, and hybrid glycans. The obtained results should facilitate future genetic and molecular approaches addressing the role of N-glycosylation in the central nervous system (CNS) of Drosophila.     

Characterization of intact N- and O-linked glycopeptides using higher energy collisional dissociation

Simultaneous elucidation of the glycan structure and the glycosylation site are needed to reveal the biological function of protein glycosylation. In this study, we employed a recent type of fragmentation termed higher energy collisional dissociation (HCD) to examine fragmentation patterns of intact glycopeptides generated from a mixture of standard glycosylated proteins. The normalized collisional energy (NCE) value for HCD was varied from 30 to 60% to evaluate the optimal conditions for the fragmentation of peptide backbones and glycoconjugates. Our results indicated that HCD with lower NCE values preferentially fragmented the sugar chains attached to the peptides to generate a ladder of neutral loss of monosaccharides, thereby enabling the putative glycan structure characterization. In addition, detection of the oxonium ions enabled unambiguous differentiation of glycopeptides from non-glycopeptides. In contrast, HCD with higher NCE values preferentially fragmented the peptide backbone and, thus, provided information needed for confident peptide identification. We evaluated the HCD approach with alternating NCE parameters for confident characterization of intact N- and O-linked glycopeptides in a single liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. In addition, we applied a novel data analysis pipeline, so-called GlycoFinder, to form a basis for automated data analysis. Overall, 38 unique intact glycopeptides corresponding to eight glycosylation sites (six N-linked and two O-linked sites) were confidently identified from a standard protein mixture. This approach provided concurrent characterization of both the peptide and the glycan, thereby enabling comprehensive structural characterization of glycoproteins in a single LC–MS/MS analysis.     

A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation

Characterization of glycoproteins using mass spectrometry ranges from determination of carbohydrate-protein linkages to the full characterization of all glycan structures attached to each glycosylation site. In a novel approach to identify N-glycosylation sites in complex biological samples, we performed an enrichment of glycosylated peptides through hydrophilic interaction liquid chromatography (HILIC) followed by partial deglycosylation using a combination of endo-beta-N-acetylglucosaminidases (EC 3.2.1.96). After hydrolysis with these enzymes, a single N-acetylglucosamine (GlcNAc) residue remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopeptides, while the remaining GlcNAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach on a mixture of known glycoproteins, and subsequently the method was applied to samples of human plasma obtained by lectin chromatography followed by 1D gel-electrophoresis for determination of 62 glycosylation sites in 37 glycoproteins.     

A review of methods for interpretation of glycopeptide tandem mass spectral data

Despite the publication of several software tools for analysis of glycopeptide tandem mass spectra, there remains a lack of consensus regarding the most effective and appropriate methods. In part, this reflects problems with applying standard methods for proteomics database searching and false discovery rate calculation. While the analysis of small post-translational modifications (PTMs) may be regarded as an extension of proteomics database searching, glycosylation requires specialized approaches. This is because glycans are large and heterogeneous by nature, causing glycopeptides to exist as multiple glycosylated variants. Thus, the mass of the peptide cannot be calculated directly from that of the intact glycopeptide. In addition, the chemical nature of the glycan strongly influences product ion patterns observed for glycopeptides. As a result, glycopeptidomics requires specialized bioinformatics methods. We summarize the recent progress towards a consensus for effective glycopeptide tandem mass spectrometric analysis.     

Glycoproteomics of Trypanosoma cruzi trypomastigotes using subcellular fractionation, lectin affinity, and stable isotope labeling

Herein we detail the first glycoproteomic analysis of a human pathogen. We describe an approach that enables the identification of organelle and cell surface N-linked glycoproteins from Trypanosoma cruzi, the causative agent of Chagas' disease. This approach is based on a subcellular fractionation protocol to produce fractions enriched in either organelle or plasma membrane/cytoplasmic proteins. Through lectin affinity capture of the glycopeptides from each subcellular fraction and stable isotope labeling of the glycan attachment sites with H(2)18O, we unambiguously identified 36 glycosylation sites on 35 glycopeptides which mapped to 29 glycoproteins. We also present the first expression evidence for 11 T. cruzi specific glycoproteins and provide experimental data indicating that the mucin associated surface protein family (MASP) and dispersed gene family (DGF-1) are post-translationally modified by N-linked glycans.     

Direct on-membrane glycoproteomic approach using MALDI-TOF mass spectrometry coupled with microdispensing of multiple enzymes

We report a novel approach for direct on-membrane glycoproteomics by digestion of membrane-blotted glycoproteins with multiple enzymes using piezoelectric chemical inkjet printing technology and on-membrane direct MALDI-TOF mass spectrometry. With this approach, both N-linked glycan analyses and peptide mass fingerprinting of several standard glycoproteins were successfully performed using PNGase F and trypsin microscale digestions of the blotted spots on membrane from an SDS-PAGE gel. In addition, we performed a similar analysis for 2-DE separated serum glycoproteins as a demonstration of how the system could be used in human plasma glycoproteomics.     

Structural studies of fertilization-associated carbohydrate-rich glycoproteins (hyosophorin) isolated from the fertilized and unfertilized eggs of flounder, Paralichthys olivaceus. Presence of a novel penta-antennary N-linked glycan chain in the tandem repeating glycopeptide unit of hyosophorin

New glycoproteins of 100-120 kDa were isolated from the unfertilized eggs of flounder, Paralichthys olivaceus. Compositionally indistinguishable glycopeptides of 6 kDa were also purified from the activated or fertilized eggs. These high and low molecular mass glycoproteins are characterized by high (about 85%) carbohydrate content. Although some heterogeneities exist in the amino acid sequences, the 6-kDa glycopeptides (decapeptides with single large N-linked glycan chains), isolated from the fertilized eggs are the repeating units of the high molecular mass glycoproteins. As judged from several distinctive features the 100-120-kDa glycoproteins are apparently major components of cortical alveoli of flounder eggs and are regarded as members of glycoproteins we have defined under the name of "hyosophorin" (Kitajima, K., Inoue, S., and Inoue, Y. (1989) Dev. Biol. 132, 544-553). Composition analysis, Smith degradation, hydrazinolysis-nitrous acid deamination, permethylation analysis, and 400-MHz 1H NMR spectroscopy provided evidence for the structure of a novel penta-antennary glycan chain attached to the repeating unit (decapeptide) of the protein core. The structure thus determined is: (Formula: see text). The presence of a unique class of carbohydrate-rich glycoproteins (H-hyosophorin) in the unfertilized eggs, their conversion to the repeating unit (L-hyosophorin) at fertilization, and the finding of a free glycan chain that was formed by scission between the GlcNAc and Asn residues of L-hyosophorin, in the fertilized eggs including embryos of 4-11-h postinsemination, support the view that these molecules may be important in fertilization and subsequent development.     

N-linked glycosylation profiling of pancreatic cancer serum using capillary liquid phase separation coupled with mass spectrometric analysis

Glycoproteins play important roles in various biological processes including intracellular transport, cell recognition, and cell-cell interactions. The change of the cellular glycosylation profile may have profound effects on cellular homeostasis and malignancy. Therefore, we have developed a sensitive screening approach for the comprehensive analysis of N-glycans and glycosylation sites on human serum proteins. Using this approach, N-linked glycopeptides were extracted by double lectin affinity chromatography. The glycans were enzymatically cleaved from the peptides and then profiled using capillary hydrophilic interaction liquid chromatography coupled online with ESI-TOF MS. The structures of the separated glycans were determined by MALDI quadrupole ion-trap TOF mass spectrometry in both positive and negative modes. The glycosylation sites were elucidated by sequencing of PNGase F modified glycopeptides using nanoRP-LC-ESI-MS/MS. Alterations of glycosylation were analyzed by comparing oligosaccharide expression of serum glycoproteins at different disease stages. The efficiency of this method was demonstrated by the analysis of pancreatic cancer serum compared to normal serum. Ninety-two individual glycosylation sites and 202 glycan peaks with 105 unique carbohydrate structures were identified from approximately 25 mug glycopeptides. Forty-four oligosaccharides were found to be distinct in the pancreatic cancer serum. Increased branching of N-linked oligosaccharides and increased fucosylation and sialylation were observed in samples from patients with pancreatic cancer. The methodology described in this study may elucidate novel, cancer-specific oligosaccharides and glycosylation sites, some of which may have utility as useful biomarkers of cancer.     

Quantitative site-specific analysis of protein glycosylation by LC-MS using different glycopeptide-enrichment strategies

A common technique for analysis of protein glycosylation is HPLC coupled to mass spectrometry (LC-MS). However, analysis is challenging due to a low abundance of glycopeptides in complex protein digests, microheterogeneity at the glycosylation site, ion suppression effects, and competition for ionization by coeluting peptides. Specific sample preparation is necessary for a comprehensive and site-specific glycosylation analysis by MS. In this study we qualitatively compared hydrophilic interaction chromatography (HILIC) and hydrazine chemistry for the enrichment of all N-linked glycopeptides and titanium dioxide for capturing sialylated glycopeptides from a complex peptide mixture. Bare silica, microcrystalline cellulose, amino-, amide- (TSKgel Amide-80), and sulfobetaine-(ZIC-HILIC) bonded phases were evaluated for HILIC enrichment. The experiments revealed that ZIC-HILIC and TSKgel Amide-80 are very specific for capturing glycopeptides under optimized conditions. Quantitative analysis of N-glycosidase F-released and 2-aminobenzamide-labeled glycans of a ZIC-HILIC-enriched monoclonal antibody demonstrated that glycopeptides could be enriched without bias for particular glycan structures and without significant losses. Sialylated glycopeptides could be efficiently enriched by titanium dioxide and in addition to HILIC both methods enable a comprehensive analysis of protein glycosylation by MS. Enrichment of N-linked glycopeptides by hydrazine chemistry resulted in lower peptide recovery using a more complex enrichment scheme.     

Investigation of cationic peanut peroxidase glycans by electrospray ionization mass spectrometry

Cationic peanut peroxidase (CP) was isolated from peanut (Arachis hypogaea) cell suspension culture medium. CP is a glycoprotein with three N-linked glycan sites at Asn60, Asn144, and Asn185. ESI-MS of the intact purified protein reveals the microheterogeneity of the glycans. Tryptic digestion of CP gave a near complete sequence coverage by ESI-MS. The glycopeptides from the tryptic digestion were separated by RP HPLC identified by ESI-MS and the structure of the glycan chains determined by ESI-MS/MS. The glycans are large structures of up to 16 sugars, but most of their non-reducing ends have been modified giving a mixture of shorter chains at each site. Good agreement was found with the one glycan previously analyzed by (1)H NMR. This work is the basis for the future studies on the role of the glycans on stability and folding of CP and is another example of a detailed structural characterization of complex glycoproteins by mass spectrometry.     

综述: N-连接完整糖肽的质谱分析策略和研究方法

蛋白质糖基化作为最普遍、最重要的蛋白质修饰,一直是组学研究的焦点之一．近十几年来,N-连接糖蛋白质组学研究普遍采用的方法是将糖链与所修饰的多肽分开进行分析．该策略虽降低了分析难度,却也丢失了糖链与蛋白质糖基化位点间重要的对应关系信息．近年来,完整糖肽的质谱分析策略和方法逐步建立起来．总体而言,要实现对完整糖肽的直接质谱分析,首先需要从复杂样品中富集完整糖肽以消除非糖基化多肽对完整糖肽分析的影响,然后在质谱分析中还需要根据糖肽特性调整相应质谱分析参数,最后在后续数据分析中还需要开发相应的分析软件以完成完整糖肽中多肽序列和糖链组成或结构的鉴定．本文即从以上三个主要方面系统阐述目前N-完整糖肽分析中常用的质谱和数据分析策略和方法,并进一步在糖肽谱图识别、母离子单同位素分子质量校正、数据库选择以及假阳性率评估和控制等方面都进行了逐一探讨．完整糖肽的直接质谱分析有助于获取糖链和糖基化位点间的对应关系信息,可为生物标志物发现和疾病致病机理等研究提供更有力的糖蛋白质组学研究工具．