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．     

Site determination of protein glycosylation based on digestion with immobilized nonspecific proteases and Fourier transform ion cyclotron resonance mass spectrometry

An improved method for site-specific characterization of protein glycosylation has been devised using nonspecific digestion with immobilized pronase combined with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). This procedure was demonstrated using ribonuclease B (RNase B) and kappa-casein (kappa-csn) as representative N-linked and O-linked glycoproteins, respectively. Immobilization of the pronase enzymes facilitated their removal from the glycopeptide preparations, and was found to prevent enzyme autolysis while leaving the proteolytic activities of pronase intact. Increased digestion efficiency, simplified sample preparation, and reduced sample complexity were consequently realized. To supplement this technique, a refined glycopeptide search algorithm was developed to aid in the accurate mass based assignment of N-linked and O-linked glycopeptides derived from nonspecific proteolysis. Monitoring the progress of glycoprotein digestion over time allowed detailed tracking of successive amino acid cleavages about the sites of glycan attachment, and provided a more complete protein glycosylation profile than any single representative time point. This information was further complemented by tandem MS experiments with infrared multiphoton dissociation (IRMPD), allowing confirmation of glycopeptide composition. Overall, the combination of immobilized pronase digestion, time course sampling, FTICR-MS, and IRMPD was shown to furnish an efficient and robust approach for the rapid and sensitive profiling of protein glycosylation.     

Site-specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC/electrospray mass spectrometry and sequential glycosidase digestion.

Site-specific structural characterization of the glycosylation of human lecithin:cholesterol acyltransferase (LCAT) was carried out using microbore reversed-phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESIMS). A recently described mass spectrometric technique involving monitoring of carbohydrate-specific fragment ions during HPLC/ESIMS was employed to locate eight different groups of glycopeptides in a digest of a human LCAT protein preparation. In addition to the four expected N-linked glycopeptides of LCAT, a di-O-linked glycopeptide was detected, as well as three additional glycopeptides. Structural information on the oligosaccharides from all eight glycopeptides was obtained by sequential glycosidase digestion of the glycopeptides followed by HPLC/ESIMS. All four potential N-linked glycosylation sites (Asn20, Asn84, Asn272, and Asn384) of LCAT were determined to contain sialylated triantennary and/or biantennary complex structures. Two unanticipated O-linked glycosylation sites were identified at Thr407 and Ser409 of the LCAT O-linked glycopeptide, each of which contain sialylated galactose beta 1-->3N-acetylgalactosamine structures. The three additional glycopeptides were determined to be from a copurifying protein, apolipoprotein D, which contains potential N-linked glycosylation sites at Asn45 and Asn78. These glycopeptides were determined to bear sialylated triantennary oligosaccharides or fucosylated sialylated biantennary oligosaccharides. Previous studies of LCAT indicated that removal of the glycosylation site at Asn272 converts this protein to a phospholipase (Francone OL, Evangelista L, Fielding CJ, 1993, Biochim Biophys Acta 1166:301-304). Our results indicate that the carbohydrate structures themselves are not the source of this functional discrimination; rather, it must be mediated by the structural environment around Asn272.     

Characterization of intact glycopeptides reveals the impact of culture media on site-specific glycosylation of EPO-Fc fusion protein generated by CHO-GS cells

With the increasing demand to provide more detailed quality attributes, more sophisticated glycan analysis tools are highly desirable for biopharmaceutical manufacturing. Here, we performed an intact glycopeptide analysis method to simultaneously analyze the site-specific N- and O-glycan profiles of the recombinant erythropoietin Fc (EPO-Fc) protein secreted from a Chinese hamster ovary glutamine synthetase stable cell line and compared the effects of two commercial culture media, EX-CELL (EX) and immediate advantage (IA) media, on the glycosylation profile of the target protein. EPO-Fc, containing the Fc region of immunoglobulin G1 (IgG1) fused to EPO, was harvested at Day 5 and 8 of a batch cell culture process followed by purification and N- and O-glycopeptide profiling. A mixed anion exchange chromatographic column was implemented to capture and enrich N-linked glycopeptides. Using intact glycopeptide characterization, the EPO-Fc was observed to maintain their individual EPO and Fc N-glycan characteristics in which the EPO region presented bi-, tri-, and tetra-branched N-glycan structures, while the Fc N-glycan displayed mostly biantennary glycans. EPO-Fc protein generated in EX medium produced more complex tetra-antennary N-glycans at each of the three EPO N-sites while IA medium resulted in a greater fraction of bi- and tri-antennary N-glycans at these same sites. Interestingly, the sialylation content decreased from sites 1–4 in both media while the fucosylation progressively increased with a maximum at the final IgG Fc site. Moreover, we observed that low amounts of Neu5Gc were detected and the content increased at the later sampling time in both EX and IA media. For O-glycopeptides, both media produced predominantly three structures, N1F1F0SOG0, N1H1F0S1G0, and N1H1F0S2G0, with lesser amounts of other structures. This intact glycopeptide method can decipher site-specific glycosylation profile and provide a more detailed characterization of N- and O-glycans present for enhanced understanding of the key product quality attributes such as media on recombinant proteins of biotechnology interest.     

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.     

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.     

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.     

Determination of site-specific glycan heterogeneity on glycoproteins

The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco)peptides are analyzed by capillary/nano-liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). If required, specific glycopeptide enrichment steps, such as hydrophilic interaction liquid chromatography (HILIC), can also be performed. Particular emphasis is placed on data interpretation and the determination of site-specific glycan heterogeneity. The described workflow takes approximately 3-5 d, including sample preparation and data analysis. The data obtained from analyzing released glycans of rHuEPO and IgG, described in the second protocol of this series (10.1038/nprot.2012.063), provide complementary detailed glycan structural information that facilitates characterization of the glycopeptides.     

Elucidation of glycoprotein structures by unspecific proteolysis and direct nanoESI mass spectrometric analysis of ZIC-HILIC-enriched glycopeptides

Protein glycosylation was explored by direct nanoESI MS and MS/MS analysis of ZIC-HILIC-enriched proteolytic glycopeptides without further separation or purification. In a previous publication, we demonstrated that a direct MS-based analysis of proteolytic glycopeptides is feasible for a number of proteins (Henning , S. J. Mass Spectrom. 2007 , 42 , 1415 - 21). This method has now been refined for two aspects: (1) separation of glycopeptides by use of ZIC-HILIC SPE and (2) the use of unspecific proteases like thermolysin, elastase, or a trypsin/chymotrypsin mixture leading per se to a mass-based separation, that is, small nonglycosylated peptides and almost exclusively glycopeptides at higher m/z values. Furthermore, the glycopeptides produced by the above proteases in general contain short peptide backbones thus improving-probably due to their higher hydrophilicity--the ZIC-HILIC-based separation. The combination of unspecific proteolysis, glycopeptide separation, and their direct MS analysis was successfully accomplished for probing glycoproteins carrying high-mannose type (ribonuclease B), neutral (asialofetuin), and acidic (haptoglobin and α1-acid glycoprotein) complex type glycans as well as for glycopeptides derived from glycoprotein mixtures and, finally, for exploring the glycosylation of a human IgG preparation. Our results show that the presented method is a fast, facile, and inexpensive procedure for the elucidation of protein N-glycosylation.     

Guinea-pig kidney beta-N-acetylgalactosaminyltransferase towards Tamm-Horsfall glycoprotein. Requirement of sialic acid in the acceptor for transferase activity.

A beta-N-acetylgalactosaminyltransferase that preferentially transferred N-acetylgalactosamine to Sd(a-) Tamm-Horsfall glycoprotein was found in guinea-pig kidney microsomal preparations. This enzyme was kidney-specific and was able to transfer the sugar to other glycoproteins, such as fetuin and alpha 1-acidic glycoprotein. The presence of sialic acid in the acceptors was essential for the transferase activity when either glycoproteins or their Pronase glycopeptides were used as acceptors. Two glycopeptides (Tamm-Horsfall glycopeptides I and II) with a different carbohydrate composition were separated by DEAE-Sephacel chromatography from Pronase-digested Tamm-Horsfall glycoprotein. The amount of N-acetylgalactosamine transferred to glycopeptides by the enzyme correlated with their degree of sialylation. Enzymic digestion of N-[14C]acetylgalactosamine-labelled Tamm-Horsfall glycopeptide II showed that the transferred sugar was susceptible to beta-N-hexosaminidase. The amount of sugar cleaved by beta-hexosaminidase was strongly increased when the labelled Tamm-Horsfall glycopeptide II was pretreated with mild acid hydrolysis, a procedure that removed the sialic acid residues. Alkaline borohydride treatment of the labelled Tamm-Horsfall glycopeptide II did not release radioactivity, thus indicating that enzymic glycosylation took place at the N-asparagine-linked oligosaccharide units of Tamm-Horsfall glycoprotein.     

A simple cellulose column procedure for selective enrichment of glycopeptides and characterization by nano LC coupled with electron-transfer and high-energy collisional-dissociation tandem mass spectrometry

In this report we describe an on-column method for glycopeptide enrichment with cellulose as a solid-phase extraction material. The method was developed using tryptic digests of several standard glycoproteins and validated with more complex standard protein digest mixtures. Glycopeptides of different masses containing neutral and acidic glycoforms of both N- and O-linked sugars were obtained in good yield by this method. Upon isolation, glycopeptides may be subjected to further glycoproteomic and glycomic workflows for the purpose of identifying glycoproteins present in the sample and characterizing their glycosylation sites, as well as their global and site-specific glycosylation profiles at the glycopeptide level. Detailed structural analysis of glycoforms may then be performed at the glycan level upon chemical or enzymatic release of the oligosaccharides. Aiming at complementing other purification methods, this technique is extremely simple, cost-effective, and efficient. Glycopeptide enrichment was verified and validated by nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) combining electron-transfer dissociation (ETD) and collision-activated dissociation (CAD) fragmentation techniques.     

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.     

Measuring change in glycoprotein structure

Biosynthetic enzymes in the secretory pathway create distributions of glycans at each glycosite that elaborate the biophysical properties and biological functions of glycoproteins. Because the biosynthetic glycosylation reactions do not go to completion, each protein glycosite is heterogeneous with respect to glycosylation. This heterogeneity means that it is not sufficient to measure protein abundance in omics experiments. Rather, it is necessary to sample the distribution of glycosylation at each glycosite to quantify the changes that occur during biological processes. On the one hand, the use of data-dependent acquisition methods to sample glycopeptides is limited by the instrument duty cycle and the missing value problem. On the other, stepped window data-independent acquisition samples all precursors, but ion abundances are limited by duty cycle. Therefore, the ability to quantify accurately the flux in glycoprotein glycosylation that occurs during biological processes requires the exploitation of emerging mass spectrometry technologies capable of deep, comprehensive sampling and selective high confidence assignment of the complex glycopeptide mixtures. This review summarizes recent technical advances and mass spectral glycoproteomics analysis strategies and how these developments impact our ability to quantify the changes in glycosylation that occur during biological processes. We highlight specific improvements to glycopeptide characterization through activated electron dissociation, ion mobility trends and instrumentation, and efficient algorithmic approaches for glycopeptide assignment. We also discuss the emerging need for unified standards to enable interlaboratory collaborations and effective monitoring of structural changes in glycoproteins.     

Advances in LC–MS/MS-based glycoproteomics: Getting closer to system-wide site-specific mapping of the N- and O-glycoproteome

Site-specific structural characterization of glycoproteins is important for understanding the exact functional relevance of protein glycosylation. Resulting partly from the multiple layers of structural complexity of the attached glycans, the system-wide site-specific characterization of protein glycosylation, defined as glycoproteomics, is still far from trivial leaving the N- and O-linked glycoproteomes significantly under-defined. However, recent years have seen significant advances in glycoproteomics driven, in part, by the developments of dedicated workflows and efficient sample preparation, including glycopeptide enrichment and prefractionation. In addition, glycoproteomics has benefitted from the continuous performance enhancement and more intelligent use of liquid chromatography and tandem mass spectrometry (LC–MS/MS) instrumentation and a wider selection of specialized software tackling the unique challenges of glycoproteomics data. Together these advances promise more streamlined N- and O-linked glycoproteome analysis. Tangible examples include system-wide glycoproteomics studies detecting thousands of intact glycopeptides from hundreds of glycoproteins from diverse biological samples. With a strict focus on the system-wide site-specific analysis of protein N- and O-linked glycosylation, we review the recent advances in LC–MS/MS based glycoproteomics. The review opens with a more general discussion of experimental designs in glycoproteomics and sample preparation prior to LC–MS/MS based data acquisition. Although many challenges still remain, it becomes clear that glycoproteomics, one of the last frontiers in proteomics, is gradually maturing enabling a wider spectrum of researchers to access this new emerging research discipline. The next milestone in analytical glycobiology is being reached allowing the glycoscientist to address the functional importance of protein glycosylation in a system-wide yet protein-specific manner.     

Site-specific characterization of N-linked glycosylation in human urinary glycoproteins and endogenous glycopeptides

Glycosylation is a very important post-translational modification involved in various cellular processes, such as cell adhesion, signal transduction and immune response. Urine is a rich source of glycoproteins and attractive biological fluid for biomarker discovery, owing to its availability, ease of collection, and correlation with pathophysiology of diseases. Although the urinary proteomics have been explored previously, the urinary glycoproteome characterization remains challenging requiring the development and optimization of analytical and bioinformatics methods for protein glycoprofiling. This study describes the high confident identification of 472 unique N-glycosylation sites covering 256 urinary glycoproteins. Besides, 202 unique N-glycosylation sites were identified in low molecular weight endogenous glycopeptides, which belong to 90 glycoproteins. Global site-specific characterization of the N-linked glycan heterogeneity was achieved by intact glycopeptide analysis, revealing 303 unique glycopeptides most of them displaying complex/hybrid glycans composed by sialic acid and fucose. These datasets consist in a valuable resource of glycoproteins and N-glycosylation sites found in healthy human urine that can be further explored in different disorders, in which the N-linked glycosylation may be aberrant.     

One-pipeline approach achieving glycoprotein identification and obtaining intact glycopeptide information by tandem mass spectrometry

A novel one-pipeline approach is reported, which can demonstrate glycoprotein identification and obtain intact glycosylation information after glycopeptide-level enrichment, without de-glycosylation. The proposed workflow has two enrichment steps plus two proteolytic processes: enriched glycoproteins were digested to peptides by Lys-C, and then enriched again and secondly digested by trypsin. In the resulting mixture, with a reasonable complexity, intact glycopeptides could be preserved and utilized informatively for glycosylation analysis, and non-glycopeptides for protein identification. In both standard protein mixture tests and real sample analysis, the resulting glycopeptides and non-glycopeptides were proved to play their expected roles, thus more confident protein glycosylation information was obtained.     

Glycopeptide microarray for autoantibody detection in cancer

Evaluation of: Pedersen JW, Blixt O, Bennett EP et al. Seromic profiling of colorectal cancer patients with novel glycopeptide microarray. Int. J. Cancer 128(8), 1860-1871 (2011). Autoantibodies to cancer-associated antigens hold promise as sensitive biomarkers for cancer detection. Based on this hypothesis, and knowing that O-glycans on proteins constitute a source of possible epitopes recognized by autoantibodies, Pedersen and colleagues have generated a glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from human mucins. The profiling of sera immunoreactivity of colon cancer patients allowed the identification of cancer-associated autoantibodies to various mucin (MUC)1 and MUC4 glycopeptides carrying aberrant glycosylation. This article provides evidence for the value of glycopeptides displaying cancer-associated glycans in diagnostic applications, and opens new avenues for the expansion to other protein glycoforms, as well as to further applications of such a microarray strategy for other post-translational modifications of proteins in the search for cancer biomarker.     

Synthetic methods of glycopeptide assembly, and biological analysis of glycopeptide products

The technology of glycopeptide synthesis has recently developed into a fully mature science capable of creating diverse glycopeptides of biological interest, even in combinatorial displays. This has allowed biochemists to investigate substrate specificity in the biosynthetic processing and immunology of various protein glycoforms. The construction of all the mucin core structures and a varietyof cancer-related glycopeptides has facilitated detailed analysis of the interaction between MHC-bound glycopeptides and T cell receptors. Novel dendritic neoglycopeptide ligands have been shown to demonstrate high affinity for carbohydrate receptors and these interactions are highly dendrimer specific. Large complex N-linked oligosaccharides have been introduced into glycopeptides using synthetic or chemoenzymatic procedures, both methods affording pure glycopeptides corresponding to a single glycoform in preparative quantities. The improved availability of glycosyl transferases has led to increased use of chemoenzymatic synthesis. Chemical ligation has been introduced as a method of attaching glycans to peptide templates. Combinatorial synthesis and the analysis of resin-bound glycopeptide libraries have been successfully carried out by applying the ladder synthesis principle. Direct quantitative glycosylation of peptide templates on solid phase has paved the way for the synthesis of templated glycopeptide mixtures as libraries of libraries.     

Quantitative analysis and process monitoring of site-specific glycosylation microheterogeneity in recombinant human interferon-γ from Chinese hamster ovary cell culture by hydrophilic interaction chromatography

A chromatographic method was developed for quantitative analysis of site-specific microheterogeneity of the two N-linked glycosylation sites in recombinant human interferon-γ produced from Chinese hamster ovary (CHO) cell culture. After the interferon-γ was harvested by affinity chromatography, the tryptic digestion was carried out. The two glycopeptide pools, isolated from reversed-phase chromatography of tryptic digestion of interferon-γ, were subjected to further separation by hydrophilic interaction chromatography. Each peak in the chromatograms was identified by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI–TOF–MS). The overall elution order of the glycopeptides was the following: neutral glycopeptides, monosialylated glycopeptides, bisialylated glycopeptides, trisialylated glycopeptide and tetrasialylated glycopeptides. Based on the integrated peak area for each compound in the chromatograms, the percentage for each glycan was utilized to quantify the glycosylation pattern of the interferon-γ. Finally, sialylation and antennarity structure percentages at the two glycosylation sites were chosen as the quality indicators in process monitoring of interferon-γ production from a serum-free suspension-batch CHO culture.     

Interactions of concanavalin A with asparagine-linked glycopeptides: formation of homogeneous cross-linked lattices in mixed precipitation systems

We have previously shown that certain oligomannose and bisected hybrid type glycopeptides are bivalent for binding to concanavalin A (Con A) [Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., & Brewer, C. F. (1987) J. Biol. Chem. 262, 1288-1293]. Each glycopeptide gives a quantitative precipitation profile with the protein which consists of a single peak that corresponds to the binding stoichiometry of glycopeptide to protein monomer (1:2). We have shown that the affinities of the primary and secondary sites of the glycopeptides influence their extent of precipitation with the lectin [Bhattacharyya, L., & Brewer, C. F. (1988) Eur. J. Biochem. (in press)]. In the present study, we demonstrate that equimolar mixtures of any two of the glycopeptides result in a quantitative precipitation profile which shows two protein peaks. Using radiolabeled glycopeptides, the precipitation profiles of the individual glycopeptides were determined. The results show that each glycopeptide forms its own precipitation profile with the protein which is independent of the profile of the other glycopeptide. For mixtures containing an equimolar ratio of two glycopeptides, the glycopeptide with lower affinity shows a precipitation maximum at a lower concentration than the one with higher affinity. However, this can be reversed by increasing the ratio of the lower affinity glycopeptide in the mixture. Thus, the relative precipitation maxima of the glycopeptides are determined by mass-action equilibria involving competitive binding of the two carbohydrates to the protein. These equilibria, in turn, are sensitive to the relative amounts and affinities of the carbohydrates at both their primary and secondary sites.(ABSTRACT TRUNCATED AT 250 WORDS)