Proteomic analysis of glycosylation: structural determination of N- and O-linked glycans by mass spectrometry

This review summarizes the methods, mainly based on mass spectrometry, for the structural determination of N- and O-linked carbohydrates that are post-translationally attached to a large number of proteins and which play a key role in determining the function and biophysical properties of these compounds. Analysis of these carbohydrates has proved difficult in the past due to their structural complexity. However, modern analytical methods such as mass spectrometry have the ability to elucidate most structural details at the concentration levels required for proteomics. This review describes methods for direct examination of glycoproteins by mass spectrometry, the release of N- and O-linked glycans from glycoproteins separated in sodium dodecyl sulfate polyacrylamide electrophoresis gels, and the analysis of these compounds by techniques such as matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry provides the most rapid method for comparing glycan profiles and is probably most appropriate for clinical studies. One of the most promising techniques for determining the structures of N-glycans in proteomic studies is negative ion fragmentation of electrosprayed ions. This technique combines high throughput with ease of structural interpretation and provides structural details that are difficult to obtain by classical methods.     

Derivatization of carbohydrates for chromatographic,electrophoretic and mass spectrometric structure analysis

Carbohydrates, either alone or as constituents of glycoproteins, proteoglycans and glycolipids, are mediators of several cellular events and (patho)physiological processes. Progress in the "glycome" project is closely related to the analytical tools used to define carbohydrate structure and correlate structure with function. Chromatography, electrophoresis and mass spectrometry are the indispensable analytical tools of the on-going research. Carbohydrate derivatization is required for most of these analytical procedures. This review article gives an overview of derivatization methods of carbohydrates for their liquid chromatographic and electrophoretic separation, as well as the mass spectrometric characterization. Pre-column and on-capillary derivatization methods are presented with special emphasis on the derivatization of large carbohydrates.     

Analysis of protein glycosylation by mass spectrometry

We present a detailed protocol for the structural analysis of protein-linked glycans. In this approach, appropriate for glycomics studies, N-linked glycans are released using peptide N-glycosidase F and O-linked glycans are released by reductive alkaline beta-elimination. Using strategies based on mass spectrometry (matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano-electrospray ionization mass spectrometry/mass spectrometry (nano-ESI-MS-MS)), chemical derivatization, sequential exoglycosidase digestions and linkage analysis, the structures of the N- and/or O-glycans are defined. This approach can be used to study the glycosylation of isolated complex glycoproteins or of numerous glycoproteins encountered in a complex biological medium (cells, tissues and physiological fluids).     

Capillary electrophoresis of carbohydrates

Capillary electrophoresis has emerged as a highly promisingtechnique for the analysis of mono- and oligosaccharides. Theapproaches developed for overcoming the lack of chromophoricand fluorophoric functions in most carbohydrates involve theuse of indirect photometric detection, amperometry, mass spectrometry,and precolumn derivatization with various tags. The merits anddrawbacks of the derivatizing agents, including 2-aminopyridine,4-amino-benzoic acid and its analogues, which for the firsttime permitted the reproducible determination of aldoses, uronicacids and even ketoses in the low femtomole range by means ofreadily available UV detection, and other agents such as 8-aminonaphthalene-1,3,6-trisulphonicacid, 1-phenyl-3-methyl-5-pyrazolone and 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde,are discussed in detail. Means to secure electromigration ofthe usually neutral carbohydrates are: (i) ionization of hydroxylgroups at high pH; (ii) complexation of vicinal or alternatehydroxyl groups with borate or other charged compounds suchas alkaline earth metal ions; (iii) derivatization with a reagentpossessing ionizable functions; and (iv) partitioning into apseudostationary phase such as sodium dodecyl sulphate micelles.Each alternative has its own analytical rewards, and combinationsof the above mechanisms allow the two-dimensional and perhapseven three-dimensional mapping of oligosaccharides. Pyridylaminatedoligosaccharides, for instance, have been separated both accordingto size by exploiting differences in the charge-to-mass ratio,with the charge being identical for each oligomer under acidicconditions due to protonation of the imino group incorporatedby precolumn derivatization, as well as on the basis of structuraldifferences, as a consequence of differences in the ease ofborate complexation of the peripheral monosaccharide residues.It is also shown that the 4-aminobenzonitrile derivatives ofmono- and disaccharides can be separated by micellar electrokineticchromatography with a resolving power superior to that achievedby capillary zone electrophoresis of sugar-borate complexes.Based on the progress made, it can be concluded that capillaryelectrophoresis represents a powerful alternative and complementto existing methodology in the area of carbohydrate analysis. borate complexation capillary zone electrophoresis micellar electrokinetic chromatography precolumn derivatization     

Introduction to ion trap mass spectrometry: Application to the structural characterization of plant oligosaccharides

This review will focus on ion trap mass spectrometry (ITMS) and the application of this technique to the structural analysis of carbohydrates. The basic principles of operation of the electrostatic ion traps are briefly described and the applicability of the technique to the structural characterization of carbohydrates is illustrated with the analysis of arabinoxylan oligosaccharides by ion trap mass spectrometry.     

Chemical derivatization and mass spectral libraries in metabolic profiling by GC/MS and LC/MS/MS

An overview is presented of gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), the two major hyphenated techniques employed in metabolic profiling that complement direct 'fingerprinting' methods such as atmospheric pressure ionization (API) quadrupole time-of-flight MS, API Fourier transform MS, and NMR. In GC/MS, the analytes are normally derivatized prior to analysis in order to reduce their polarity and facilitate chromatographic separation. The electron ionization mass spectra obtained are reproducible and suitable for library matching, mass spectral collections being readily available. In LC/MS, derivatization and library matching are at an early stage of development and mini-reviews are provided. Chemical derivatization can dramatically increase the sensitivity and specificity of LC/MS methods for less polar compounds and provides additional structural information. The potential of derivatization for metabolic profiling in LC/MS is demonstrated by the enhanced analysis of plant extracts, including the potential to measure volatile acids such as formic acid, difficult to achieve by GC/MS. The important role of mass spectral library creation and usage in these techniques is discussed and illustrated by examples.     

Derivatization of carbohydrates for analysis by chromatography; electrophoresis and mass spectrometry

Carbohydrates display a large diversity of structures and their analysis presents many obstacles as the result of properties such as isomeric diversity, existence of branched structures and the lack of chromophores or fluorophores. Consequently, many analytical approaches depend on the application of chemical modifications such as hydrolysis or derivative formation. This review covers various aspects of derivatization that are used for such approaches as improving thermal stability and volatility for gas-phase analyses, introduction of fluorophores for optical detectors, introduction of charge for mass spectral analyses and attachment of bioaffinity tags for bioactivity studies. Reducing carbohydrates contain, in addition to multiple hydroxyl groups, several other sites for derivatization such as the single anomeric site that has been used in numerous methods for attaching various property-enhancing tags. Other sites are restricted to specific carbohydrates but include carboxy groups in sialic acids and amino groups in glycosylamines. All of these groups have been the targets of derivatization and this review attempts to summarise the main methods used for these various functional groups.     

A bioanalytical method to determine the cell wall composition of Mycobacterium tuberculosis grown in vivo

Mycobacterium tuberculosis bacilli exhibit cell wall alterations during in vivo growth. Development of ultrasensitive analytical techniques with high specificities is required to analyze the cell wall of M. tuberculosis isolated from experimental animals because of the low amounts of bacteria available and contamination by host tissue. Here we present a novel methodology to analyze all three major components (mycolic acids, arabinogalactan, and peptidoglycan) of the mycobacterial cell wall from mycobacteria isolated from animal tissue. In this procedure, the cell wall carbohydrates are analyzed by gas chromatography tandem mass spectrometry (GC/MS/MS) of alditol acetates, the peptidoglycan by GC/MS (mass spectrometry) analysis of the unique amino acid diaminopimelic acid (after derivatization with isopropyl chloroformate), and the mycolic acids by liquid chromatography (LC)/MS (negative ion) without derivatization. The procedure was designed so that all three analyses could be performed starting with a single sample given the difficulty of preparing multiple aliquots in known ratios. Linkage analysis, including an enantiomeric specific procedure, of the arabinogalactan polymer is also presented. These procedures will enable the determination of the cell wall alterations known to occur in the important nongrowing "dormant" M. tuberculosis present during disease. With some adaptations, the methodology is also applicable to the analysis of small amounts of in vivo grown bacteria of other species.     

Carbohydrates in the functions of natural killer cells.

There is little doubt that carbohydrates are crucial to the functions of NK cells. Exogenous carbohydrates alter the cytolytic capabilities of these cells, treatment of NK cells with glycosidases or inhibitors of glycosylation affect function, NK cells can bind selective sugars, and NK cells can be identified and subdivided into functionally distinct subsets on the basis of cell surface carbohydrates. Yet, despite the large number of observations which have been made concerning carbohydrates and NK cells, there is little consensus regarding these studies and few investigations which satisfactorily demonstrate specific mechanisms for the observed effect of the carbohydrates. Almost certainly, the number of diverse observations implies roles for carbohydrates in multiple areas of NK function, probably including target recognition, tissue distribution and post-binding events in the lytic cascade. Hopefully, these observations made to date will be viewed as exciting preliminary studies which will entice more sophisticated investigations designed to elucidate the precise roles of carbohydrates in the functions of NK cells.     

Microanalysis of N-linked oligosaccharides in a glycoprotein by capillary liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

We have studied rapid and simple sugar mapping using liquid chromatography/electrospray ionization mass spectrometry (LC/MS) equipped with a graphitized carbon column. The oligosaccharide mixture was separated on the basis of the sequence, branching structure, and linkage, and each oligosaccharide was characterized based on its molecular mass. In this study we demonstrated the usefulness of capillary LC/MS (CapLC/MS) and capillary liquid chromatography/tandem mass spectrometry (CapLC/MS/MS) as sensitive means for accomplishing the structural analysis of oligosaccharides in a low-abundance glycoprotein. The carbohydrate heterogeneity and molecular mass information of each oligosaccharide can be readily obtained from CapLC/MS of a small amount of glycoprotein. CapLC/MS/MS provided b-ion series, which is informative with regard to monosaccharide sequence. Exoglycosidase digestion followed by CapLC/MS elucidated a carbohydrate residue linkage. Using this method, we characterized N-linked oligosaccharides in hepatocyte growth factor produced in mouse myeloma NS0 cells as the complex-type bi-, tri-, and tetraantennary terminated with N-glycolylneuraminic acids and alpha-linked galactose residues. Sugar mapping with CapLC/MS and CapLC/MS/MS is useful for monitoring glycosylation patterns and for structural analysis of carbohydrates in a low-abundance glycoprotein and thus will become a powerful tool in biological, pharmaceutical, and clinical studies.     

Analysis of protein glycosylation by mass spectrometry

There is a growing pharmaceutical market for protein-based drugs for use in therapy and diagnosis. The rapid developments in molecular and cell biology have resulted in production of expression systems for manufacturing of recombinant proteins and monoclonal antibodies. These proteins are glycosylated when expressed in cell systems with glycosylation ability. For glycoproteins intended for therapeutic administration it is important to have knowledge about the structure of the carbohydrate side chains to avoid cell systems that produce structures, which in humans can cause undesired reactions, e.g., immunological and unfavorable serum clearance rate. Structural analysis of glycoprotein oligosaccharides requires sophisticated instruments like mass spectrometers and nuclear magnetic resonance spectrometers. However, before the structural analysis can be conducted, the carbohydrate chains have to be released from the protein and purified to homogeneity, and this is often the most time-consuming step. Mass spectrometry has played and still plays an important role in analysis of protein glycosylation. The superior sensitivity compared to other spectroscopic methods is its main asset. Structural analysis of carbohydrates faces several problems, however, due to the chemical nature of the constituent monosaccharide residues. For oligosaccharides or glycoconjugates, the structural information from mass spectrometry is essentially limited to monosaccharide sequence, molecular weight, and only in exceptional cases glycosidic linkage positions can be obtained. In order to completely establish an oligosaccharide structure, several other structural parameters have to be determined, e.g., linkage positions, anomeric configuration and identification of the monosaccharide building blocks. One way to address some of these problems is to work on chemical pretreatment of the glycoconjugate, to specifically modify the carbohydrate chain. In order to introduce specific modifications, we have used periodate oxidation and trifluoroacetolysis with the objective of determining glycosidic linkage positions by mass spectrometry.     

Application of hydrogen/deuterium exchange mass spectrometry to study protein tyrosine phosphatase dynamics, ligand binding, and substrate specificity

Protein tyrosine phosphatases (PTPs) are signaling enzymes that control a diverse array of cellular processes. Further insight into the specific functional roles of PTPs in cellular signaling requires detailed understanding of the molecular basis for substrate recognition by the PTPs. A central question is how PTPs discriminate between multiple structurally diverse substrates that they encounter in the cell. Although X-ray crystallography is capable of revealing the intimate structural details for molecular interaction, structures of higher order PTP.substrate complexes are often difficult to obtain. Hydrogen/deuterium exchange mass spectrometry (H/DX-MS) is a powerful tool for mapping protein-protein interfaces, as well as identifying conformational and dynamic perturbations in proteins. In addition, H/DX-MS enables analysis of large protein complexes at physiological concentrations and provides insight into the solution behavior of these complexes that can not be gleaned from crystal structures. We have utilized H/DX-MS to probe PTP dynamics, ligand binding, and the structural basis of substrate recognition. In this article, we review general principles of H/DX-MS technology as applied to study protein-protein interactions and dynamics. We also provide protocols for H/DX-MS successfully used in our laboratory to define the molecular basis of ERK2 substrate recognition by MKP3. Many of the aspects that we cover in detail should be applicable to the study of other PTPs with their specific targets.     

Online mass spectrometric analysis of proteins/peptides following electrolytic cleavage of disulfide bonds

The disulfide bond bridge is an important post-translational modification for proteins. This study presents a structural analysis of biologically active peptides and proteins containing disulfide bonds using electrochemistry (EC) online combined with desorption electrospray ionization mass spectrometry (DESI-MS), in which the sample undergoes electrolytic disulfide cleavage in an electrochemical flow cell followed by MS detection. Using this EC/DESI-MS method, the disulfide-containing peptides can be quickly identified from enzymatic digestion mixtures, simply based on the abrupt decrease in their relative ion abundances after electrolysis. Peptide mass mapping and tandem MS analysis of the ions of the resulting free peptide chains can possibly establish the disulfide linkage pattern and sequence the precursor peptides. In this regard, the method provides much more chemical information than previous analogous electrochemical analyses. In addition, derivatization of thiols by selective selenamide reagents is useful for easy recognition of reduced peptide ions and the number of their free thiols. Furthermore, electrolytic reduction of proteins (e.g., α-lactalbumin) leads to increased charges on the detected protein ions, revealing the role of disulfide bonds on maintaining protein conformation. This electrochemical mass spectrometric method is fast (completed in few minutes) and does not need chemical reductants, potentially having valuable applications in proteomics research.     

Carbohydrate-binding proteins in cancer, and their ligands as therapeutic agents

Experimental evidence directly implicates complex carbohydrates in recognition processes, including adhesion between cells, adhesion of cells to the extracellular matrix, and specific recognition of cells by one another. In addition, carbohydrates are recognized as differentiation markers and as antigenic determinants. Lectins are nonenzymatic proteins present in plants and animals, which preferentially bind to specific carbohydrate structures and play an important role in cell recognition. Modified carbohydrates and oligosaccharides have the ability to interfere with carbohydrate-protein interactions and therefore, inhibit the cell-cell recognition and adhesion processes, which play an important role in cancer growth and progression. Carbohydrate ligands therefore, are candidates to play important roles in cancer therapeutics.     

Structural classification of carbohydrates in glycoproteins by mass spectrometry and high-performance anion-exchange chromatography

A general strategy has been developed for determining the structural class (oligomannose, hybrid, complex), branching types (biantennary, triantennary, etc.), and molecular microheterogeneity of N-linked oligosaccharides at specific attachment sites in glycoproteins. This methodology combines mass spectrometry and high-performance anion-exchange chromatography with pulsed amperometric detection to take advantage of their high sensitivity and the capability for analysis of complex mixtures of oligosaccharides. Glycopeptides are identified and isolated by comparative HPLC mapping of proteolytic digests of the protein prior to, and after, enzymatic release of carbohydrates. Oligosaccharides are enzymatically released from each isolated glycopeptide, and the attachment site peptide is identified by fast atom bombardment mass spectrometry (FAB-MS) of the mixture. Part of each reaction mixture is then permethylated and analyzed by FAB-MS to identify the composition and molecular heterogeneity of the carbohydrate moiety. Fragment ions in the FAB mass spectra are useful for detecting specific structural features such as polylactosamine units and bisecting N-acetylhexosamine residues, and for locating inner-core deoxyhexose residues. Methylation analysis of these fractions provides the linkages of monomers. Based on the FAB-MS and methylation analysis data, the structural classes of carbohydrates at each attachment site can be proposed. The remaining portions of released carbohydrates from specific attachment sites are preoperatively fractionated by high-performance anion-exchange chromatography, permethylated, and analyzed by FAB-MS. These analyses yield the charge state and composition of each peak in the chromatographic map, and provide semiquantitative information regarding the relative amounts of each molecular species. Analytically useful data may be obtained with as little as 10 pmol of derivatized carbohydrate, and fmol sensitivity has been achieved. The combined carbohydrate mapping and structural fingerprinting procedures are illustrated for a recombinant form of the CD4 receptor glycoprotein.     

Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).

Ceramide (CER) is an important signaling molecule involved in a variety of cellular processes, including differentiation, cell growth, and apoptosis. Currently, different techniques are applied for CER quantitation, some of which are relatively insensitive and/or time consuming. Tandem mass spectrometry with its high selectivity and sensitivity is a very useful technique for detection of low abundant metabolites without prior purification or derivatization. In contrast to existing mass spectrometry methods, the developed electrospray tandem mass spectrometry (ESI-MS/MS) technique is capable of quantifying different CER species from crude cellular lipid extracts. The ESI-MS/MS is performed with a continuous flow injection and the use of an autosampler, resulting in a high throughput capability. The collision-induced fragmentation of CER produced, in addition to others, a characteristic fragment of m/z 264, making a precursor ion scan of 264 well suited for CER quantitation. Quantitation is achieved by use of a constant concentration of a non-naturally occurring internal standard C8-CER, together with a calibration curve established by spiking different concentrations of naturally occurring CER. The calibration curves showed linearity over a wide concentration range and sample volumes equivalent to 10 microg of cell protein corresponding to about 20, 000 fibroblasts were sufficient for CER analysis. Moreover this assay showed a detection limit at the subpicomole level. In summary, this methodology enables accurate and rapid analysis of CER from small samples without prior separation steps, thus providing a useful tool for signal transduction research.     

Identification of keratinocyte-specific markers using phage display and mass spectrometry

Specific molecular markers for various normal and pathogenic cell states and cell types provide knowledge of basic biological systems and have a direct application in targeted therapy. We describe a proteomic method based on the combination of new and improved phage display antibody technologies and mass spectrometry that allows identification of cell type-specific protein markers. The most important features of the method are (i) reduction of experimental noise originating from background binding of phage particles and (ii) isolation of affinity binders after a single round of selection, which assures a high diversity of binders. The method demonstrates, for the first time, the ability to detect, identify, and analyze both secreted and membrane-associated extracellular proteins as well as a variety of different cellular structures including proteins and carbohydrates. The optimized phage display method was applied to analysis of human skin keratinocytes resulting in the isolation of a panel of antibodies. Fourteen of these antibodies were further characterized, half of which predominantly recognized keratinocytes in a screen of a range of different cell types. Three cognate keratinocyte antigens were subsequently identified by mass spectrometry as laminin-5, plectin, and fibronectin. The combination of phage display technology with mass spectrometry methods for protein identification is a general and promising approach for proteomic analysis of cell surface complexity.     

Chemical ionization mass spectrometry of trimethylsilylated carbohydrates and organic acids retained in uremic serum

After appropriate sample pretreatment and derivatization, uremic serum was investigated by combined high resolution gas chromatography and mass spectrometry, using both electron impact and chemical ionization methods. Electron impact and chemical ionization spectra of a number of identified (trimethylsilylated) carbohydrates and organic acids are compared. The utilization of chemical ionization mass spectrometry, with isobutane as the reagent gas, is discussed in detail. The influence of the reagent gas pressure on the total ion current and on the spectral appearance was studied. The identification of compounds, based on electron impact mass spectral data, was confirmed and often aided appreciably by using this technique. The chemical ionization spectra of trimethylsilyated alditols and aldonic acids, as well as of other organic acids showed protonated molecular ions, whereas aldoses did not. Differences with electron impact spectra are found mainly in the high mass region. The loss of one or more trimethylsilanol groups becomes the predominating fragmentation route at higher reagent gas pressures.     

不同结构类型糖类物质的荧光标记策略

为阐明糖链结构与功能的关系,寻找高灵敏度和高分离度的微量分析方法对糖 类物质的分析至关重要.近几年来,荧光标记方法作为糖类物质的色谱定量及辅助结 构分析的最佳选择,日益受到人们的广泛关注.荧光标记法分为柱前标记和柱后标记 两种,且灵敏度高、分离度好、有多种标记试剂可供选择.本文根据不同糖类物质的 结构特点,分别对中性糖和氨基糖以及含有唾液酸、糖醛酸、硫酸基的酸性糖的荧光 标记方法及其应用进行了系统的综述,并对近年来报道的荧光标记试剂的特点和反应 机理进行了比较和总结,以期为糖类物质微量分析方法的研究提供参考.     

Application of mass spectrometry to structural identification of flavonoid monoglycosides isolated from shoot of lupin (Lupinus luteus L.).

Flavonoid glycosides constitute important group of plant secondary metabolites. This class of natural products play significant role in different physiological processes. A new methodological approach where mass spectrometric techniques are applied to structural studies of this class of compounds is presented. Four flavonoid O-monoglycosides and one C-monoglycoside were isolated from green parts of lupin (Lupinus luteus L.). Several different mass spectrometric techniques were applied to structural elucidation of isolated compounds. Desorption ionization mass spectrometry was used for registration of mass spectra of intact and derivatized (permethylated) flavonoid glycosides. In some cases electron impact mass spectra of permethylated compounds were also recorded. Methylated samples after methanolysis and further derivatization of free hydroxyl groups (methylation or acetylation) were analyzed with gas chromatography-mass spectrometry. Combined information drawn from the registered mass spectra enabled us to define molecular mass, structure of aglycones and sugars, and positions of glycosidic bonds on the aglycon. Structures of four flavonoid monoglycosides were elucidated as follows: genistein 7-O-glucoside (1), genistein 4'-O-glucoside (2), 2'-hydroxygenistein 7-O-glucoside (3), and apigenin or genistein 8-C-glycoside (5). For the fourth O-glycoside (4) only molecular mass and masses of the aglycone and sugar were estimated.