Identification of N-linked glycosylation sites using glycoprotein digestion with pronase prior to MALDI tandem time-of-flight mass spectrometry

Glycopeptides are typically prepared by cleaving the proteins with specific proteolytic enzymes, such as trypsin. The resulting glycopeptides tend to have weak mass spectrometry ion signals (ESI or MALDI) due to their relatively large molecular weight. The identification of glycosylation sites with tandem mass spectrometry is further complicated by fragmentation of both the peptide backbone and the glycan moiety. We explored a method using a nonspecific enzyme, pronase, to generate small glycopeptides (between two and six amino acids). These glycopeptides were enriched and desalted using a microscale hydrophilic interaction chromatography extraction device prior to MALDI QTof MS analysis. MALDI matrix, 2, 5-dihydroxybenzoic acid, doped with ammonium triscitrate, was utilized for analysis. Sodiated ions were observed as minor ions, while protonated ions were enhanced dramatically with this matrix. Collision-induced dissociation was performed on both the protonated and sodiated ions. MS/MS fragmentation spectra reveal that proton has greater affinity for the peptide moiety, while the sodium cation tends to associate with the sugar moiety. Characteristic fragment patterns allowed for identifications of glycosylation sites for both the protonated and the sodiated precursor ions. Model proteins, horseradish peroxidase and alpha1-acid glycoproteins, were analyzed to illustrate the identification of N-linked glycosylation sites and data interpretation algorithm.     

Enhanced detection and identification of glycopeptides in negative ion mode mass spectrometry

A combined mass spectrometry (MS) and tandem mass spectrometry (MS/MS) approach implemented with matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FTICR MS) in the negative ion mode is described for enhanced glycopeptide detection and MS/MS analysis. Positive ion mode MS analysis is widely used for glycopeptide characterization, but the analyses are hampered by potential charge-induced fragmentation of the glycopeptides and poor detection of the glycopeptides harboring sialic acids. Furthermore, tandem MS analysis (MS/MS) via collision-induced dissociation (CID) of glycopeptides in the positive ion mode predominantly yields glycan fragmentation with minimal information to verify the connecting peptide moiety. In this study, glycoproteins such as, bovine lactoferrin (b-LF) for N-glycosylation and kappa casein (k-CN) for O-glycosylation were analyzed in both the positive- and negative ion modes after digestion with bead-immobilized Pronase. For the b-LF analysis, 44 potential N-linked glycopeptides were detected in the positive ion mode while 61 potential N-linked glycopeptides were detected in the negative ion mode. By the same token, more O-linked glycopeptides mainly harboring sialic acids from k-CN were detected in the negative ion mode. The enhanced glycopeptide detection allowed improved site-specific analysis of protein glycosylation and superior to positive ion mode detection. Overall, the negative ion mode approach is aimed toward enhanced N- and O-linked glycopeptide detection and to serve as a complementary tool to positive ion mode MS/MS analysis.     

Determination of N-glycosylation sites and site heterogeneity in glycoproteins

An approach for the characterization of glycosylation sites and oligosaccharide heterogeneity in glycoproteins based on a combination of nonspecific proteolysis, deglycosylation, and matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FT MS) is described. Glycoproteins were digested with Pronase yielding primarily glycopeptides and amino acids. Nonglycosylated peptide fragments were susceptible to complete Pronase digestion to their constituent amino acids. Steric hindrance prohibited the digestion of the peptide moiety attached to the glycan. Glycopeptides were desalted and concentrated using solid-phase extraction and analyzed by MALDI MS. The oligosaccharides were also analyzed by MALDI MS after releasing the glycans from glycoproteins using PNGase F. The peptide moiety of the glycopeptides was identified by subtracting the masses of the glycans derived from PNGase F treatment from the masses of the glycopeptides. The experimental strategy was validated using glycoproteins with known oligosaccharide structures, ribonuclease B and chicken ovalbumin. This procedure was then used to determine the N-glycosylation sites and site heterogeneity of a glycoprotein whose glycosylation pattern was unknown, namely, the Xenopus laevis egg cortical granule lectin. This procedure is useful for determining protein site heterogeneity and structural heterogeneities of the oligosaccharide moiety of glycoproteins.     

Nano-LC-MS/MS of glycopeptides produced by nonspecific proteolysis enables rapid and extensive site-specific glycosylation determination

Given the biological importance of glycosylation on proteins, the identification of protein glycosylation sites is integral to understanding broader biological structure and function. Unfortunately, the determination of the microheterogeneity at the site of glycosylation still remains a significant challenge. Nanoflow liquid chromatography with tandem mass spectrometry provides both separation of glycopeptides and the ability to determine glycan composition and site-specific glycosylation. However, because of the size of glycopeptides, they are not often amenable to tandem MS. In this work, proteins are digested with multiple proteases to produce glycopeptides that are of suitable size for tandem MS analysis. The conditions for collision-induced dissociation are optimized to obtain diagnostic ions that maximize glycan and peptide information. The method is applied to glycoproteins with contrasting glycans and multiple sites of glycosylation and identifies multiple glycan compositions at each individual glycosylation site. This method provides an important improvement in the routine determination of glycan microheterogeneity by mass spectrometry.     

Determination and characterization of site-specific N-glycosylation using MALDI-Qq-TOF tandem mass spectrometry: case study with a plant protease

MALDI tandem mass spectrometry analysis on a hybrid quadrupole-quadrupole time-of-flight (Qq-TOF) instrument was used in combination with two-dimensional gel electrophoresis, proteolytic digestion, and liquid chromatography for identification and structural characterization of glycosylation in a novel glycoprotein, pathogenesis-related subtilisin-like proteinase P69B from tomato. Glycopeptide fractions from microcolumn reversed-phase HPLC deposited on MALDI targets were identified from MS by their specific m/z spacing patterns (203, 162, 146 u) between glycoforms. In most cases, MS/MS spectra of [M + H]+ ions of glycopeptides featured peaks useful for determining sugar compositions, peptide sequences, and thus probable glycosylation sites. Furthermore, peptide-related product ions could readily be used in database search procedures to identify the glycoprotein. Four out of five predicted glycosylation sites were biologically relevant and occupied by five N-linked glycan side chains each. In addition, the fragmentation efficiency allowed detection of further modification of methionine-containing glycoforms with either oxidized or iodoacetamide alkylated methionine. The high resolution furnished by MALDI-Qq-TOF allowed rapid and sensitive structural characterization of site-specific N-glycosylation from a limited quantity of material and revealed heterogeneity at different levels, including different glycan side-chain modifications, and heterogeneity of oligosaccharide structures on the same glycosylation site.     

Ultrasensitive characterization of site-specific glycosylation of affinity-purified haptoglobin from lung cancer patient plasma using 10 μm i.d. porous layer open tubular liquid chromatography-linear ion trap collision-induced dissociation/electron transfer dissociation mass spectrometry

Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography-collision-induced dissociation/electron transfer dissociation mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of the glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization, 200-500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrixes. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultranarrow porous layer open tubular (PLOT) LC column (2.5 m × 10 μm i.d.) was coupled to a linear ion trap (LTQ) collision-induced dissociation/electron transfer dissociation mass spectrometer to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low picomole amounts of protein, we affinity purified it from 1 μL of pooled lung cancer patient plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol of Hpt digest (13 ng of protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS system allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the ～100 amol level. The PLOT LC-MS system is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace levels of glycosylated proteins.     

Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics

In glycoproteomics, key structural issues, protein identification, locations of glycosylation sites, and evaluation of the glycosylation site microheterogeneity should be easily evaluated in a large number of glycoproteins, while mass spectrometry (MS) provides substantial information about individual purified glycoproteins. Considering that structural issues are elucidated by studying glycopeptides and that the tandem MS of a tryptic peptide composed of several amino acid residues is enough for protein identification, construction of an MS-based method handling tryptic glycopeptides would be of considerable benefit in research. To this end, a simple and efficient method, utilizing hydrophilic binding of carbohydrate matrixes such as cellulose and Sepharose to oligosaccharides, was successfully applied to the isolation of tryptic glycopeptides. Both peptide and oligosaccharide structures were elucidated by multiple-stage tandem MS (MS(n)) of the ions generated by matrix-assisted laser desorption/ionization (MALDI), as follows. The MALDI ion trap mass spectrum of a tryptic glycopeptide mixture from N-linked glycoproteins was composed of the [M + H]+ ions of component glycopeptides. Collision-induced dissociation (CID) of the glycopeptide [M + H]+ ion generated saccharide-spaced peaks, with an interval of, for example, 146, 162, and 203 Da, and their fragment ions corresponding to the peptide and peptide + N-acetylglucosamine (GlcNAc) species in the MS2 spectrum. The saccharide-spaced ladder served to outline oligosaccharide structures, which were then selected as precursors for subsequent MS(n) analyses. The peptide or peptide + GlcNAc ions in the MS2 spectrum or the corresponding ions abundant in the MS1 spectrum were subjected to CID for determination of peptide sequences, to identify proteins and their glycosylation sites. The strategy, isolation of glycopeptides followed by MS(n) analysis, efficiently characterized the structures of beta2-glycoprotein I with four N-glycosylation sites and was applied to an analysis of total serum glycoproteins.     

Rapidly alternating transmission mode electron-transfer dissociation and collisional activation for the characterization of polypeptide ions

Cation transmission/electron-transfer reagent anion storage mode electron-transfer ion/ion reactions and beam-type collisional activation of the polypeptide ions are performed in rapid succession in the high-pressure collision cell (Q2) of a quadrupole/time-of-flight tandem mass spectrometer (QqTOF), where the electron-transfer reagent anions are accumulated. Duty cycles for both electron-transfer dissociation (ETD) and collision-induced dissociation (CID) experiments are improved relative to ion trapping approaches since there are no discrete ion storage and reaction steps for ETD experiments and no discrete ion storage step and frequency tuning for CID experiments. For this technique, moderately high resolution and mass accuracy are also obtained due to mass analysis via the TOF analyzer. This relatively simple approach has been demonstrated with a triply charged tryptic peptide, a triply charged tryptic phosphopeptide, and a triply charged tryptic N-linked glycopeptide. For the tryptic peptide, the sequence is identified with more certainty than would be available from a single method alone due to the complementary information provided by these two dissociation methods. Because of the complementary information derived from both ETD and CID dissociation methods, peptide sequence and post-translational modification (PTM) sites for the phosphopeptide are identified. This combined ETD and CID approach is particularly useful for characterizing glycopeptides because ETD generates information about both peptide sequence and locations of the glycosylation sites, whereas CID provides information about the glycan structure.     

Separation and identification of isomeric glycopeptides by high field asymmetric waveform ion mobility spectrometry

The analysis of intact glycopeptides by mass spectrometry is challenging due to the numerous possibilities for isomerization, both within the attached glycan and the location of the modification on the peptide backbone. Here, we demonstrate that high field asymmetric wave ion mobility spectrometry (FAIMS), also known as differential ion mobility, is able to separate isomeric O-linked glycopeptides that have identical sequences but differing sites of glycosylation. Two glycopeptides from the glycoprotein mucin 5AC, GT(GalNAc)TPSPVPTTSTTSAP and GTTPSPVPTTST(GalNAc)TSAP (where GalNAc is O-linked N-acetylgalactosamine), were shown to coelute following reversed-phase liquid chromatography. However, FAIMS analysis of the glycopeptides revealed that the compensation voltage ranges in which the peptides were transmitted differed. Thus, it is possible at certain compensation voltages to completely separate the glycopeptides. Separation of the glycopeptides was confirmed by unique reporter ions produced by supplemental activation electron transfer dissociation mass spectrometry. These fragments also enable localization of the site of glycosylation. The results suggest that glycan position plays a key role in determining gas-phase glycopeptide structure and have implications for the application of FAIMS in glycoproteomics.     

Sequencing of O-glycopeptides derived from an S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a containing up to 51 monosaccharide residues at a single glycosylation site by fourier transform ion cyclotron resonance infrared multiphoton dissociation mass spectrometry

The microheterogeneity of large sugar chains in glycopeptides from S-layer glycoproteins containing up to 51 monosaccharide residues at a single O-attachment site on a 12 amino acid peptide backbone was investigated by Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Structural elucidation of glycopeptides with the same amino acid sequence and different glycoforms, having such a high saccharide-to-peptide ratio, was achieved by applying infrared multiphoton dissociation (IRMPD) MS/MS for the first time. A 100% sequence coverage of the glycan chain and a 50% coverage of the peptide backbone fragmentation were obtained. The microheterogeneity of carbohydrate chains at the same glycosylation site, containing largely rhamnose, could have been reliably assessed.     

Isolation and identification of sialylated glycopeptides from bovine alpha1-acid glycoprotein by off-line capillary electrophoresis MALDI-TOF mass spectrometry

Sialylated glycopeptides contained in liquid chromatographic fractions of bovine alpha1-glycoprotein tryptic digests were isolated from asialo peptides using capillary electrophoresis (CE). CE effluents were deposited directly onto a metallic target and analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. This method allowed the characterization of four N-glycosylation sites in the glycoprotein, and each site was observed as a set of sialylated peptide glycoforms. Tandem mass spectrometry was used to confirm peptide sequences and glycan content in glycoforms. The CE method developed for this study resulted in a very clear separation of the sialylated from the asialo content of glycoprotein digests and proved very useful in the determination of the nature and location of sialylated glycans along the protein chain. This article is the first report describing the use of on-target CE fraction collection using a MALDI removable sample concentrator.     

Factors that influence fragmentation behavior of N-linked glycopeptide ions

The investigation of site-specific glycosylation is essential for further understanding the many biological roles that glycoproteins play; however, existing methods for characterizing site-specific glycosylation either are slow or yield incomplete information. Mass spectrometry (MS) is being applied to investigate site-specific glycosylation with bottom-up proteomic type strategies. When using these approaches, tandem mass spectrometry techniques are often essential to verify glycopeptide composition, minimize false positives, and investigate structure. The fragmentation behavior of glycopeptide ions has previously been investigated with multiple techniques including collision induced dissociation (CID), infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD); however, due to the almost exclusive analysis of multiply protonated tryptic glycopeptide ions, some dissociation behaviors of N-linked glycopeptide ions have not been fully elucidated. In this study, IRMPD of N-linked glycopeptides has been investigated with a focus on the effects of charge state, charge carrier, glycan composition, and peptide composition. Each of these parameters was shown to influence the fragmentation behavior of N-linked glycopeptide ions. For example, in contrast to previously reported accounts that IRMPD results only in glycosidic bond cleavage, the fragmentation of singly protonated glycopeptide ions containing a basic amino acid residue almost exclusively resulted in peptide backbone cleavage. The fragmentation of the doubly protonated glycopeptide ion exhibited fragmentation similar to that previously reported; however, when the same glycopeptide was sodium coordinated, a previously inaccessible series of glycan fragments were observed. Molecular modeling calculations suggest that differences in the site of protonation and metal ion coordination may direct glycopeptide ion fragmentation.     

Top-down sequencing of O-glycoproteins by in-source decay matrix-assisted laser desorption ionization mass spectrometry for glycosylation site analysis

The sites of mucin-type O-glycosylation are largely unpredictable, making structural analysis by mass spectrometry (MS) indispensible. On the peptide level, a site localization and characterization of O-linked glycans in situ using tandem MS with electron-transfer dissociation or matrix-assisted laser desorption ionization (MALDI) MS with postsource decay have been reported. The top-down sequencing on the protein level by MALDI-MS is based on the in-source decay (ISD) of intact glycoproteins induced by hydrogen radical transfer from the matrix. It allows a ladder sequencing from both termini with assignment of O-glycosylation sites based on intense c-, y-, and z-type ions. The feasibility of ISD-MALDI-MS in the localization of O-glycosylation sites was demonstrated with synthetic O-glycopeptides, the tandem repeat domain of recombinant MUC1, and the natural bovine glycoproteins asialofetuin and desialylated κ-casein. Ladder sequencing of the 17-18.5 kD MUC1 hexarepeat domains revealed (1) cell-specific glycosylation site patterns on comparison of probes expressed in human HEK-293 or Drosophila S2 cells, and (2) a site-specific microheterogeneity at the Thr/Ser sites with variations of the glycan compositions from zero to four monosaccharides. Novel O-glycosylation sites in the C-terminal domains of fetuin (T334) and κ-caseinoglycopeptide (S154 and T156) were assigned, the former representing a sequence conflict with the published T154.     

MALDI quadrupole time-of-flight mass spectrometry: a powerful tool for proteomic research

A MALDI QqTOF mass spectrometer has been used to identify proteins separated by one-dimensional or two-dimensional gel electrophoresis at the femtomole level. The high mass resolution and the high mass accuracy of this instrument in both MS and MS/MS modes allow identification of a protein either by peptide mass fingerprinting of the protein digest or from tandem mass spectra acquired by collision-induced dissociation of individual peptide precursors. A peptide mass map of the digest and tandem mass spectra of multiple peptide precursor ions can be acquired from the same sample in the course of a single experiment. Database searching and acquisition of MS and MS/MS spectra can be combined in an interactive fashion, increasing the information value of the analytical data. The approach has demonstrated its usefulness in the comprehensive characterization of protein in-gel digests, in the dissection of complex protein mixtures, and in sequencing of a low molecular weight integral membrane protein. Proteins can be identified in all types of sequence databases, including an EST database. Thus, MALDI QqTOF mass spectrometry promises to have remarkable potential for advancing proteomic research.     

Simplification of mass spectral analysis of acidic glycopeptides using GlycoPep ID

Mass spectral analysis is an increasingly common method used to characterize glycoproteins. When more than one glycosylation site is present on a protein, obtaining MS data of glycopeptides is a highly effective way of obtaining glycosylation information because this approach can be used to identify not only what the carbohydrates are but also at which glycosylation site they are attached. Unfortunately, this is not yet a routine analytical approach, in part because data analysis can be quite challenging. We are developing strategies to simplify this analysis. Presented herein is a novel mass spectrometry technique that identifies the peptide moiety of either sulfated, sialylated, or both sialylated and sulfated glycopeptides. This technique correlates product ions in collision-induced dissociation (CID) experiments of suspected glycopeptides to a peptide composition using a newly developed web-based tool, GlycoPep ID. After identifying the peptide portion of glycopeptides with GlycoPep ID, the process of assigning the rest of the glycopeptide composition to the MS data is greatly facilitated because the "unknown" portion of the mass assignment that remains can be directly attributed to the carbohydrate component. Several examples of the utility and reliability of this method are presented herein.     

Prediction of collision-induced dissociation spectra of common N-glycopeptides for glycoform identification

Confident identification of the glycan moieties in glycopeptides by collision-induced dissociation (CID) requires accurate prediction of the CID spectrum of the glycopeptides. In this Article, the kinetic model for the prediction of peptide CID spectra is extended to predict the CID spectra of N-glycopeptides. The model was trained with 1831 ion-trap CID spectra of N-glycopeptides and is able to predict ion-trap CID spectra with excellent accuracy in ion intensities for N-glycopeptides up to 8000 u in mass. A total of 524 common glycoforms including complex N-glycans with 2-4 antennas, plus high-mannose type and hybrid type, can be predicted.     

The characteristics of peptide collision-induced dissociation using a high-performance MALDI-TOF/TOF tandem mass spectrometer

A new matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometer is described for sequencing peptides. This instrument combines the advantages of high sensitivity for peptide analysis associated with MALDI and comprehensive fragmentation information provided by high-energy collision-induced dissociation (CID). Unlike the postsource decay technique that is widely used with MALDI-TOF instruments and typically combines as many as 10 separate spectra of different mass regions, this instrument allows complete fragment ion spectra to be obtained in a single acquisition at a fixed reflectron voltage. To achieve optimum resolution and focusing over the whole mass range, it may be desirable to acquire and combine three separate sections. Different combinations of MALDI matrix and collision gas determine the amount of internal energy deposited by the MALDI process and the CID process, which provide control over the extent and nature of the fragment ions observed. Examples of peptide sequencing are presented that identify sequence-dependent features and demonstrate the value of modifying the ionization and collision conditions to optimize the spectral information.     

Atmospheric pressure MALDI Fourier transform mass spectrometry of labile oligosaccharides

An atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) source coupled to Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS) under UV laser and solid matrix conditions has been demonstrated to analyze a variety of labile oligosaccharides including O-linked and N-linked complex glycans released from glycoproteins. Spectra were acquired by both AP MALDI and vacuum MALDI and directly compared. The results presented here confirm that AP MALDI can generate significantly less energetic ions than vacuum MALDI and is able to produce the intact molecular ions with little or no fragmentation in both positive and negative ion mode analyses. Under certain conditions, noncovalent complexes of sialylated oligosaccharides were observed. The sensitivity attainable by AP MALDI was found to be comparable to conventional MALDI, and tandem mass spectrometry of oligosaccharides ionized by AP MALDI was shown to allow detailed structural analysis. Analysis of N-glycan mixtures derived from human fibrinogen further demonstrated that AP MALDI-FT ICR MS is ideal for the study of complex glycan samples as it provides high-accuracy, high-resolution mass analysis with no difficulty in distinguishing sample constituents from fragment ions.     

Identification of low-abundance cancer biomarker candidate TIMP1 from serum with lectin fractionation and peptide affinity enrichment by ultrahigh-resolution mass spectrometry

As investigating a proteolytic target peptide originating from the tissue inhibitor of metalloproteinase 1 (TIMP1) known to be aberrantly glycosylated in patients with colorectal cancer (CRC), we first confirmed that TIMP1 is to be a CRC biomarker candidate in human serum. For this, we utilized matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) showing ultrahigh-resolution and high mass accuracy. This investigation used phytohemagglutinin-L(4) (L-PHA) lectin, which shows binding affinity to the β-1,6-N-acetylglucosamine moiety of N-linked glycan on a protein, to compare fractionated aberrant protein glycoforms from both noncancerous control and CRC serum. Each lectin-captured fraction containing aberrant glycoforms of TIMP1 was digested by trypsin, resulting in the tryptic target peptide, representative of the serum glycoprotein TIMP1. The resulting target peptide was enriched using a stable isotope standard and capture by the antipeptide antibody (SISCAPA) technique and analyzed by a 15 T MALDI FTICR mass spectrometer with high mass accuracy (Δ < 0.5 ppm to the theoretical mass value of the target peptide). Since exact measurement of multiplex isotopic peaks of the target peptide could be accomplished by virtue of high mass resolution (Rs > 400,000), robust identification of the target peptide is only achievable with 15 T FTICR MS. Also, MALDI data obtained in this study showed that the L-PHA-captured glycoforms of TIMP1 were measured in the pooled CRC serum with about 5 times higher abundance than that in the noncancerous serum, and were further proved by MRM mass analysis. These results confirm that TIMP1 in human serum is a potent CRC biomarker candidate, demonstrating that ultrahigh-resolution MS can be a powerful tool toward identifying and verifying potential protein biomarker candidates.     

Method for investigation of oligosaccharides from glycopeptides: direct determination of glycosylation sites in proteins

Characterization of glycopeptides has become an important tool toward a better understanding of the molecular details in carbohydrate-protein interactions. In this approach, oligosaccharides are commonly not detectable under mass spectrometric conditions because of ionization suppression by deglycosylated peptides. Their composition is only deduced from the mass differences between glycopeptides and corresponding deglycosylated peptides. Here, we describe how carbohydrates can be easily detected in the PNGase-treated samples and structurally investigated next to the peptides. The efficacy of this method is demonstrated through the analysis of tryptic glycopeptides obtained from human IgG. Following deglycosylation with PNGaseF and derivatization with phenylhydrazine, MALDI spectra produced ion peaks of labeled oligosaccharides and deglycosylated peptides. The relative abundances of individual oligosaccharides were consistent with those of the glycopeptides. MALDI-MS/MS provided useful data for the structural elucidation of oligosaccharides, including the assignment of dominant isomers and glycosylation sites in peptides. MALDI-MS/MS fragmentation patterns of deglycosylated peptide ions indicated glycosylation sites at asparagine 297 and 299. The observed peptide of the composition ADQTVYR, described for the first time in this study, indicated new glycosylation sites in IgG1 human myeloma plasma.