Two-step derivatization and mass spectral distinction of α2,3 and α2,6 sialic acid linkages on N-glycans by MALDI-TOF

Sialic acid linkages on N-glycans were distinguished by MALDI-TOF MS after two steps derivatization by dimethylamine and ammonium hydroxide. By using this method, more than 20 kinds of sialic acid with detailed linkage information were detected on A549 cells.     

GC–MS Measurement of 13C-Enrichment of Lactic Acid in Sepsis Plasma

Sepsis is a systemic inflammatory response syndrome arising from infection. The plasma lactic acid level is a reliable marker of sepsis. A novel procedure based on microwave-assisted derivatization followed by gas chromatography–mass spectrometry (GC–MS) has been developed for the rapid measurement of ¹³C-isotope enrichment of lactic acid in plasma. The derivatization conditions and method validation were studied. The method was applied to the measurements of isotopic enrichment of lactic acid in the plasma of a rabbits which had received the tracer ([1,2,3-¹³C₃] lactic acid) by carotid infusion 2.5 h before blood sampling from the portal vein. These results show that the proposed method has excellent precision (RSD less than 4.57%), linearity (slope = 0.9936; r ² = 0.9998), accuracy and selectivity. The analytical results show that microwave-assisted derivatization coupled to GC–MS is a rapid method for the direct isotopic measurement of deproteinised plasma. The method could be of great interest for metabolic studies in animal models and human patients.     

Highly specific purification of N-glycans using phosphate-based derivatization as an affinity tag in combination with Ti-SPE enrichment for mass spectrometric analysis

N-linked protein glycosylation is involved in regulation of a wide variety of cellular processes and associated with numerous diseases. Highly specific identification of N-glycome remains a challenge while its biological significance is acknowledged. The relatively low abundance of glycan in complex biological mixtures, lack of basic sites for protonation, and suppression by other highly abundant proteins/peptides lead to the particularly poor detection sensitivity of N-glycans in the MS analysis. Therefore, the highly specific purification procedure becomes a crucial step prior to MS analysis of the N-glycome. Herein, a novel N-glycans enrichment approach based on phosphate derivatization combined with Ti⁴⁺-SPE (solid phase extraction) was developed. Briefly, in this strategy, N-glycans were chemically labeled with a phospho-group at their reducing ends, such that the Ti⁴⁺-SPE microspheres were able to capture the phospho-containing glycans. The enrichment method was developed and optimized using model oligosaccharides (maltoheptaose DP7 and sialylated glycan A1) and also glycans from a standard glycoprotein (asialofetuin, ASF). This method experimentally showed high derivatization efficiency (almost 100%), excellent selectivity (analyzing DP7 in the digests of bovine serum albumin at a mass ratio of 1:100), high enriching recovery (90%), good reproducibility (CV<15%) as well as high sensitivity (LOD at fmol level). At last, the proposed method was successfully applied in the profiling of N-glycome in human serum, in which a total of 31 N-glycan masses were identified.     

Hydrophobic Derivatization of <Emphasis Type="Italic">N</Emphasis>-linked Glycans for Increased Ion Abundance in Electrospray Ionization Mass Spectrometry

A library of neutral, hydrophobic reagents was synthesized for use as derivatizing agents in order to increase the ion abundance of N-linked glycans in electrospray ionization mass spectrometry (ESI MS). The glycans are derivatized via hydrazone formation and are shown to increase the ion abundance of a glycan standard more than 4-fold. Additionally, the data show that the systematic addition of hydrophobic surface area to the reagent increases the glycan ion abundance, a property that can be further exploited in the analysis of glycans. The results of this study will direct the future synthesis of hydrophobic reagents for glycan analysis using the correlation between hydrophobicity and theoretical non-polar surface area calculation to facilitate the development of an optimum tag for glycan derivatization. The compatibility and advantages of this method are demonstrated by cleaving and derivatizing N-linked glycans from human plasma proteins. The ESI-MS signal for the tagged glycans are shown to be significantly more abundant, and the detection of negatively charged sialylated glycans is enhanced.     

Mass spectrometric analysis of the glycosphingolipid‐derived glycans from miniature pig endothelial cells and islets: identification of NeuGc epitope in pig islets

Glycosphingolipid (GSL) is a major component of the plasma membrane in eukaryotic cells that is involved directly in a variety of immunological events via cell‐to‐cell or cell‐to‐protein interactions. In this study, qualitative and quantitative analyses of GSL‐derived glycans on endothelial cells and islets from a miniature pig were performed and their glycosylation patterns were compared. A total of 60 and 47 sialylated and neutral GSL‐derived glycans from the endothelial cells and islets, respectively, were characterized by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and collision‐induced fragmentation using positive‐ion electrospray ionization (ESI) ion‐trap tandem mass spectrometry (MS/MS). In accordance with previous immunohistochemistry studies, the α‐Gal‐terminated GSL was not detected but NeuGc‐terminated GSLs were newly detected from miniature pig islets. In addition, the neutral GSL‐derived glycans were relatively quantified by derivatization with carboxymethyl trimethylammonium hydrazide (so called Girard's T reagent) and MALDI‐TOF MS. The structural information of the GSL‐derived glycans from pig endothelial cells and islets suggests that special attention should be paid to all types of glycoconjugates expressed on pig tissues or cells for successful clinical xenotransplantation. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of<Emphasis Type="Italic"> N</Emphasis>-glycosylation sites of the murine neural cell adhesion molecule NCAM by MALDI-TOF and MALDI-FTICR mass spectrometry

Mass spectrometry has been shown in recent years to be a powerful tool to determine accurate molecular masses and sequences of peptides and proteins and post-translational modifications such as glycosylation, phosphorylation, and sulfation. For glycosylation, it has been increasingly recognized to be of pivotal importance to identify whether potential glycosylation sites are actually modified by glycans, because functions of proteins may be modulated or depend on the presence of glycans at specific sites. Several recent reports have established that mass spectrometric techniques such as matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry (MALDI-TOF or ESI-MS, respectively) with or without preceding HPLC and in combination with PNGase F treatment are suited to analyze whether consensus sequences for N-glycosylation are glycosylated or not. Here we report the mass spectrometric analysis of the six potential N-glycosylation sites of the neural cell adhesion molecule NCAM from adult mouse brain. Unmodified peptides and glycopeptides each carrying a single glycosylation site were generated from NCAM by AspN and trypsin treatment and submitted to reversed-phase HPLC with or without prior enzymatic release of N-glycans. The resulting peptides were analyzed by MALDI-TOF-MS. In addition, high-resolution Fourier transform–ion cyclotron resonance (MALDI-FTICR) mass spectrometry was performed after in-gel deglycosylation and subsequent trypsin digestion. By using these procedures all six consensus sequences were shown to be glycosylated; the observation of an unmodified peptide with the consensus sequence N-1 indicates only partial glycosylation at this site.Abbreviations amu atomic mass units - AspN endoproteinase AspN - CAM cell adhesion molecule - ESI electrospray ionization - FTICR Fourier transform–ion cyclotron resonance - IgSF immunoglobulin superfamily - MALDI-TOF matrix-assisted laser desorption ionization–time of flight - MS mass spectrometry - NCAM neural cell adhesion molecule - PNGase F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase - PSA polysialic acid - TFA trifluoroacetic acid     

Gas chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry in quantifying fatty acids

This critical overview covers current analytical methods and future developments in quantitative determination of fatty acids (FAs), emphasizing sample extraction, derivatization and instrumental analysis with gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS²). We compare the benefits and the drawbacks of these two analytical techniques.We consider the well-established GC/MS method with pre-derivatization to be a traditional technique in terms of highly standardized sample-preparation procedures, affordability and readily available library searching for compound identification. However, the complicated derivatization steps required prior to instrumental analysis with GC/MS take a long time, with loss and transformation of FAs, low recovery and poor reproducibility.HPLC/MS² without derivatization shows the benefits of simple, mild sample-processing conditions, satisfactory recovery, short running time and high selectivity and sensitivity, which may allow it to become a viable alternative to GC/MS for the analysis of FAs in the years ahead.     

An efficient synthesis of a novel analog of octreotide with an unnatural l-lysine-like tetrazolyl amino acid

A new cyclic octreotide-like octapeptide was prepared by incorporation of an unnatural tetrazolyl amino acid, an analog of Fmoc-l-lysine, into the peptide chain. The new tetrazolyl amino acid, (S)-2-{[2-(9H-fluoren-9-yl)acetoxy]amino}-6-(1H-tetrazol-1-yl)hexanoic acid, was obtained by azidation of Fmoc-l-lysine trifluoroacetate with sodium azide in the presence of triethyl orthoformate in glacial acetic acid. The linear peptide sequence was prepared using efficient Fmoc/t-Bu solid-phase peptide synthesis (SPPS). Cyclization of the octapeptide was carried out via oxidation with iodine. The structure and purity of the cyclic octapeptide were confirmed by LC–MS, MALDI-TOF MS/MS analysis as well as 1D/2D ¹H and ¹³C NMR spectroscopy.     

Ion Mobility Mass Spectrometry for Extracting Spectra <Emphasis Type="Italic">of N</Emphasis>-Glycans Directly from Incubation Mixtures Following Glycan Release: Application to Glycans from Engineered Glycoforms of Intact,Folded HIV gp120

The analysis of glycosylation from native biological sources is often frustrated by the low abundances of available material. Here, ion mobility combined with electrospray ionization mass spectrometry have been used to extract the spectra of N-glycans released with PNGase F from a serial titration of recombinantly expressed envelope glycoprotein, gp120, from the human immunodeficiency virus (HIV). Analysis was also performed on gp120 expressed in the α-mannosidase inhibitor, and in a matched mammalian cell line deficient in GlcNAc transferase I. Without ion mobility separation, ESI spectra frequently contained no observable ions from the glycans whereas ions from other compounds such as detergents and residual buffer salts were abundant. After ion mobility separation on a Waters T-wave ion mobility mass spectrometer, the N-glycans fell into a unique region of the ion mobility/m/z plot allowing their profiles to be extracted with good signal:noise ratios. This method allowed N-glycan profiles to be extracted from crude incubation mixtures with no clean-up even in the presence of surfactants such as NP40. Furthermore, this technique allowed clear profiles to be obtained from sub-microgram amounts of glycoprotein. Glycan profiles were similar to those generated by MALDI-TOF MS although they were more susceptible to double charging and fragmentation. Structural analysis could be accomplished by MS/MS experiments in either positive or negative ion mode but negative ion mode gave the most informative spectra and provided a reliable approach to the analysis of glycans from small amounts of glycoprotein.     

Application of negative ion MS/MS to the identification of N-glycans released from carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1)

Structures of N-glycans released from rat CEACAM1 expressed in human embryonic kidney cells were determined by MALDI and negative ion nanospray MS/MS techniques. The major carbohydrates were bi-, tri- and tetra-antennary complex glycans with and without sialic acid, fucose and bisecting GlcNAc residues. High-mannose glycans, predominantly Man(5)GlcNAc(2), were also found. The negative ion fragmentation technique easily identified the branching pattern of the triantennary glycans (mainly branched on the 6-antenna) and the presence of 'bisecting' GlcNAc residues (attached to the 4-position of the core mannose), features that are difficult to determine by traditional techniques. Sialic acids were in both alpha2-3 and alpha2-6 linkage as determined by MALDI-TOF MS following linkage-specific derivatization.     

Compositional monosaccharide analysis of transgenic corn glycoproteins by HPLC with fluorescence detection and LC-MS with sonic spray ionization

Transgenic corn offers an attractive, cost-effective means for the large-scale production of engineered glycoproteins suitable for pharmaceutical purposes. A glycoprotein expressed in transgenic corn theoretically should not contain glycans because glycosylation sites have been genetically altered. A sensitive and reliable analytical method is developed to investigate this particular protein for the presence of glycans by monitoring the monosaccharide composition. Identification and quantitation of low-level monosaccharides in the glycoprotein hydrolyzate are accomplished by derivatization prior to high-performance liquid chromatography (HPLC)-fluorescence and liquid chromatography (LC)-sonic spray ionization (SSI)-mass spectrometry (MS) analyses. LC-SSI-MS is used to confirm the results from HPLC-fluorescence analysis and to positively identify the compositional monosaccharides. Glucosamine, glucose, mannose, arabinose, xylose, and sialic acid are found in the transgenic corn derived glycoprotein at less than one moiety per protein, which indicates heterogeneity of the particular glycoprotein. In addition to the compositional analysis of low-level monosaccharides in glycoprotein by HPLC-fluorescence, the utility of SSI for the LC-MS analysis of derivatized monosaccharides is demonstrated in this paper.     

N-Glycosylamine-mediated isotope labeling for mass spectrometry-based quantitative analysis of N-linked glycans

N-Linked glycosylation is a major protein modification involved in many essential cellular functions. Methods capable of quantitative glycan analysis are highly valuable and have been actively pursued. Here we describe a novel N-glycosylamine-based strategy for isotopic labeling of N-linked glycans for quantitative analysis by use of mass spectrometry (MS). This strategy relies on the primary amine group on the reducing end of freshly released N-linked glycans for labeling, and eliminates the need for the harsh labeling reaction conditions and/or tedious cleanup procedures required by existing methods. By using NHS-ester amine chemistry we used this strategy to label N-linked glycans from a monoclonal antibody with commercially available tandem mass tags (TMT). Only duplex experiments can be performed with currently available TMT reagents, because quantification is based on the intensity of intact labeled glycans. Under mild reaction conditions, greater than 95 % derivatization was achieved in 30 min and the labeled glycans, when kept at ?20 °C, were stable for more than 10 days. By performing glycan release, TMT labeling, and LC–MS analysis continuously in a single volatile aqueous buffer without cleanup steps, we were able to complete the entire analysis in less than 2 h. Quantification was highly accurate and the dynamic range was large. Compared with previously established methods, N-glycosylamine-mediated labeling has the advantages of experimental simplicity, efficient labeling, and preserving glycan integrity.

LC-MS/MS Peptide Mapping with Automated Data Processing for Routine Profiling of N-Glycans in Immunoglobulins

Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.

Quantification of monosaccharides through multiple‐reaction monitoring liquid chromatography/mass spectrometry using an aminopropyl column

A simple, sensitive, and reproducible quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was designed for the simultaneous quantification of monosaccharides derived from glycoprotein and blood serum using a multiple‐reaction monitoring (MRM) approach. Sialic acids and neutral monosaccharides were efficiently separated using an amino‐bonded silica phase column. Neutral monosaccharide molecules were detected as their aldol acetate anion adducts [M + CH₃CO₂]^? using electrospray ionization in negative ion MRM mode, while sialic acids were detected as deprotonated ions [M–H]^?. The new method did not require a reduction step, and exhibited very high sensitivity to carbohydrates with limits of detection of 1 pg for the sugars studied. The linearity of the described approach spanned over three orders of magnitude (pg to ng). The method was validated for monosaccharides originating from N‐linked glycans attached to glycoproteins and glycoproteins found in human blood serum. The method effectively quantified monosaccharides originating from as little as 1 µg of glycoprotein and 5 µL of blood serum. The method was robust, reproducible, and highly sensitive. It did not require reduction, derivatization or postcolumn addition of reagents. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of Minor N-linked Glycans on Antibodies Using Endo H Release and MALDI–Mass Spectrometry

Abstract

A MALDI mass spectrometry method using Bruker Daltonic's LIFT technology for MS/MS analysis has been developed for profiling and characterizing low abundant N-glycans from recombinant immunoglobulin G (IgG) antibodies. In this method, Endoglycosidase H (Endo H) released N-glycans are derivatized at their reducing end with 2-aminobenzamide (2-AB) and separated by normal phase chromatography. Endo H hydrolyses the bond between the two GlcNAc residues of the trimannosyl core of high mannose and hybrid N-linked glycans, leaving the core GlcNAc attached to the protein. High mannose and hybrid type N-glycans are released from the glycoprotein whereas the more abundant, complex biantennary type oligosaccharide structures are unaffected. Analysis of Endo H treated glycan moieties by MALDI mass spectrometry identified several minor species of high mannose and hybrid type glycans. Subsequent MALDI TOF MS/MS analysis of the resulting products yielded information about structural features of the high mannose and hybrid type glycans. This study involving Endo H treatment followed by MALDI mass spectrometry coupled with LIFT technology for MS/MS analysis offers a specific and sensitive technique for visualizing, and characterizing minor glycan species.     

LC-MS/MS biopharmaceutical glycoanalysis: identification of desirable reference material characteristics

Glycosylation, the enzymatic addition of carbohydrates to a protein, is one of the most abundant post-translational modifications found in nature. There is variability in the number, location, and identity of glycans attached. As a result, a glycoprotein consists of a number of glycoforms with different combinations of glycans, potentially resulting in different stability, toxicity, and activity. This is especially important in the biopharmaceutical industry where product consistency and safety are vital. Glycoprotein analysis involves numerous mass spectrometry based techniques, each of which provides various aspects of characterization. The current paper describes two commonly used analytical techniques for glycoprotein characterization. In one experiment, nonspecific proteolysis is combined with a two-tiered mass spectrometry approach (MALDI-TOF and LC-MS/MS) to gain glycosylation site and glycan identity. In a second approach, glycans were enzymatically released, labeled with a fluorescent dye, and analyzed using LC-Fluorescence-MS/MS to give glycan identification and relative quantification. The type and degree of information yielded by each method is assessed in an effort to identify desired reference material characteristics for improving biopharmaceutical glycoanalysis.     

Glycan analysis: scope and limitations of different techniques—a case for integrated use of LC-MS(/MS) and NMR techniques

The structure of glycans from glycoproteins is highly relevant for their function. We tightly integrate liquid chromatography–mass spectrometry (LC-MS), MS/MS, and nuclear magnetic resonance (NMR) data to achieve a complete characterization of even isobaric glycans differing in only one linkage position or in the substitution in one branch. As example, we analyzed ten desialylated underivatized glycans from bovine fibrinogen. The molecules were separated on a PGC column, and LC-MS data allowed an assignment of the compositions of the glycans. MS/MS data of the same glycans allowed elucidation of sequence and to some extent of branching and linkage. All MS/MS fragmentation methods led to multiple dissociations, resulting in several cases in ambiguous data. The MS/MS data were interpreted both by scientists and automatically by software, and the differential results are compared. Additional data from a tight integration of LC-MS and NMR data resulted in a complete structural characterization of the glycans. The acquisition of simple 1D ¹H NMR data led—in combination with LC-MS and MS/MS data—to an unambiguous assignment of the isobaric glycans. Compounds that were not separated in the chromatography could easily be assigned structurally by applying the 3D cross-correlation (3DCC) technology to arrive at NMR spectra of the pure components—without actually separating them. By applying LC-MS, MS/MS, 1D ¹H NMR, and 3DCC together, one can assign glycan structures from glycoconjugates with high confidence affording only 200 pmol of glycan material.     

Quantitative mapping of glycoprotein micro‐heterogeneity and macro‐heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro‐heterogeneity) and evaluate the molar site occupancy (macro‐heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N‐glycans was chemically synthesised by solid‐phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N‐acetylglucosamine‐linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well‐defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI‐IT, ESI‐Q‐TOF, MALDI‐TOF, ESI/MALDI‐FT‐ICR‐MS). Depending on the ion source/mass analyser, glycopeptides carrying complex‐type N‐glycans exhibited clearly lower signal strengths (10–50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano‐ESI and medium‐pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro‐heterogeneity and macro‐heterogeneity by label‐free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitation of the glutathione in human peripheral blood by matrix-assisted laser desorption ionization time-of-flight mass spectrometry coupled with micro-scale derivatization

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been broadly applied to analyze high-molecular-weight compound (such as polymer or proteomic research) but seldom used for low-molecular-weight compound analysis. The objective of this study is the development of a simple analytical method for the determination of the concentration of tripeptide glutathione (GSH) by MALDI-TOF MS. Unfortunately, GSH could not be detected directly by MALDI-TOF MS. Our method is based on the derivatization of GSH with 4-bromomethyl-6,7-dimethoxycoumarin (BrDMC) in acetonitrile using potassium hydroxide (KOH) as a base catalyst. After simple extraction step, the supernatant is spotted on a target plate, mixed with matrix α-cyano-4-hydroxycinnamic acid (CHCA) and then detected by MALDI-TOF MS.Some parameters affecting the derivatization of GSH were investigated, such as the concentration of BrDMC, KOH, different base catalyst, and reaction time, etc. The regression equations of GSH derivative possessed good linearity (r ≧ 0.995) over the range of 1.0–100.0 μM. The relative standard deviation (R.S.D.) and relative error (R.E.) values in intra- and inter-day assays were below 13%, which showed good precision and accuracy. This proposed method was successfully applied to monitor the concentration of GSH in human blood at micro-scale level.     

Mass spectrometric study of N‐glycans from serum of woodchucks with liver cancer

Woodchucks have been a preferred lab animal model of chronic hepatitis B viral infection. The model recapitulates the disease progression of HBV infection to hepatocellular carcinoma (HCC) and has documented similarities in protein glycosylation with human HCC. This study examined N‐glycans in serum of animals with(out) HCC. Oligosaccharides were released enzymatically using PNGaseF from total serum or from serum partially fractionated by extraction. Two different extraction procedures – reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and solid‐phase extraction (SPE) on a cation‐exchange/reversed‐phase STRATA‐XC cartridge – were used with the purpose of confirming glycosylation profiles. Oligosaccharides were analyzed by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) after derivatization with phenylhydrazine and/or permethylation. Characteristic fragment ions produced under MS/MS conditions allowed discrimination between isomeric structures of oligosaccharides, including those sialylated with two types of acidic residues. The complementary methods allowed structural characterization of oligosaccharides from various N‐glycan classes. Furthermore, to validate results, glycosylation profiles of woodchuck sera were compared to glycans obtained from mouse serum on the same conditions. In summary, we have identified 40 N‐glycan structures in the serum of woodchucks and some types of oligosaccharide structures appeared to increase in HCC samples following protease digest. The study provides improved tools for the characterization of N‐glycans from total serum in the progression of liver disease. Copyright © 2009 John Wiley & Sons, Ltd.