Rapid glycopeptide enrichment and N-glycosylation site mapping strategies based on amine-functionalized magnetic nanoparticles

Glycoproteins secreted or expressed on the cell surface at specific pathophysiological stages are well-recognized disease biomarkers and therapeutic targets. While mapping of specific glycan structures can be performed at the level of released glycans, site-specific glycosylation and identification of specific protein carriers can only be determined by analysis of glycopeptides. A key enabling step in mass spectrometry (MS)-based glycoproteomics is the ability to selectively or non-selectively enrich for the glycopeptides from a total pool of a digested proteome for MS analysis since the highly heterogeneous glycopeptides are usually present at low abundance and ionize poorly compared with non-glycosylated peptides. Among the most common approaches for non-destructive and non-glycan-selective glycopeptide enrichment are strategies based on various forms of hydrophilic interaction liquid chromatography (HILIC). We present here a variation of this method using amine-derivatized Fe₃O₄ nanoparticles, in concert with in situ peptide N-glycosidase F digestion for direct matrix-assisted laser desorption/ionization–mass spectrometry analysis of N-glycosylation sites and the released glycans. Conditions were also optimized for efficient elution of the enriched glycopeptides from the nanoparticles for on-line nanoflow liquid chromatography–MS/MS analysis. Successful applications to single glycoproteins as well as total proteomic mixtures derived from biological fluids established the unrivaled practical versatility of this method, with enrichment efficiency comparable to other HILIC-based methods.     

Comparison of HPLC/ESI-FTICR MS versus MALDI-TOF/TOF MS for glycopeptide analysis of a highly glycosylated HIV envelope glycoprotein

Defining the structures and locations of the glycans attached on secreted proteins and virus envelope proteins is important in understanding how glycosylation affects their biological properties. Glycopeptide mass spectrometry (MS)-based analysis is a very powerful, emerging approach to characterize glycoproteins, in which glycosylation sites and the corresponding glycan structures are elucidated in a single MS experiment. However, to date there is not a consensus regarding which mass spectrometric platform provides the best glycosylation coverage information. Herein, we employ two of the most widely used MS approaches, online high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI-MS) and offline HPLC followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), to determine which of the two approaches provides the best glycosylation coverage information of a complex glycoprotein, the group M consensus HIV-1 envelope, CON-S gp140DeltaCFI, which has 31 potential glycosylation sites. Our results highlight differences in the informational content obtained between the two methods such as the overall number of glycosylation sites detected, the numbers of N-linked glycans present at each site, and the type of confirmatory information obtained about the glycopeptide using MS/MS experiments. The two approaches are quite complementary, both in their coverage of glycopeptides and in the information they provide in MS/MS experiments. The information in this study contributes to the field of mass spectrometry by demonstrating the strengths and limitations of two widely used MS platforms in glycoprotein analysis.     

Zirconia layer coated mesoporous silica microspheres as HILIC SPE materials for selective glycopeptide enrichment

Characterization of protein glycosylation requires highly specific methods for the enrichment of glycopeptides because of their sub-stoichiometric glycosylation-site occupancy. The hydrophilic affinity based strategy has attracted more attention, owing to its broad glycan specificity, good reproducibility, and compatibility with mass spectrometric (MS) analysis. Several polar matrices have emerged for hydrophilic interaction chromatography (HILIC) approaches, including sepharose, cellulose, ZIC-HILIC and titania. Here, we present the solid-phase extraction (SPE) utility of zirconia coated mesoporous silica (ZrO(2)/MPS) microspheres for glycopeptide isolation prior to MS analysis. The high specificity of this SPE approach was demonstrated by the enrichment of glycopeptides from the digests of model glycoproteins in HILIC mode. ZrO(2)/MPS microspheres show superior selectivity and glycosylation heterogeneity coverage for glycopeptide enrichment to conventional sepharose. Furthermore, digested mixtures of the phosphoprotein α-casein and IgG were also treated with ZrO(2)/MPS HILIC SPE materials, which exhibited that glycopeptides could be effectively enriched with interference from phosphorylated peptides.     

Rapid and efficient glycoprotein identification through microwave-assisted enzymatic digestion

Identification of protein glycosylation sites is analytically challenging due to the diverse glycan structures associated with a glycoprotein. Mass spectrometry (MS)-based identification and characterization of glycoproteins has been achieved predominantly with the bottom-up approach, which typically involves the enzymatic cleavage of proteins to peptides prior to LC/MS or LC/MS/MS analysis. However, the process can be challenging due to the structural variations and steric hindrance imposed by the attached glycans. Alternatives to conventional heating protocols, that increase the rate of enzymatic cleavage of glycoproteins, may aid in addressing these challenges. An enzymatic digestion of a glycoprotein can be accelerated and made more efficient through microwave-assisted digestion. In this paper, a systematic study was conducted to explore the efficiency of microwave-assisted enzymatic (trypsin) digestion (MAED) of glycoproteins as compared with the conventional method. In addition, the optimum experimental parameters for the digestion such as temperature, reaction time, and microwave radiation power were investigated. It was determined that efficient tryptic digestion of glycoproteins was attained in 15 min, allowing comparable if not better sequence coverage through LC/MS/MS analysis. Optimum tryptic cleavage was achieved at 45°C irrespective of the size and complexity of the glycoprotein. Moreover, MAED allowed the detection and identification of more peptides and subsequently higher sequence coverage for all model glycoprotein. MAED also did not appear to prompt a loss or partial cleavage of the glycan moieties attached to the peptide backbones.     

HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals

In contrast with conventional drugs, biopharmaceuticals are highly complex molecules with remarkable heterogeneity. Protein glycosylation is an inherent source of this heterogeneity and also affects the safety, efficacy, and serum half-life of therapeutic glycoproteins. Therefore analysis of the glycan pattern is an important issue for characterization and quality control in the biopharmaceutical industry. In this publication we describe a complete workflow for the analysis of protein N-glycans. The sample-preparation procedure, consisting of the release of the N-glycans by PNGase-F, followed by fluorescence labeling with 2-aminobenzamide and removal of excess label, was optimized to avoid alteration of the glycan sample. Subsequently, labeled glycans were analyzed by hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection. The developed method was validated for analysis of antibody N-glycans. To demonstrate the accuracy of the method an antibody sample was additionally analyzed by an orthogonal method. The antibody was digested with lysyl endopeptidase and the (glyco-)peptides were analyzed by RP-HPLC–MS. The consistency of the results between these two methods demonstrates the reliability of the glycan analysis method introduced herein.     

Site-specific protein glycosylation analysis with glycan isomer differentiation

Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single N-glycosylation site on bovine ribonuclease B, 59 distinct glycans at five N-glycosylation sites on bovine lactoferrin, 13 distinct glycans at one N-glycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five O-glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis.     

Glycoform characterization of intact erythropoietin by capillary electrophoresis-electrospray-time of flight-mass spectrometry

Glycosylated proteins often show a large variation in their glycosylation pattern, complicating their structural characterization. In this paper, we present a method for the accurate mass determination of intact isomeric glycoproteins based on capillary electrophoresis-electrospray-time of flight-mass spectrometry. Human recombinant erythropoietin has been chosen as a showcase. The approach enables the on-line removal of nonglycosylated proteins, salts, and neutral and negatively charged species. More important, different glycosylation forms are separated both on the base of differences in the number of negatively charged sialic acid residues and the size of the glycans. Thus, 44 glycoforms and in total about 135 isoforms of recombinant human erythropoietin, taking also acetylation into account, could be distinguished for the reference material from the European Pharmacopeia. Distinct glycosylation differences for samples from different suppliers are clearly observed. Based on the accurate mass an overall composition of each single isoform is proposed, perfectly in agreement with data on glycan and glycopeptide analysis. This method is an ideal complement to the established techniques for glycopeptide and glycan analysis, not differentiating branching or linkage isoforms, but leading to an overall composition of the glycoprotein. The presented strategy is expected to improve significantly the ability to characterize and quantify isomeric glycoforms for a large variety of glycoproteins.     

Site-specific N-glycosylation analysis: matrix-assisted laser desorption/ionization quadrupole-quadrupole time-of-flight tandem mass spectral signatures for recognition and identification of glycopeptides

The identification of glycosylation sites in proteins is often possible through a combination of proteolytic digestion, separation, mass spectrometry (MS) and tandem MS (MS/MS). Liquid chromatography (LC) in combination with MS/MS has been a reliable method for detecting glycopeptides in digestion mixtures, and for assigning glycosylation sites and glycopeptide sequences. Direct interfacing of LC with MS relies on electrospray ionization, which produces ions with two, three or four charges for most proteolytic peptides and glycopeptides. MS/MS spectra of such glycopeptide ions often lead to ambiguous interpretation if deconvolution to the singly charged level is not used. In contrast, the matrix-assisted laser desorption/ionization (MALDI) technique usually produces singly charged peptide and glycopeptide ions. These ions require an extended m/z range, as provided by the quadrupole-quadrupole time-of-flight (QqTOF) instrument used in these experiments, but the main advantages of studying singly charged ions are the simplicity and consistency of the MS/MS spectra. A first aim of the present study is to develop methods to recognize and use glycopeptide [M+H]+ ions as precursors for MS/MS, and thus for glycopeptide/glycoprotein identification as part of wider proteomics studies. Secondly, this article aims at demonstrating the usefulness of MALDI-MS/MS spectra of N-glycopeptides. Mixtures of diverse types of proteins, obtained commercially, were prepared and subjected to reduction, alkylation and tryptic digestion. Micro-column reversed-phase separation allowed deposition of several fractions on MALDI plates, followed by MS and MS/MS analysis of all peptides. Glycopeptide fractions were identified after MS by their specific m/z spacing patterns (162, 203, 291 u) between glycoforms, and then analyzed by MS/MS. In most cases, MS/MS spectra of [M+H]+ ions of glycopeptides featured peaks useful for determining sugar composition, peptide sequence, and thus probable glycosylation site. Peptide-related product ions could be used in database search procedures and allowed the identification of the glycoproteins.     

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.

Reliable Determination of Site-Specific In Vivo Protein N-Glycosylation Based on Collision-Induced MS/MS and Chromatographic Retention Time

Site-specific glycopeptide mapping for simultaneous glycan and peptide characterization by MS is difficult because of the heterogeneity and diversity of glycosylation in proteins and the lack of complete fragmentation information for either peptides or glycans with current fragmentation technologies. Indeed, multiple peptide and glycan combinations can readily match the same mass of glycopeptides even with mass errors less than 5 ppm providing considerably ambiguity and analysis of complex mixtures of glycopeptides becomes quite challenging in the case of large proteins. Here we report a novel strategy to reliably determine site-specific N-glycosylation mapping by combining collision-induced dissociation (CID)-only fragmentation with chromatographic retention times of glycopeptides. This approach leverages an experimental pipeline with parallel analysis of glyco- and deglycopeptides. As the test case we chose ABCA4, a large integral membrane protein with 16 predicted sites for N-glycosylation. Taking advantage of CID features such as high scan speed and high intensity of fragment ions together combined with the retention times of glycopeptides to conclusively identify the non-glycolytic peptide from which the glycopeptide was derived, we obtained virtually complete information about glycan compositions and peptide sequences, as well as the N-glycosylation site occupancy and relative abundances of each glycoform at specific sites for ABCA4. The challenges provided by this example provide guidance in analyzing complex relatively pure glycoproteins and potentially even more complex glycoprotein mixtures.

Analysis of glycans derived from glycoconjugates by capillary electrophoresis-mass spectrometry

The high structural variation of glycan derived from glycoconjugates, which substantially increases with the molecular size of a protein, contributes to the complexity of glycosylation patterns commonly associated with glycoconjugates. In the case of glycoproteins, such variation originates from the multiple glycosylation sites of proteins and the number of glycan structures associated with each site (microheterogeneity). The ability to comprehensively characterize highly complex mixture of glycans has been analytically stimulating and challenging. Although the most powerful MS and MS/MS techniques are capable of providing a wealth of structural information, they are still not able to readily identify isomeric glycan structures without high-order MS/MS (MS(n) ). The analysis of isomeric glycan structures has been attained using several separation methods, including high-pH anion-exchange chromatography, hydrophilic interaction chromatography and GC. However, CE and microfluidics CE (MCE) offer high separation efficiency and resolutions, allowing the separation of closely related glycan structures. Therefore, interfacing CE and MCE to MS is a powerful analytical approach, allowing potentially comprehensive and sensitive analysis of complex glycan samples. This review describes and discusses the utility of different CE and MCE approaches in the structural characterization of glycoproteins and the feasibility of interfacing these approaches to MS.     

A rapid sample preparation method for mass spectrometric characterization of N-linked glycans

A rapid method for analysis of glycans of glycoproteins is presented. This method comprised deglycosylation, sample cleanup and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of glycans. The enzymatic deglycosylation of N-linked glycoproteins was enhanced in terms of speed and reproducibility using an enzyme-friendly surfactant. The released glycans were desalted using a micro-scale solid phase extraction (SPE) device packed with a hydrophilic interaction chromatography (HILIC) sorbent. Hydrophilic glycans were well retained by SPE, while salts and surfactants were removed from the sample. The glycans were eluted using 25-50 microL of solvent and analyzed directly without derivatization using MALDI-MS. MALDI quadrupole time-of-flight (Q-Tof) instrumentation was utilized for glycan profiling and structure characterization by tandem mass spectrometry (MS/MS). The presented method allows sensitive analysis of glycans benefiting from optimized deglycosylation reactions and efficient sample cleanup.     

Improvement of mass spectrometry analysis of glycoproteins by MALDI-MS using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid

Protein glycosylation analysis is important for elucidating protein function and molecular mechanisms in various biological processes. We previously developed a glycan analysis method using a 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix (3-AQ/CHCA LM) and applied it to the quantitative glycan profiling of glycoproteins. However, information concerning glycosylation sites is lost; glycopeptide analysis is therefore required to identify the glycosylation sites in glycoproteins. Human epidermal growth factor receptor 2 (HER2) is a glycoprotein that plays a role in the regulation of cell proliferation, differentiation, and migration. Several reports have described the structure of HER2, but the structures of N-glycans attached to this protein remain to be fully elucidated. In this study, 3-AQ/CHCA LM was applied to tryptic digests of HER2 to reveal its N-glycosylation state and to evaluate the utility of this LM in characterizing glycopeptides. Peptide sequence coverage was considerably improved compared to analysis of HER2 using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. Most of the peaks observed using only this LM were localized at the inner or outer regions of sample spots. Furthermore, five of the peptide peaks that were enriched within the inner region were confirmed to be glycosylated by MS/MS analysis. Three glycosylation sites were identified and their glycan structures were elucidated. The reduction in sample complexity by on-target separation allowed for higher sequence coverage, resulting in effective detection and characterization of glycopeptides. In conclusion, these results demonstrate that MS-based glycoprotein analysis using 3-AQ/CHCA is an effective method to identify glycosylation sites in proteins and to elucidate the glycan structures of glycoproteins in complex samples.     

Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS

Site-specific characterisation of mucin-type O-linked glycosylation is an analytical challenge due to glycan heterogeneity, lack of glycosylation site consensus sequence and high density of occupied glycosylation sites. Here, we report the use of electron transfer dissociation (ETD) for the site-specific characterisation of densely glycosylated mucin-type O-linked glycopeptides using ESI-IT-MS/MS. Synthetic glycopeptides from the human mucin-1 (MUC-1) tandem repeat region containing a range of O-linked, tumour-associated carbohydrate antigens, namely Tn, T and sialyl T, with different glycosylation site occupancies and an increasing number of tandem repeats were studied. In addition, a glycopeptide from the anti-freeze glycoprotein of Antarctic and Arctic notothenoids, bearing four O-linked, per-acetylated T antigens was characterised. ETD MS/MS of infused or capillary LC-separated glycopeptides provided broad peptide sequence coverage (c/z·-type fragment ions) with intact glycans still attached to the Ser/Thr residues. Thus, the glycosylation sites were unambiguously determined, while simultaneously obtaining information about the attached glycan mass and peptide identity. Highly sialylated O-glycopeptides showed less efficient peptide fragmentation, but some sequence and glycosylation site information was still obtained. This study demonstrates the capabilities of ETD MS/MS for site-specific characterisation of mucin-type glycopeptides containing high-density O-linked glycan clusters, using accessible and relative low-resolution/low-mass accuracy IT MS instrumentation.     

Mass spectrometry in the detection and diagnosis of congenital disorders of glycosylation

Mass spectrometry (MS) of large molecules such as proteins and oligosaccharides has not been employed in clinical practices, while that of small metabolites is widely used for the screening and diagnosis of various congenital diseases. Congenital disorders of glycosylation (CDG) is a newly recognized group of diseases derived from defects in the biosynthetic pathway of protein glycosylation and the patients are never decisively diagnosed unless the glycoprotein molecules are analyzed. We have constructed a diagnostic system where MS of glycoproteins and glycopeptides identifies abnormalities in their glycan moieties. This program is anticipated to reveal the prevalence of CDG and to demonstrate the essential role of MS in the emerging field of medicine, disease glycomics and glycoproteomics.     

Poly(amidoamine) dendrimer‐coated magnetic nanoparticles for the fast purification and selective enrichment of glycopeptides and glycans

Glycosylated proteins modulate various important functions of organisms. To reveal the functions of glycoproteins, in‐depth characterization studies are necessary. Although mass spectrometry is a very efficient tool for glycoproteomic and glycomic studies, efficient sample preparation methods are required prior to analyses. In the study, poly(amidoamine) dendrimer‐coated magnetic nanoparticles were presented for the specific enrichment and fast purification of glycopeptides and glycans. The enrichment and purification performance of the developed method was evaluated both at the glycopeptide, and the glycan level using several standard glycoprotein digests and released glycan samples. The poly(amidoamine) dendrimer‐coated magnetic nanoparticles not only showed selective affinity (Immunoglobulin G/Bovine Serum Albumin, 1/10 by weight) to glycopeptides and released glycans but also good sensitivity (0.4 ng/µL for Immunoglobulin G) for glycoproteomic and glycomic applications. Thirty‐five glycopeptides of Immunoglobulin G were detected after enrichment with poly(amidoamine) dendrimer‐coated magnetic nanoparticles. In addition, 55 ¹⁸O tagged deamidated glycopeptides belonging to human plasma glycoproteome were confirmed. Finally, fifty 2‐aminobenzoic acid, and 30 procainamide‐labelled human plasma N‐glycans released from human plasma glycoproteins were determined after purifications. The results indicate that the proposed enrichment and purification method using poly(amidoamine) dendrimer‐coated magnetic nanoparticles could be simply adjusted to sample preparation methods.     

Mass spectrometry and the emerging field of glycomics

The biological significance of protein and lipid glycosylation is well established. For example, cells respond to environmental stimuli by altering glycan structures on their surfaces, and cancer cells evade normal growth regulation in part by remodeling their surface glycans. In general, glycan chemical properties differ significantly from those of proteins, lipids, nucleic acids, and small molecule metabolites. Thus, advances in glycomics, a comprehensive study to identify all glycans in an organism, rely on the development of specialized analytical methods. Mass spectrometry (MS) is emerging as an enabling technology in the field of glycomics. This review summarizes recent developments in mass spectrometric analysis methods for protein-based glycomics and glycoproteomics workflows.     

A Unified Strategy for the Synthesis of Mucin Cores 1–4 Saccharides and the Assembled Multivalent Glycopeptides

By displaying different O‐glycans in a multivalent mode, mucin and mucin‐like glycoproteins are involved in a plethora of protein binding events. The understanding of the roles of the glycans and the identification of potential glycan binding proteins are major challenges. To enable future binding studies of mucin glycan and glycopeptide probes, a method that gives flexible and efficient access to all common mucin core‐glycosylated amino acids was developed. Based on a convergent synthesis strategy starting from a shared early stage intermediate by differentiation in the glycoside acceptor reactivity, a common disaccharide building block allows for the creation of extended glycosylated amino acids carrying the mucin type‐2 cores 1–4 saccharides. Formation of a phenyl‐sulfenyl‐N‐Troc (Troc=trichloroethoxycarbonyl) byproduct during N‐iodosuccinimide‐promoted thioglycoside couplings was further characterized and a new methodology for the removal of the Troc group is described. The obtained glycosylated 9‐fluorenylmethoxycarbonyl (Fmoc)‐protected amino acid building blocks are incorporated into peptides for multivalent glycan display.     

Characterization of the N-linked glycosylation site of recombinant pectate lyase

Recombinant pectate lyase from Aspergillus niger was overexpressed in Aspergillus nidulans. The two recombinant proteins produced differed in molecular mass by 1200 Da, which suggested that the larger molecular weight protein was glycosylated. The deduced amino acid sequence was searched for potential N-linked glycosylation sites, and one potential site was identified at residue 64. The proteins were analyzed for their ability to bind various lectins as an assay for the presence of carbohydrates. The proteins were then digested with trypsin to facilitate the isolation of the potential glycosylation site. The resulting digestion products were subsequently analyzed by liquid chromatography/mass spectrometry using in-source collision induced dissociation to detect glycopeptides. Once the glycopeptide had been identified, treatment with an endoglycosidase both verified the location of glycosylation and identified the mass of the glycan. The Complex Carbohydrate Structural Database was searched for possible N-linked structures with the same mass, and the suggested primary sequence was confirmed by an exoglycosidase digestion. The data demonstrated that the larger recombinant protein contained a high mannose N-linked structure (Man(5)GlcNAc(2)) attached to N-64, while this site was not occupied in the smaller protein.     

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.