Nonglycosylated Peptides Research Articles

Comparative study of sialyl glycoprotein with multiple glycosylation sites using isotope labeling and capillary liquid chromatography/mass spectrometry

A comparative strategy has been demonstrated using RNase B, a single-site N-linked high-mannose glycoprotein. Glycoproteins are more common with multiple glycosylation sites and with complex glycans. A strategy capable of differentiating the changes caused by glycoprotein concentration, glycosylation site occupancy, and a glycoform profile of complex glycoproteins would be beneficial. Tryptic-digested glycoproteins were labeled using 12 C,H-formaldehyde and 13 C, D-formaldehyde, purified, and then analyzed using capillary reversed-phase liquid chromatography/mass spectrometry (RPLC/MS). The relative intensity of non-glycosylated peptides provided information on glycoprotein concentration variation. A site-specific glycoform profile variation was obtained by comparing the glycoform profile of CH2 and 13 CD2 glycopeptides. Determining the protein concentration and glycoform profile variations allows the glycosylation site occupancy variation to be calculated. A strong correlation between the observed and prepared ratios for fetuin glycopeptides from 0.2 to 5 was obtained. Two fetuin samples with different glycoprotein concentrations (4-fold change), glycoform profiles (normal and modified), and glycosylation site occupancies (100% and 50%) were prepared, labeled, mixed, purified, and analyzed using RPLC/MS. The results of the comparative study had a strong correlation with the prepared values. In this report, we demonstrated a comparative analysis of fetuin, a glycoprotein with multiple glycosylation sites and complex sialyl glycans. Compared to our previous approach, we made several modifications including the use of RPLC, a larger mass difference isotope tag, and isotope overlapping correction. The modified approach is expected to be applicable to most glycoproteins. Copyright © 2013 John Wiley & Sons, Ltd.

Concurrent Automated Sequencing of the Glycan and Peptide Portions of O-Linked Glycopeptide Anions by Ultraviolet Photodissociation Mass Spectrometry

O-Glycopeptides are often acidic owing to the frequent occurrence of acidic saccharides in the glycan, rendering traditional proteomic workflows that rely on positive mode tandem mass spectrometry (MS/MS) less effective. In this report, we demonstrate the utility of negative mode ultraviolet photodissociation (UVPD) MS for the characterization of acidic O-linked glycopeptide anions. This method was evaluated for a series of singly and multiply deprotonated glycopeptides from the model glycoprotein kappa casein, resulting in production of both peptide and glycan product ions that afforded 100% sequence coverage of the peptide and glycan moieties from a single MS/MS event. The most abundant and frequent peptide sequence ions were a/x-type products which, importantly, were found to retain the labile glycan modifications. The glycan-specific ions mainly arose from glycosidic bond cleavages (B, Y, C, and Z ions) in addition to some less common cross-ring cleavages. On the basis of the UVPD fragmentation patterns, an automated database searching strategy (based on the MassMatrix algorithm) was designed that is specific for the analysis of glycopeptide anions by UVPD. This algorithm was used to identify glycopeptides from mixtures of glycosylated and nonglycosylated peptides, sequence both glycan and peptide moieties simultaneously, and pinpoint the correct site(s) of glycosylation. This methodology was applied to uncover novel site-specificity of the O-linked glycosylated OmpA/MotB from the "superbug" A. baumannii to help aid in the elucidation of the functional role that protein glycosylation plays in pathogenesis.

Identification of a family of four UDP-polypeptide N-acetylgalactosaminyl transferases in Cryptosporidium species

Although mucin-type O-glycans are critical for Cryptosporidium infection, the enzymes catalyzing their synthesis have not been studied. Here, we report four UDP N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl transferases (ppGalNAc-Ts) from the genomes of C. parvum, C. hominis and C. muris. All are Type II membrane proteins which include a cytoplasmic tail, a transmembrane domain, a stem region, a glycosyltransferase family 2 domain and a C-terminal ricin B lectin domain. All are expressed during C. parvum infection in vitro, with Cp-ppGalNAc-T1 and -T4 expressed at 24h and Cp-ppGalNAc-T2 and -T3 at 48 and 72h post-infection, suggesting that their expression may be developmentally regulated. C. parvum sporozoite lysates display ppGalNAc-T enzymatic activity against non-glycosylated and pre-glycosylated peptides suggesting that they contain enzymes capable of glycosylating both types of substrates. The importance of mucin-type O-glycans in Cryptosporidium–host cell interactions raises the possibility that Cp-ppGalNAc-Ts may serve as targets for intervention in cryptosporidiosis.

Microarray analysis of the human antibody response to synthetic Cryptosporidium glycopeptides

Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopeptides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.

The GlycoFilter: A Simple and Comprehensive Sample Preparation Platform for Proteomics, N-Glycomics and Glycosylation Site Assignment

Current strategies to study N-glycoproteins in complex samples are often discrete, focusing on either N-glycans or N-glycosites enriched by sugar-based techniques. In this study we report a simple and rapid sample preparation platform, the GlycoFilter, which allows a comprehensive characterization of N-glycans, N-glycosites, and proteins in a single workflow. Both PNGase F catalyzed de-N-glycosylation and trypsin digestions are accelerated by microwave irradiation and performed sequentially in a single spin filter. Both N-glycans and peptides (including de-N-glycosylated peptides) are separately collected by filtration. The condition to effectively collect complex and heterogeneous N-glycans was established on model glycoproteins, bovine ribonuclease B, bovine fetuin, and human serum IgG. With this platform, the N-glycome, N-glycoproteome and proteome of human urine and plasma were characterized. Overall, a total of 865 and 295 N-glycosites were identified from three pairs of urine and plasma samples, respectively. Many sites were defined unambiguously as partially occupied by the detection of their nonsugar-modified peptides (128 from urine and 61 from plasma), demonstrating that partial occupancy of N-glycosylation occurs frequently. Given the likely high prevalence and variability of partial occupancy, glycoprotein quantification based exclusively on deglycosylated peptides may lead to inaccurate quantification.

The Lectin Domain of the Polypeptide GalNAc Transferase Family of Glycosyltransferases (ppGalNAc Ts) Acts as a Switch Directing Glycopeptide Substrate Glycosylation in an N- or C-terminal Direction, Further Controlling Mucin Type O-Glycosylation

Mucin type O-glycosylation is initiated by a large family of polypeptide GalNAc transferases (ppGalNAc Ts) that add α-GalNAc to the Ser and Thr residues of peptides. Of the 20 human isoforms, all but one are composed of two globular domains linked by a short flexible linker: a catalytic domain and a ricin-like lectin carbohydrate binding domain. Presently, the roles of the catalytic and lectin domains in peptide and glycopeptide recognition and specificity remain unclear. To systematically study the role of the lectin domain in ppGalNAc T glycopeptide substrate utilization, we have developed a series of novel random glycopeptide substrates containing a single GalNAc-O-Thr residue placed near either the N or C terminus of the glycopeptide substrate. Our results reveal that the presence and N- or C-terminal placement of the GalNAc-O-Thr can be important determinants of overall catalytic activity and specificity that differ between transferase isoforms. For example, ppGalNAc T1, T2, and T14 prefer C-terminally placed GalNAc-O-Thr, whereas ppGalNAc T3 and T6 prefer N-terminally placed GalNAc-O-Thr. Several transferase isoforms, ppGalNAc T5, T13, and T16, display equally enhanced N- or C-terminal activities relative to the nonglycosylated control peptides. This N- and/or C-terminal selectivity is presumably due to weak glycopeptide binding to the lectin domain, whose orientation relative to the catalytic domain is dynamic and isoform-dependent. Such N- or C-terminal glycopeptide selectivity provides an additional level of control or fidelity for the O-glycosylation of biologically significant sites and suggests that O-glycosylation may in some instances be exquisitely controlled.

Bacterial expression, purification and angiogenesis-promoting activity of human thymosin β4

Thymosin β4 (Tβ4) is an actin-binding peptide involved in tissue regeneration and angiogenesis. This 43-amino acid peptide is chemically synthesized for research or clinical trials. To overcome the high costs of solid phase synthesis, we developed a genetic engineering procedure of Tβ4 expression in a protease-deficient host strain, Escherichia coli BL21(DE3), transformed with different expression vectors (pRSETA, pET-15b and pEcoli-Cterm6×HN). The recombinant, non-glycosylated peptide was overexpressed in soluble form and purified by two-step immobilized metal ion affinity chromatography. Use of the pET vector expression system allowed for easy removal of the polyhistidine tag by thrombin. Functional studies revealed that recombinant Tβ4 stimulated angiogenesis via activation of the endothelial proteolytic systems, inhibition of endothelial cell adhesion, promotion of migration and capillary tube formation in Matrigel, and that its activity was similar to that observed for the synthetic peptide. The presented study comprises the first evidence that recombinant Tβ4 promotes angiogenesis in an in vitro endothelial cell model.

Application of a strong anion exchange material in electrostatic repulsion–hydrophilic interaction chromatography for selective enrichment of glycopeptides

Glycoproteins are involved in various cellular activities, including inter- and extracellular signaling. However, glycopeptide signals are significantly suppressed by coeluting non-glycosylated peptides in mass spectrometry-based analysis. For detailed elucidation of the biological functions of glycoproteins, selective enrichment of glycopeptides from non-glycosylated peptides is crucial. In the present study, a SAX material, XCharge SAX, was used in a column in the ERLIC mode with the aim of specifically enriching glycopeptides. Enrichment conditions were initially optimized, and selectivity, glycosylation heterogeneity coverage and detection sensitivity of XCharge SAX were subsequently assessed. In the selectivity assessment, glycopeptides were effectively isolated from a peptide mixture (human serum immunoglobulin G (IgG) and human serum albumin digests) and a tryptic digest of human serum using XCharge SAX. In the evaluation of glycosylation heterogeneity coverage, five glycosites and eleven glycopeptides from horseradish peroxidase were identified after enrichment with XCharge SAX. In detection sensitivity assessment, glycopeptides within four orders of magnitude were identified after enrichment with XCharge SAX. In addition, volatile solvents were used in the loading and eluting buffers so that desalting was not necessary for ERLIC fractions. Our results collectively support the utility of XCharge SAX as a suitable chromatographic material for global glycosylation site analysis.

Development of an Online Microbore Hollow Fiber Enzyme Reactor Coupled with Nanoflow Liquid Chromatography-Tandem Mass Spectrometry for Global Proteomics

In this study, we report the development of a microbore hollow fiber enzyme reactor (mHFER) coupled to nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) for the online digestion or selective enrichment of glycopeptides and analysis of proteins. With mHFER, enzymatic digestion of protein could be achieved by continuous flow within a very small volume (~10 μL) of mHF inserted in a PEEK tube. Digested peptides exited through the pores of the hollow fiber membrane wall to external single or multiplexed trap columns for nLC-ESI-MS/MS analysis. Evaluation of online mHFER-nLC-ESI-MS/MS system was made with bovine serum albumin (BSA) by varying the temperature of digestion and the amount of protein injected. We evaluated the ability of the mHFER system to enrich glycopeptides by injecting a mixture of lectin (concanavalin A) and digested peptides from α-1-acid glycoprotein (AGP) into the mHFER, followed by delivery of PNGase F for endoglycosidic digestion. Nonglycosylated peptides unbound to lectins eluted at the first breakthrough run while N-linked glycopeptides eluted after the endoglycosidic digestion. The developed method was applied to urine samples from patients with prostate cancer and controls; 67 N-linked glycopeptides were identified and relative differences in glycopeptide content between patient and control samples were determined.

Association between leptin and systemic lupus erythematosus

Leptin, the product of ob gene, is a 16-kDa nonglycosylated peptide hormone produced by adipocytes that regulates appetite and energy expenditure at the hypothalamic level. As is known to be a satiety factor that can regulate body weight by inhibiting food intake and stimulating energy expenditure, leptin is a pleiotropic hormone whose multiple effects include regulation of endocrine function, reproduction and immunity. Since leptin has been considered to be a pro-inflammatory cytokine, investigations of leptin in the pathogenesis of autoimmune diseases have been detected, such as systemic lupus erythematosus, rheumatoid arthritis and osteoarthritis. Recently, the role of leptin in the modulation of immune response and inflammation has been discussed in the autoimmune diseases increasingly but less in systemic lupus erythematosus (SLE). Therefore, this article will focus on the current understanding of the role of leptin with such similar pathogenic mechanism in SLE in order to provide insights which may assist in the development of leptin-based approaches for the treatment of SLE.

A Classifier Based on Accurate Mass Measurements to Aid Large Scale, Unbiased Glycoproteomics

Determining which glycan moieties occupy specific N-glycosylation sites is a highly challenging analytical task. Arguably, the most common approach involves LC-MS and LC-MS/MS analysis of glycopeptides generated by proteases with high cleavage site specificity; however, the depth achieved by this approach is modest. Nonglycosylated peptides are a major challenge to glycoproteomics, as they are preferentially selected for data-dependent MS/MS due to higher ionization efficiencies and higher stoichiometric levels in moderately complex samples. With the goal of improving glycopeptide coverage, a mass defect classifier was developed that discriminates between peptides and glycopeptides in complex mixtures based on accurate mass measurements of precursor peaks. By using the classifier, glycopeptides that were not fragmented in an initial data-dependent acquisition run may be targeted in a subsequent analysis without any prior knowledge of the glycan or protein species present in the mixture. Additionally, from probable glycopeptides that were poorly fragmented, tandem mass spectra may be reacquired using optimal glycopeptide settings. We demonstrate high sensitivity (0.892) and specificity (0.947) based on an in silico dataset spanning >100,000 tryptic entries. Comparable results were obtained using chymotryptic species. Further validation using published data and a fractionated tryptic digest of human urinary proteins was performed, yielding a sensitivity of 0.90 and a specificity of 0.93. Lists of glycopeptides may be generated from an initial proteomics experiment, and we show they may be efficiently targeted using the classifier. Considering the growing availability of high accuracy mass analyzers, this approach represents a simple and broadly applicable means of increasing the depth of MS/MS-based glycoproteomic analyses.

Hydrophilic interaction/cation-exchange chromatography for glycopeptide enrichment by using a modified strong-cation exchange material

Protein glycosylation analysis based on mass spectrometry (MS) remains a major analytical challenge, because of the low proportions of glycopeptides in the glycoprotein digest and the suppression effect of the coexisting non-glycosylated peptides. Therefore, selective enrichment of glycopeptides prior to MS is of great significance. In this study, we presented hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) for glycopeptide enrichment by applying a customized material named MEX. MEX is a silica-based material bonded with two types of functional groups, benzenesulfonic acid and cyano-propyl groups. Compared with traditional strong-cation exchange (SCX) columns, moderate retention of analytes can be obtained on MEX due to the decreased ion-exchange capacity caused by the introduction of the cyano-propyl groups. Meanwhile, superior HILIC selectivity was also exhibited on MEX. In glycosylation analysis, MEX exhibited high selectivity and broad coverage for glycopeptides, which indicated the promising potential of the MEX material in glycosylation analysis.

Detection of the Heterogeneous O-Glycosylation Profile of MT1-MMP Expressed in Cancer Cells by a Simple MALDI-MS Method

BackgroundGlycosylation is an important and universal post-translational modification for many proteins, and regulates protein functions. However, simple and rapid methods to analyze glycans on individual proteins have not been available until recently.Methods/Principal FindingsA new technique to analyze glycopeptides in a highly sensitive manner by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using the liquid matrix 3AQ/CHCA was developed recently and we optimized this technique to analyze a small amount of transmembrane protein separated by SDS-PAGE. We used the MALDI-MS method to evaluate glycosylation status of membrane-type 1 matrix metalloproteinase (MT1-MMP). O-glycosylation of MT1-MMP is reported to modulate its protease activity and thereby to affect cancer cell invasion. MT1-MMP expressed in human fibrosarcoma HT1080 cells was immunoprecipitated and resolved by SDS-PAGE. After in-gel tryptic digestion of the protein, a single droplet of the digest was applied directly to the liquid matrix on a MALDI target plate. Concentration of hydrophilic glycopeptides within the central area occurred due to gradual evaporation of the sample solution, whereas nonglycosylated hydrophobic peptides remained at the periphery. This specific separation and concentration of the glycopeptides enabled comprehensive analysis of the MT1-MMP O-glycosylation.Conclusions/SignificanceWe demonstrate, for the first time, heterogeneous O-glycosylation profile of a protein by a whole protein analysis using MALDI-MS. Since cancer cells are reported to have altered glycosylation of proteins, this easy-to-use method for glycopeptide analysis opens up the possibility to identify specific glycosylation patterns of proteins that can be used as new biomarkers for malignant tumors.

Investigation of ovarian cancer associated sialylation changes in N-linked glycopeptides by quantitative proteomics

BackgroundIn approximately 80% of patients, ovarian cancer is diagnosed when the patient is already in the advanced stages of the disease. CA125 is currently used as the marker for ovarian cancer; however, it lacks specificity and sensitivity for detecting early stage disease. There is a critical unmet need for sensitive and specific routine screening tests for early diagnosis that can reduce ovarian cancer lethality by reliably detecting the disease at its earliest and treatable stages.ResultsIn this study, we investigated the N-linked sialylated glycopeptides in serum samples from healthy and ovarian cancer patients using Lectin-directed Tandem Labeling (LTL) and iTRAQ quantitative proteomics methods. We identified 45 N-linked sialylated glycopeptides containing 46 glycosylation sites. Among those, ten sialylated glycopeptides were significantly up-regulated in ovarian cancer patients’ serum samples. LC-MS/MS analysis of the non-glycosylated peptides from the same samples, western blot data using lectin enriched glycoproteins of various ovarian cancer type samples, and PNGase F (+/−) treatment confirmed the sialylation changes in the ovarian cancer samples.ConclusionHerein, we demonstrated that several proteins are aberrantly sialylated in N-linked glycopeptides in ovarian cancer and detection of glycopeptides with abnormal sialylation changes may have the potential to serve as biomarkers for ovarian cancer.

Dual Lectin-Based Size Sorting Strategy to Enrich Targeted N-Glycopeptides by Asymmetrical Flow Field-Flow Fractionation: Profiling Lung Cancer Biomarkers

A dual lectin-based size sorting and simultaneous enrichment strategy for selectively isolating N-linked glycopeptides was developed using asymmetrical flow field-flow fractionation (AF4). AF4 is an elution-based method for separating biological macromolecules that has been utilized for the separation of lectin-glycopeptide complexes formed by mixing serum peptides with lectin cocktails according to the difference in diffusion coefficients. It has also been used for simultaneous depletion of nonglycosylated peptides. The dual lectin-based enrichment method was applied to proteolytic peptides from lung cancer serum samples with two lectins (WGA, GlcNAc-specific, and SNA, Sia-specific), and the whole mixture was separated by AF4. The lectin-glycopeptide complex fractions collected during AF4 separation were endoglycosidically digested with PNGase F. The resulting deamidated glycopeptides were analyzed by nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS) to semiquantitatively profile the N-linked glycopeptides from the sera of lung cancer patients and healthy controls. The AF4 enrichment strategy coupled with nLC-ESI-MS-MS identified 16/24 (up/down-regulated by at least 10-fold compared to normal sera) N-linked glycopeptides from a WGA complex fraction of lung cancer sera and 18/3 from a SNA fraction.

A comparative study of glycoprotein concentration, glycoform profile and glycosylation site occupancy using isotope labeling and electrospray linear ion trap mass spectrometry

A strategy is presented for comparative analysis of glycoproteins in which the variation of protein concentration, variation of glycosylation site occupancy and variation of glycoform profile can be determined. A comparative study was performed using stable isotope labeling of glycopeptides and peptides by formaldehyde-H2 and formaldehyde-D2 and analysis by ESI-MS analysis. The relative intensity of the nonglycosylated peptide provided information about protein concentration variation. Variation of the glycoform profile was obtained by comparing the glycoform profile of d0- and d4-dimethyl labeled glycopeptides. By knowing the variation of protein concentration and the variation of glycoform profile, the variation of glycosylation site occupancy could be calculated. The utility of the proposed strategy was demonstrated with ribonuclease B with different protein concentrations, different levels of glycosylation site occupancy and different glycoform profiles.

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 Fe(3)O(4) 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.

ITRAQ plus 18O: A new technique for target glycoprotein analysis

A novel strategy combining iTRAQ with 18O stable isotope labeling (iTRAQ plus 18O) was established to identify N-glycosylation site, quantify the glycopeptides and non-glycosylated peptides, and obtain N-glycosylation site ratio on the target glycoprotein. In this approach, all peptides of four biological samples are labeled with four iTRAQ reagents in parallel, followed by PNGase F catalyzed labeling of N-glycosylation sites with H216O and H218O. Two sample groups are labeled with H216O and the other two are labeled with H218O. After the modification of MS precursor ion isolation window, tagged peptides are identified by LC–MS/MS, both glycopeptides and non-glycopeptides are quantified simultaneously using ProteinPilot™ Software. With four samples to be maximally analyzed in parallel, this workflow supports accurate identification and quantification of glycopeptides in a site-specific fashion. Furthermore, N-glycosylation site ratios on serum haptoglobin (Hp) β chain in healthy individuals as well as patients with hepatitis B virus (HBV), liver cirrhosis (LC) and hepatocellular carcinoma (HCC) were quantified to validate the novel ‘iTRAQ plus 18O’ method. Glycosite ratios of VVLHPN#YSQVDIGLIK were observed to change significantly in HCC patients compared with LC and HBV patients. This novel approach supports the screening of the target glycoproteins as biomarkers in clinical application.

Magnetic bead-based hydrophilic interaction liquid chromatography for glycopeptide enrichments

Purification of glycopeptides prior to the analysis by mass spectrometry (MS) is demanded due to ion suppression effect during ionization caused by the co-presence of non-glycosylated peptides. Among various purification methods, hydrophilic interaction liquid chromatography (HILIC) has become a popular method in recent years. In this work, we reported a novel magnetic bead-based zwitterionic HILIC (ZIC-HILIC) material which was fabricated by coating a zwitterionic polymer synthesized by spontaneous acid-catalyzed polymerization of 4-vinyl-pyridinium ethanesulfonate monomer on iron oxide magnetic nanoparticles. The resulting magnetic ZIC-HILIC nanoparticles were shown to provide high specificity and high recovery yield (95–100%) for the enrichment of glycopeptides from a standard glycoprotein, fetuin, using a simple magnetic bar. In addition, we proposed a two-step HILIC enrichment strategy using magnetic ZIC-HILIC nanoparticles for a large scale analysis of glycoproteins in complex biological samples. Using this approach, we identified 85 N-glycosylation sites in 53 glycoproteins from urine samples. Two novel glycosylation sites on N 513 of uromodulin and N 470 of lysosomal alpha-glucosidase which have not yet been reported were identified by two-step HILIC approach. Furthermore, all these identified sites were confirmed by studies conducted using PNGase F deglycosylation and 18O enzymatic labeling.

Immune recognition of tumor-associated mucin MUC1 is achieved by a fully synthetic aberrantly glycosylated MUC1 tripartite vaccine

The mucin MUC1 is typically aberrantly glycosylated by epithelial cancer cells manifested by truncated O-linked saccharides. The resultant glycopeptide epitopes can bind cell surface major histocompatibility complex (MHC) molecules and are susceptible to recognition by cytotoxic T lymphocytes (CTLs), whereas aberrantly glycosylated MUC1 protein on the tumor cell surface can be bound by antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Efforts to elicit CTLs and IgG antibodies against cancer-expressed MUC1 have not been successful when nonglycosylated MUC1 sequences were used for vaccination, probably due to conformational dissimilarities. Immunizations with densely glycosylated MUC1 peptides have also been ineffective due to impaired susceptibility to antigen processing. Given the challenges to immuno-target tumor-associated MUC1, we have identified the minimum requirements to consistently induce CTLs and ADCC-mediating antibodies specific for the tumor form of MUC1 resulting in a therapeutic response in a mouse model of mammary cancer. The vaccine is composed of the immunoadjuvant Pam(3)CysSK(4), a peptide T(helper) epitope and an aberrantly glycosylated MUC1 peptide. Covalent linkage of the three components was essential for maximum efficacy. The vaccine produced CTLs, which recognized both glycosylated and nonglycosylated peptides, whereas a similar nonglycosylated vaccine gave CTLs which recognized only nonglycosylated peptide. Antibodies elicited by the glycosylated tripartite vaccine were significantly more lytic compared with the unglycosylated control. As a result, immunization with the glycosylated tripartite vaccine was superior in tumor prevention. Besides its own aptness as a clinical target, these studies of MUC1 are likely predictive of a covalent linking strategy applicable to many additional tumor-associated antigens.