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

ABSTRACT: BACKGROUND: In 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. Results: In 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. Conclusion: Herein, 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.     

Identification and quantification of N-linked glycoproteins using hydrazide chemistry,stable isotope labeling and mass spectrometry

Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. Glycosylation is the most common post-translational modification. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the specific release of formerly N-linked glycosylated peptides via peptide- N-glycosidase F (PNGase F). The recovered peptides are then identified and quantified by MS/MS. We applied the approach to the analysis of plasma membrane proteins and proteins contained in human blood serum.     

Tools for glycoproteomic analysis: size exclusion chromatography facilitates identification of tryptic glycopeptides with N-linked glycosylation sites

Proteomic techniques, such as HPLC coupled to tandem mass spectrometry (LC-MS/MS), have proved useful for the identification of specific glycosylation sites on glycoproteins (glycoproteomics). Glycosylation sites on glycopeptides produced by trypsinization of complex glycoprotein mixtures, however, are particularly difficult to identify both because a repertoire of glycans may be expressed at a particular glycosylation site, and because glycopeptides are usually present in relatively low abundance (2% to 5%) in peptide mixtures compared to nonglycosylated peptides. Previously reported methods to facilitate glycopeptide identification require either several pre-enrichment steps, involve complex derivatization procedures, or are restricted to a subset of all the glycan structures that are present in a glycoprotein mixture. Because the N-linked glycans expressed on tryptic glycopeptides contribute substantially to their mass, we demonstrate that size exclusion chromatography (SEC) provided a significant enrichment of N-linked glycopeptides relative to nonglycosylated peptides. The glycosylated peptides were then identified by LC-MS/MS after treatment with PNGase-F by the monoisotopic mass increase of 0.984 Da caused by the deglycosylation of the peptide. Analyses performed on human serum showed that this SEC glycopeptide isolation procedure results in at least a 3-fold increase in the total number of glycopeptides identified by LC-MS/MS, demonstrating that this simple, nonselective, rapid method is an effective tool to facilitate the identification of peptides with N-linked glycosylation sites.     

Misfolding of glycoproteins is a prerequisite for peptide: N-glycanase mediated deglycosylation

Peptide:N-glycanase (PNGase) is a deglycosylating enzyme that catalyzes the hydrolysis of the beta-aspartylglycosylamine bond of aspargine-linked glycopeptides and glycoproteins. Earlier studies from our laboratory indicated that PNGase catalyzed de-N-glycosylation was limited to glycopeptide substrates, but recent reports have demonstrated that it also acts upon full-length misfolded glycoproteins. In this study, we utilized two glycoprotein substrates, yeast carboxypeptidase and chicken egg albumin (ovalbumin), to study the deglycosylation activity of yeast PNGase and its mutants. Our results provide further evidence that PNGase acts upon full-length glycoprotein substrates and clearly establish that PNGase acts only on misfolded or denatured glycoproteins.     

Changes in protein glycosylation during chick embryo development

To investigate the molecular changes in cell-surface glycoproteins during chick embryo development, fibroblasts from 8- and 16-day embryos were extensively digested by pronase after (i) metabolic labeling with radioactive precursors and (ii) external labeling. Two main classes of glycopeptide pronase digestion product were distinguished by Sephadex G-50 column chromatography. The large material excluded was mostly composed of glycosaminoglycans. The small retarded glycopeptides underwent age-related modifications. Those in the 8-day cells were mainly N-linked, whereas 16-day cells contained both O- and N-linked glycopeptides. The evolution of high-mannose chains in younger cells to complex-type chains in the older cells is suggested by (i) the decrease in the mannose-to-galactose and mannose-to-N-acetylglucosamine ratio with embryo development, and (ii) the fact that endo-β-N-acetylglucosaminidase H treatment released more oligomannosyls from younger than from older embryo cell glycopeptides. Small glycopeptides were also more highly sialylated in 16-day cells than in 8-day cells. The present results provide the first biochemical evidence that both quantitative and qualitative modifications occur in cell-surface glycoconjugates during the late stages of chick embryo development.     

N-linked glycosylation does not impair proteasomal degradation but affects class I major histocompatibility complex presentation

The addition of N-linked glycans to nascent polypeptides occurs cotranslationally in the endoplasmic reticulum (ER). For many proteins the state of the glycans serves as an indicator, which allows the ER quality control system to monitor the conformation of polypeptides upon folding. Proteins that fail to fold in the ER are often dislocated to the cytoplasm, where they are subjected to proteasomal degradation. Although the addition of N-linked glycans occurs within the ER, non-lysosomal removal of the glycans occurs in the cytosol by the action of peptide N-glycanase (PNGase). In this study, we investigated the interplay between PNGase action and proteasomal degradation of ER misfolded proteins (i.e. whether PNGase acts prior to or following proteasomal degradation). Interestingly, we found that glycan removal from N-terminally extended peptides modulates the presentation of class I major histocompatibility complex-restricted epitopes. Our findings provide direct evidence that the proteasome is capable of degrading glycoproteins without prior removal of their glycans. This degradation is independent of either the identity of the glycosylated protein or the type and number of N-linked glycans it harbors. We also captured and characterized glycopeptides generated following proteasomal degradation of RNaseB. Although the carbohydrate moiety reduced the variability of the degradation products that include the glycosylated residue (local effect), the overall global digestion pattern of RNaseB was unaffected. Together with earlier findings by others, our data support a model in which PNGase may act both upstream and downstream to proteasomal degradation and demonstrates its important role in class I major histocompatibility complex antigen presentation.     

Enhanced analysis of the mouse plasma proteome using cysteine-containing tryptic glycopeptides

A comprehensive understanding of the mouse plasma proteome is important for studies using mouse models to identify protein markers of human disease. To enhance our analysis of the mouse plasma proteome, we have developed a method for isolating low-abundance proteins using a cysteine-containing glycopeptide strategy. This method involves two orthogonal affinity capture steps. First, glycoproteins are coupled to an azlactone copolymer gel using hydrazide chemistry and cysteine residues are then biotinylated. After trypsinization and extensive washing, tethered N-glycosylated tryptic peptides are released from the gel using PNGase F. Biotinylated cysteinyl-containing glycopeptides are then affinity selected using a monomeric avidin gel and analyzed by LC-MS/MS. We have applied the method to a proteome analysis of mouse plasma. In two independent analyses using 200 muL each of C57BL mouse plasma, 51 proteins were detected. Only 42 proteins were seen when the same plasma sample was analyzed by glycopeptides only. A total of 104 N-glycosylation sites were identified. Of these, 17 sites have hitherto not been annotated in the Swiss-Prot database whereas 48 were considered probable, potential, or by similarity - i.e., based on little or no experimental evidence. We show that analysis by cysteine-containing glycopeptides allows detection of low-abundance proteins such as the epidermal growth factor receptor, the Vitamin K-dependent protein Z, the hepatocyte growth factor activator, and the lymphatic endothelium-specific hyaluronan receptor as these proteins were not detected in the glycopeptide control analysis.     

Site-specific labeling of cytoplasmic peptide:N-glycanase by N,N'-diacetylchitobiose-related compounds

Peptide:N-glycanase (PNGase) is the deglycosylating enzyme, which releases N-linked glycan chains from N-linked glycopeptides and glycoproteins. Recent studies have revealed that the cytoplasmic PNGase is involved in the degradation of misfolded/unassembled glycoproteins. This enzyme has a Cys, His, and Asp catalytic triad, which is required for its enzymatic activity and can be inhibited by "free" N-linked glycans. These observations prompted us to investigate the possible use of haloacetamidyl derivatives of N-glycans as potent inhibitors and labeling reagents of this enzyme. Using a cytoplasmic PNGase from budding yeast (Png1), Man9GlcNAc2-iodoacetoamide was shown to be a strong inhibitor of this enzyme. The inhibition was found to be through covalent binding of the carbohydrate to a single Cys residue on Png1, and the binding was highly selective. The mutant enzyme in which Cys191 of the catalytic triad was changed to Ala did not bind to the carbohydrate probe, suggesting that the catalytic Cys is the binding site for this compound. Precise determination of the carbohydrate attachment site by mass spectrometry clearly identified Cys191 as the site of covalent attachment. Molecular modeling of N,N'-diacetylchitobiose (chitobiose) binding to the protein suggests that the carbohydrate binding site is distinct from but adjacent to that of Z-VAD-fmk, a peptide-based inhibitor of this enzyme. These results suggest that cytoplasmic PNGase has a separate binding site for chitobiose and other carbohydrates, and haloacetamide derivatives can irreversibly inhibit that catalytic Cys in a highly specific manner.     

Comparison of N-linked Glycoproteins in Human Whole Saliva, Parotid, Submandibular, and Sublingual Glandular Secretions Identified using Hydrazide Chemistry and Mass Spectrometry

INTRODUCTION: Saliva is a body fluid that holds promise for use as a diagnostic fluid for detecting diseases. Salivary proteins are known to be heavily glycosylated and are known to play functional roles in the oral cavity. We identified N-linked glycoproteins in human whole saliva, as well as the N-glycoproteins in parotid, submandibular, and sublingual glandular fluids. MATERIALS AND METHODS: We employed hydrazide chemistry to affinity enrich for N-linked glycoproteins and glycopeptides. PNGase F releases the N-peptides/proteins from the agarose-hydrazide resin, and liquid chromatography-tandem mass spectrometry was used to identify the salivary N-glycoproteins. RESULTS: A total of 156 formerly N-glycosylated peptides representing 77 unique N-glycoproteins were identified in salivary fluids. The total number of N-glycoproteins identified in the individual fluids was: 62, 34, 44, and 53 in whole saliva, parotid fluid, submandibular fluid, and sublingual fluid, respectively. The majority of the N-glycoproteins were annotated as extracellular proteins (40%), and several of the N-glycoproteins were annotated as membrane proteins (14%). A number of glycoproteins were differentially found in submandibular and sublingual glandular secretions. CONCLUSIONS: Mapping the N-glycoproteome of parotid, submandibular, and sublingual saliva is important for a thorough understanding of biological processes occurring in the oral cavity and to realize the role of saliva in the overall health of human individuals. Moreover, identifying glycoproteins in saliva may also be valuable for future disease biomarker studies.     

Identification of N-linked glycoproteins in human saliva by glycoprotein capture and mass spectrometry

Glycoproteins make up a major and important part of the salivary proteome and play a vital role in maintaining the health of the oral cavity. Because changes in the physiological state of a person are reflected as changes in the glycoproteome composition, mapping the salivary glycoproteome will provide insights into various processes in the body. Salivary glycoproteins were identified by the hydrazide coupling and release method. In this approach, glycoproteins were coupled onto a hydrazide resin, the proteins were then digested and formerly N-glycosylated peptides were selectively released with the enzyme PNGase F and analyzed by LC-MS/MS. Employing this method, coupled with in-solution isoelectric focusing separation as an additional means for pre-fractionation, we identified 84 formerly N-glycosylated peptides from 45 unique N-glycoproteins. Of these, 16 glycoproteins have not been reported previously in saliva. In addition, we identified 44 new sites of N-linked glycosylation on the proteins.     

N-linked glycosylation profiling of pancreatic cancer serum using capillary liquid phase separation coupled with mass spectrometric analysis

Glycoproteins play important roles in various biological processes including intracellular transport, cell recognition, and cell-cell interactions. The change of the cellular glycosylation profile may have profound effects on cellular homeostasis and malignancy. Therefore, we have developed a sensitive screening approach for the comprehensive analysis of N-glycans and glycosylation sites on human serum proteins. Using this approach, N-linked glycopeptides were extracted by double lectin affinity chromatography. The glycans were enzymatically cleaved from the peptides and then profiled using capillary hydrophilic interaction liquid chromatography coupled online with ESI-TOF MS. The structures of the separated glycans were determined by MALDI quadrupole ion-trap TOF mass spectrometry in both positive and negative modes. The glycosylation sites were elucidated by sequencing of PNGase F modified glycopeptides using nanoRP-LC-ESI-MS/MS. Alterations of glycosylation were analyzed by comparing oligosaccharide expression of serum glycoproteins at different disease stages. The efficiency of this method was demonstrated by the analysis of pancreatic cancer serum compared to normal serum. Ninety-two individual glycosylation sites and 202 glycan peaks with 105 unique carbohydrate structures were identified from approximately 25 mug glycopeptides. Forty-four oligosaccharides were found to be distinct in the pancreatic cancer serum. Increased branching of N-linked oligosaccharides and increased fucosylation and sialylation were observed in samples from patients with pancreatic cancer. The methodology described in this study may elucidate novel, cancer-specific oligosaccharides and glycosylation sites, some of which may have utility as useful biomarkers of cancer.     

Identification of Glycoproteins from Mouse Skin Tumors and Plasma

Plasma has been the focus of testing different proteomic technologies for the identification of biomarkers due to its ready accessibility. However, it is not clear if direct proteomic analysis of plasma can be used to discover new marker proteins from tumor that are associated with tumor progression. Here, we reported that such proteins can be detected in plasma in a chemical induced skin cancer mouse model. We analyzed glycoproteins from both benign papillomas and malignant carcinomas from mice using our recently developed platform, solid-phase extraction of glycopeptides (SPEG) and mass spectrometry, and identified 463 unique N-linked glycosites from 318 unique glycoproteins. These include most known extracellular proteins that have been reported to play roles in skin cancer development such as thrombospondin, cathepsins, epidermal growth factor receptor, cell adhesion molecules, cadherins, integrins, tuberin, fibulin, TGFβ receptor, etc. We further investigated whether these tumor proteins could be detected in plasma from tumor bearing mice using isotope labeling and 2D-LC-MALDI-MS/MS. Two tumor glycoproteins, Tenascin-C and Arylsulfatase B, were identified and quantified successfully in plasma from tumor bearing mice. This result indicates that analysis of tumor associated proteins in tumors and plasma by method using glycopeptide capture, isotopic labeling, and mass spectrometry can be used as a discovery tool to identify candidate tumor proteins that may be detected in plasma.     

Glycoproteomics of Trypanosoma cruzi trypomastigotes using subcellular fractionation, lectin affinity, and stable isotope labeling

Herein we detail the first glycoproteomic analysis of a human pathogen. We describe an approach that enables the identification of organelle and cell surface N-linked glycoproteins from Trypanosoma cruzi, the causative agent of Chagas' disease. This approach is based on a subcellular fractionation protocol to produce fractions enriched in either organelle or plasma membrane/cytoplasmic proteins. Through lectin affinity capture of the glycopeptides from each subcellular fraction and stable isotope labeling of the glycan attachment sites with H(2)18O, we unambiguously identified 36 glycosylation sites on 35 glycopeptides which mapped to 29 glycoproteins. We also present the first expression evidence for 11 T. cruzi specific glycoproteins and provide experimental data indicating that the mucin associated surface protein family (MASP) and dispersed gene family (DGF-1) are post-translationally modified by N-linked glycans.     

Elucidation of glycoprotein structures by unspecific proteolysis and direct nanoESI mass spectrometric analysis of ZIC-HILIC-enriched glycopeptides

Protein glycosylation was explored by direct nanoESI MS and MS/MS analysis of ZIC-HILIC-enriched proteolytic glycopeptides without further separation or purification. In a previous publication, we demonstrated that a direct MS-based analysis of proteolytic glycopeptides is feasible for a number of proteins (Henning , S. J. Mass Spectrom. 2007 , 42 , 1415 - 21). This method has now been refined for two aspects: (1) separation of glycopeptides by use of ZIC-HILIC SPE and (2) the use of unspecific proteases like thermolysin, elastase, or a trypsin/chymotrypsin mixture leading per se to a mass-based separation, that is, small nonglycosylated peptides and almost exclusively glycopeptides at higher m/z values. Furthermore, the glycopeptides produced by the above proteases in general contain short peptide backbones thus improving-probably due to their higher hydrophilicity--the ZIC-HILIC-based separation. The combination of unspecific proteolysis, glycopeptide separation, and their direct MS analysis was successfully accomplished for probing glycoproteins carrying high-mannose type (ribonuclease B), neutral (asialofetuin), and acidic (haptoglobin and α1-acid glycoprotein) complex type glycans as well as for glycopeptides derived from glycoprotein mixtures and, finally, for exploring the glycosylation of a human IgG preparation. Our results show that the presented method is a fast, facile, and inexpensive procedure for the elucidation of protein N-glycosylation.     

Extracellular matrix-derived peptides and myocardial repair

Repairing cardiac tissue remains one of the most challenging goals in tissue engineering. Here, we discuss ways whereby we sought to treat myocardial infarctions using extracellular-matrix derived peptides. Using an ischemia/reperfusion myocardial infarction rodent model, we targeted these extracellular matrix-derived peptides to the myocardial infarct site and were able to induce angiogenesis and alter the negative remodeling seen after an acute myocardial infarction. Our results indicate a potentially new strategy for repairing damaged tissue.     

Performance of isobaric and isotopic labeling in quantitative plant proteomics

Mass spectrometry has become indispensable for peptide and protein quantification in proteomics studies. When proteomics technologies are applied to understand the biology of plants, two-dimensional gel electrophoresis is still the prevalent method for protein fractionation, identification, and quantitation. In the present work, we have used LC-MS to compare an isotopic (ICPL) and isobaric (iTRAQ) chemical labeling technique to quantify proteins in the endosperm of Ricinus communis seeds at three developmental stages (IV, VI, and X). Endosperm proteins of each stage were trypsin-digested in-solution, and the same amount of peptides was labeled with ICPL and iTRAQ tags in two orders (forward and reverse). Each sample was submitted to nanoLC coupled to an LTQ-Orbitrap high-resolution mass spectrometer. Comparing labeling performance, iTRAQ was able to label 99.8% of all identified unique peptides, while 94.1% were labeled by ICPL. After statistical analysis, it was possible to quantify 309 (ICPL) and 321 (iTRAQ) proteins, from which 95 are specific to ICPL, 107 to iTRAQ, and 214 common to both labeling strategies. We noted that the iTRAQ quantification could be influenced by the tag. Even though the efficiency of the iTRAQ and ICPL in protein quantification depends on several parameters, both labeling methods were able to successfully quantify proteins present in the endosperm of castor bean during seed development and, when combined, increase the number of quantified proteins.     

Human urinary glycoproteomics; attachment site specific analysis of N- and O-linked glycosylations by CID and ECD

Urine is a complex mixture of proteins and waste products and a challenging biological fluid for biomarker discovery. Previous proteomic studies have identified more than 2800 urinary proteins but analyses aimed at unraveling glycan structures and glycosylation sites of urinary glycoproteins are lacking. Glycoproteomic characterization remains difficult because of the complexity of glycan structures found mainly on asparagine (N-linked) or serine/threonine (O-linked) residues. We have developed a glycoproteomic approach that combines efficient purification of urinary glycoproteins/glycopeptides with complementary MS-fragmentation techniques for glycopeptide analysis. Starting from clinical sample size, we eliminated interfering urinary compounds by dialysis and concentrated the purified urinary proteins by lyophilization. Sialylated urinary glycoproteins were conjugated to a solid support by hydrazide chemistry and trypsin digested. Desialylated glycopeptides, released through mild acid hydrolysis, were characterized by tandem MS experiments utilizing collision induced dissociation (CID) and electron capture dissociation fragmentation techniques. In CID-MS(2), Hex(5)HexNAc(4)-N-Asn and HexHexNAc-O-Ser/Thr were typically observed, in agreement with known N-linked biantennary complex-type and O-linked core 1-like structures, respectively. Additional glycoforms for specific N- and O-linked glycopeptides were also identified, e.g. tetra-antennary N-glycans and fucosylated core 2-like O-glycans. Subsequent CID-MS(3), of selected fragment-ions from the CID-MS(2) analysis, generated peptide specific b- and y-ions that were used for peptide identification. In total, 58 N- and 63 O-linked glycopeptides from 53 glycoproteins were characterized with respect to glycan- and peptide sequences. The combination of CID and electron capture dissociation techniques allowed for the exact identification of Ser/Thr attachment site(s) for 40 of 57 putative O-glycosylation sites. We defined 29 O-glycosylation sites which have, to our knowledge, not been previously reported. This is the first study of human urinary glycoproteins where "intact" glycopeptides were studied, i.e. the presence of glycans and their attachment sites were proven without doubt.     

Comparison of glycoproteins by two-dimensional mapping of glycosylated peptides

We describe here a two-dimensional mapping procedure which is capable of resolving glycopeptides isolated by lectin affinity chromatography from radioiodinated tryptic digests of glycoproteins. Glycopeptide maps were successfully produced for the model proteins alpha 1-acid glycoprotein and fetuin, as well as for the two surface glycoproteins gp90 and gp45 from equine infectious anemia virus (EIAV). Differences were detected in the glycopeptide maps obtained for the gp90 and gp45 components from two antigenically distinct strains of EIAV, demonstrating the ability of this procedure to detect variations in glycosylation in closely related glycoproteins. Thus this glycopeptide mapping technique provides a simple, rapid method to study changes in glycopeptides requiring only micrograms of glycoprotein.     

Extracellular matrix-derived peptides and myocardial repair

Repairing cardiac tissue remains one of the most challenging goals in tissue engineering. Here, we discuss ways whereby we sought to treat myocardial infarctions using extracellular-matrix derived peptides. Using an ischemia/reperfusion myocardial infarction rodent model, we targeted these extracellular matrix-derived peptides to the myocardial infarct site and were able to induce angiogenesis and alter the negative remodeling seen after an acute myocardial infarction. Our results indicate a potentially new strategy for repairing damaged tissue.Key words: extracellular matrix, myocardial infarction, tissue engineering, cardiac repair, angiogenesis     

Sub-proteomic fractionation,iTRAQ, and OFFGEL-LC–MS/MS approaches to cardiac proteomics

Using an in solution based approach with a sub-proteomic fraction enriched in cardiac sarcomeric proteins; we identified protein abundance in ischemic and non-ischemic regions of rat hearts stressed by acute myocardial ischemia by ligating the left-anterior descending coronary artery in vivo for 1 h without reperfusion. Sub-cellular fractionation permitted more in depth analysis of the proteome by reducing the sample complexity. A series of differential centrifugations produced nuclear, mitochondrial, cytoplasmic, microsomal, and sarcomeric enriched fractions of ischemic and non-ischemic tissues. The sarcomeric enriched fractions were labeled with isobaric tags for relative quantitation (iTRAQ), and then fractionated with an Agilent 3100 OFFGEL fractionator. The OFFGEL fractions were run on a Dionex U-3000 nano LC coupled to a ThermoFinnigan LTQ running in PQD (pulsed Q dissociation) mode. The peptides were analyzed using two search engines MASCOT (MatrixScience), and MassMatrix with false discovery rate of < 5%. Compared to no fractionation prior to LC–MS/MS, fractionation with OFFGEL improved the identification of proteins approximately four-fold. We found that approximately 22 unique proteins in the sarcomeric enriched fraction had changed at least 20%. Our workflow provides an approach for discovery of unique biomarkers or changes in the protein profile of tissue in disorders of the heart.