Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry

Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked β-N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. Although sensitive methods exist for the enrichment and identification of protein phosphorylation sites, those for the enrichment of O-GlcNAc-containing peptides are lacking. Reported here is highly efficient methodology for the enrichment and characterization of O-GlcNAc sites from complex samples. In this method, O-GlcNAc-modified peptides are tagged with a novel biotinylation reagent, enriched by affinity chromatography, released from the solid support by photochemical cleavage, and analyzed by electron transfer dissociation mass spectrometry. Using this strategy, eight O-GlcNAc sites were mapped from a tau-enriched sample from rat brain. Sites of GlcNAcylation were characterized on important neuronal proteins such as tau, synucleins, and methyl CpG-binding protein 2.Numerous cytoplasmic and nuclear proteins are post-translationally modified with O-linked β-N-acetylglucosamine (O-GlcNAc).¹ GlcNAcylation is involved in almost all aspects of cellular metabolism (1) and is highly dependent on the nutrient status of the cell (2). The O-GlcNAc modification rivals phosphorylation in both abundance and protein distribution. Recent studies indicate that signaling pathways can be regulated by the interplay of these two modifications at the same or proximal sites on numerous protein substrates (3).Current understanding of the functions of O-GlcNAc and of the function of O-GlcNAcylation and its relationship to phosphorylation is severely hampered by the difficulties in detecting this labile monosaccharide modification. Problems associated with the identification of O-GlcNAc sites include the following. (a) O-GlcNAc is quickly removed by hydrolases during cell lysis. (b) Like phosphorylation, O-GlcNAc is usually present in less than stoichiometric amounts at given sites on protein substrates. (c) O-GlcNAc is readily lost as an oxonium ion during conventional peptide sequence analysis by collision-activated dissociation (CAD) (supplemental Fig. 1). (d) Modified and unmodified forms of the peptide often co-elute during reverse phase HPLC (supplemental Fig. 2), and the preferential ionization of the unmodified peptide suppresses the signal observed for the corresponding O-GlcNAc-modified peptide (supplemental Fig. 2, b and c).Several attempts have been made to enrich samples for O-GlcNAc-modified proteins and peptides. Immunoaffinity purification of O-GlcNAc-modified peptides with an antibody (CTD 110.6) has been largely unsuccessful because of low binding avidity (4). Long, wheat germ agglutinin lectin columns (∼39 ft) provide some enrichment but also bind strongly to complex glycans (5). A mutant galactosyltransferase (GalT1) has been used to label GlcNAcylated proteins with a ketone-containing galactose analog (6). Following proteolytic digestion, O-GlcNAc-modified peptides were biotinylated with hydrazine chemistry, isolated on a column packed with avidin beads, eluted with free biotin, and sequenced by ETD mass spectrometry. Failure to elute peptides with high efficiency from the avidin column and an inability to direct the fragmentation to the peptide backbone limit the usefulness of this approach. Reported here is an enrichment methodology that (a) is highly specific for O-GlcNAc-modified peptides, (b) provides for efficient release of the captured peptides from an affinity support, and (c) facilitates complete characterization of the released peptides by ETD mass spectrometry.     

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, ''hook-effect'').¹ An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).² In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules ^{3, 4} and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.^5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.^8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.     

Facile synthesis of magnetic metal organic frameworks for the enrichment of low‐abundance peptides for MALDI‐TOF MS analysis

In this work, core‐shell magnetic metal organic framework (MOF) microspheres were successfully synthesized by coating magnetite particles with mercaptoacetic acid and subsequent reactions with ethanol solutions of Cu(OAc)₂ and benzene‐1,3,5‐tricarboxylic acid (designated as H₃btc) alternately. The resulting Fe₃O₄@[Cu₃(btc)₂] possess strong magnetic responsiveness. We applied the novel nanocomposites in the enrichment of low‐concentration standard peptides, peptides in MYO and BSA tryptic digests and in human urine in combination with MALDI‐TOF MS analysis for the first time. In addition, the Cu₃(btc)₂ MOF shells exhibit strong affinity to peptides, thus providing a rapid and convenient approach to the concentration of low‐abundance peptides. Notably, peptides at an extremely low concentration of 10 pM could be detected by MALDI‐TOF MS after enrichment with the magnetic MOF composites. In brief, the facile synthesis and efficient enrichment process of the Fe₃O₄@[Cu₃(btc)₂] microspheres make them promising candidates for the isolation of peptides in even complex biological environments.     

Selective separation and enrichment of peptides for MS analysis using the microspheres composed of Fe3O4@nSiO2 core and perpendicularly aligned mesoporous SiO2 shell

In this work, we report the development of a novel enrichment protocol for peptides by using the microspheres composed of Fe₃O₄@nSiO₂ Core and perpendicularly aligned mesoporous SiO₂ shell (designated Fe₃O₄@nSiO₂@mSiO₂). The Fe₃O₄@nSiO₂@mSiO₂ microspheres possess useful magnetic responsivity which makes the process of enrichment fast and convenient. The highly ordered nanoscale pores (2 nm) and high‐surface areas of the microspheres were demonstrated to have good size‐exclusion effect for the adsorption of peptides. An increase of S/N ratio over 100 times could be achieved by using the microspheres to enrich a standard peptide, and the application of the microspheres to enrich universal peptides was performed by using myoglobin tryptic digest solution. The enrichment efficiency of re‐used Fe₃O₄@nSiO₂@mSiO₂ microspheres was also studied. Large‐scale enrichment of endogenous peptides in rat brain extract was achieved by the microspheres. Automated nano‐LC‐ESI‐MS/MS was applied to analyze the sample after enrichment, and 60 unique peptides were identified in total. The facile and low‐cost synthesis as well as the convenient and efficient enrichment process of the novel Fe₃O₄@nSiO₂@mSiO₂ microspheres makes it a promising candidate for selectively isolation and enrichment of endogenous peptides from complex biological samples.     

Chapter 9: Analyses Using Disease Ontologies

Advanced statistical methods used to analyze high-throughput data such as gene-expression assays result in long lists of “significant genes.” One way to gain insight into the significance of altered expression levels is to determine whether Gene Ontology (GO) terms associated with a particular biological process, molecular function, or cellular component are over- or under-represented in the set of genes deemed significant. This process, referred to as enrichment analysis, profiles a gene-set, and is widely used to makes sense of the results of high-throughput experiments. The canonical example of enrichment analysis is when the output dataset is a list of genes differentially expressed in some condition. To determine the biological relevance of a lengthy gene list, the usual solution is to perform enrichment analysis with the GO. We can aggregate the annotating GO concepts for each gene in this list, and arrive at a profile of the biological processes or mechanisms affected by the condition under study. While GO has been the principal target for enrichment analysis, the methods of enrichment analysis are generalizable. We can conduct the same sort of profiling along other ontologies of interest. Just as scientists can ask “Which biological process is over-represented in my set of interesting genes or proteins?” we can also ask “Which disease (or class of diseases) is over-represented in my set of interesting genes or proteins?“. For example, by annotating known protein mutations with disease terms from the ontologies in BioPortal, Mort et al. recently identified a class of diseases—blood coagulation disorders—that were associated with a 14-fold depletion in substitutions at O-linked glycosylation sites. With the availability of tools for automatic annotation of datasets with terms from disease ontologies, there is no reason to restrict enrichment analyses to the GO. In this chapter, we will discuss methods to perform enrichment analysis using any ontology available in the biomedical domain. We will review the general methodology of enrichment analysis, the associated challenges, and discuss the novel translational analyses enabled by the existence of public, national computational infrastructure and by the use of disease ontologies in such analyses.

What to Learn in This Chapter

• Review the commonly used approach of Gene Ontology based enrichment analysis
• Understand the pitfalls associated with current approaches
• Understand the national infrastructure available for using alternative ontologies for enrichment analysis
• Learn about a generalized enrichment analysis workflow and its application using disease ontologies

This article is part of the “Translational Bioinformatics” collection for PLOS Computational Biology.

     

Large-Scale Differential Proteome Analysis in Plasmodium falciparum under Drug Treatment

Proteome studies contribute markedly to our understanding of parasite biology, host-parasite interactions, and mechanisms of drug action. For most antimalarial drugs neither mode of action nor mechanisms of resistance development are fully elucidated although this would be important prerequisites for successfully developing urgently required novel antimalarials. Here, we establish a large-scale quantitative proteomic approach to examine protein expression changes in trophozoite stages of the malarial parasite Plasmodium falciparum following chloroquine and artemisinin treatment. For this purpose SIL (stable isotope labeling) using ¹⁴N-isoleucine and ¹³C₆,¹⁵N₁-isoleucine was optimized to obtain 99% atomic percent enrichment. Proteome fractionation with anion exchange chromatography was used to reduce sample complexity and increase quantitative coverage of protein expression. Tryptic peptides of subfractions were subjected to SCX/RP separation, measured by LC-MS/MS and quantified using the novel software tool Census. In drug treated parasites, we identified a total number of 1,253 proteins, thus increasing the overall number of proteins identified in the trophozoite stage by 30%. A relative quantification was obtained for more than 800 proteins. Under artemisinin and chloroquine treatment 41 and 38 proteins respectively were upregulated (>1.5) whereas 14 and 8 proteins were down-regulated (<0.5). Apart from specifically regulated proteins we also identified sets of proteins which were regulated as a general response to drug treatment. The proteomic data was confirmed by Western blotting. The methodology described here allows for the efficient large-scale differential proteome analysis of P. falciparum to study the response to drug treatment or environmental changes. Only 100 µg of protein is required for the analysis suggesting that the method can also be transferred to other apicomplexan parasites.     

Phosphopeptide Enrichment by Covalent Chromatography after Derivatization of Protein Digests Immobilized on Reversed-Phase Supports

A rugged sample-preparation method for comprehensive affinity enrichment of phosphopeptides from protein digests has been developed. The method uses a series of chemical reactions to incorporate efficiently and specifically a thiol-functionalized affinity tag into the analyte by barium hydroxide catalyzed β-elimination with Michael addition using 2-aminoethanethiol as nucleophile and subsequent thiolation of the resulting amino group with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate. Gentle oxidation of cysteine residues, followed by acetylation of α- and ε-amino groups before these reactions, ensured selectivity of reversible capture of the modified phosphopeptides by covalent chromatography on activated thiol sepharose. The use of C18 reversed-phase supports as a miniaturized reaction bed facilitated optimization of the individual modification steps for throughput and completeness of derivatization. Reagents were exchanged directly on the supports, eliminating sample transfer between the reaction steps and thus, allowing the immobilized analyte to be carried through the multistep reaction scheme with minimal sample loss. The use of this sample-preparation method for phosphopeptide enrichment was demonstrated with low-level amounts of in-gel-digested protein. As applied to tryptic digests of α-S1- and β-casein, the method enabled the enrichment and detection of the phosphorylated peptides contained in the mixture, including the tetraphosphorylated species of β-casein, which has escaped chemical procedures reported previously. The isolates proved highly suitable for mapping the sites of phosphorylation by collisionally induced dissociation. β-Elimination, with consecutive Michael addition, expanded the use of the solid-phase-based enrichment strategy to phosphothreonyl peptides and to phosphoseryl/phosphothreonyl peptides derived from proline-directed kinase substrates and to their O-sulfono- and O-linked β-N-acetylglucosamine (O-GlcNAc)-modified counterparts. Solid-phase enzymatic dephosphorylation proved to be a viable tool to condition O-GlcNAcylated peptide in mixtures with phosphopeptides for selective affinity purification. Acetylation, as an integral step of the sample-preparation method, precluded reduction in recovery of the thiolation substrate caused by intrapeptide lysine-dehydroalanine cross-link formation. The solid-phase analytical platform provides robustness and simplicity of operation using equipment readily available in most biological laboratories and is expected to accommodate additional chemistries to expand the scope of solid-phase serial derivatization for protein structural characterization.     

Novel post-digest isotope coded protein labeling method for phospho- and glycoproteome analysis

In the field of proteomics there is an apparent lack of reliable methodology for quantification of posttranslational modifications. Present study offers a novel post-digest ICPL quantification strategy directed towards characterization of phosphorylated and glycosylated proteins. The value of the method is demonstrated based on the comparison of two prostate related metastatic cell lines originating from two distinct metastasis sites (PC3 and LNCaP). The method consists of protein digestion, ICPL labeling, mixing of the samples, PTM enrichment and MS-analysis. Phosphorylated peptides were isolated using TiO₂, whereas the enrichment of glycosylated peptides was performed using hydrazide based chemistry. Isolated PTM peptides were analyzed along with non enriched sample using 2D-(SCX-RP)-Nano-HPLC–MS/MS instrumentation. Taken together the novel ICPL labeling method offered a significant improvement of the number of identified (∼ 600 individual proteins) and quantified proteins (> 95%) in comparison to the classical ICPL method. The results were validated using alternative protein quantification strategies as well as label-free MS quantification method. On the biological level, the comparison of PC3 and LNCaP cells has shown specific modulation of proteins implicated in the fundamental process related to metastasis dissemination. Finally, a preliminary study involving clinically relevant autopsy cases reiterated the potential biological value of the discovered proteins.     

Optimization of the β-Elimination/Michael Addition Chemistry on Reversed-Phase Supports for Mass Spectrometry Analysis of O-Linked Protein Modifications

We previously adapted the β-elimination/Michael addition chemistry to solid-phase derivatization on reversed-phase supports, and demonstrated the utility of this reaction format to prepare phosphoseryl peptides in unfractionated protein digests for mass spectrometric identification and facile phosphorylation-site determination. Here, we have expanded the use of this technique to β-N-acetylglucosamine peptides, modified at serine/threonine, phosphothreonyl peptides, and phosphoseryl/phosphothreonyl peptides, followed in sequence by proline. The consecutive β-elimination with Michael addition was adapted to optimize the solid-phase reaction conditions for throughput and completeness of derivatization. The analyte remained intact during derivatization and was recovered efficiently from the silica-based, reversed-phase support with minimal sample loss. The general use of the solid-phase approach for enzymatic dephosphorylation was demonstrated with phosphoseryl and phosphothreonyl peptides and was used as an orthogonal method to confirm the identity of phosphopeptides in proteolytic mixtures. The solid-phase approach proved highly suitable to prepare substrates from low-level amounts of protein digests for phosphorylation-site determination by chemical-targeted proteolysis. The solid-phase protocol provides for a simple, robust, and efficient tool to prepare samples for phosphopeptide identification in MALDI mass maps of unfractionated protein digests, using standard equipment available in most biological laboratories. The use of a solid-phase analytical platform is expected to be readily expanded to prepare digest from O-glycosylated- and O-sulfonated proteins for mass spectrometry-based structural characterization.     

A new chemical approach to differentiate carboxy terminal peptide fragments in cyanogen bromide digests of proteins

We present a novel approach to perform C-terminal sequence analysis by discriminating the C-terminal peptide in a mass spectral analysis of a CNBr digest. During CNBr cleavage, all Met-Xxx peptide bonds are cleaved and the generated internal peptides all end with a homoserine lactone (hsl)-derivative. The partial opening of the hsl-derivatives, by using a slightly basic buffer solution, results in the formation of m/z doublets (Δm = 18 Da) for all internal peptides and allows to identify the C-terminal peptide which appears as a singlet in the mass spectra. Using two model proteins we demonstrate that this approach can be applied to study proteins purified in gel or in solution. The chemical opening of the hsl-derivative does not require any sample clean-up and therefore, the sensitivity of the C-terminal sequencing approach is increased significantly. Finally, the new protocol was applied to characterize the C-terminal sequence of two recombinant proteins. Tandem mass spectrometry by MALDI-TOF/TOF allowed to identify the sequence of the C-terminal peptides. This novel approach will allow to perform a proteome-wide study of C-terminal proteolytic processing events in a high-throughput fashion.     

Availability of MudPIT data for classification of biological samples

Background

Mass spectrometry is an important analytical tool for clinical proteomics. Primarily employed for biomarker discovery, it is increasingly used for developing methods which may help to provide unambiguous diagnosis of biological samples. In this context, we investigated the classification of phenotypes by applying support vector machine (SVM) on experimental data obtained by MudPIT approach. In particular, we compared the performance capabilities of SVM by using two independent collection of complex samples and different data-types, such as mass spectra (m/z), peptides and proteins.

Results

Globally, protein and peptide data allowed a better discriminant informative content than experimental mass spectra (overall accuracy higher than 87% in both collection 1 and 2). These results indicate that sequencing of peptides and proteins reduces the experimental noise affecting the raw mass spectra, and allows the extraction of more informative features available for the effective classification of samples. In addition, proteins and peptides features selected by SVM matched for 80% with the differentially expressed proteins identified by the MAProMa software.

Conclusions

These findings confirm the availability of the most label-free quantitative methods based on processing of spectral count and SEQUEST-based SCORE values. On the other hand, it stresses the usefulness of MudPIT data for a correct grouping of sample phenotypes, by applying both supervised and unsupervised learning algorithms. This capacity permit the evaluation of actual samples and it is a good starting point to translate proteomic methodology to clinical application.     

CysTRAQ - A combination of iTRAQ and enrichment of cysteinyl peptides for uncovering and quantifying hidden proteomes

Shotgun proteomics is capable of characterizing differences in both protein quality and quantity, and has been applied in various biomedical applications. Unfortunately, the high complexity and dynamic range of proteins in studied samples, clinical in particular, often hinders the identification of relevant proteins. Indeed, information-rich, low abundance proteins often remain undetected, whereas repeatedly reported altered concentrations in high abundance proteins are often ambiguous and insignificant. Several techniques have therefore been developed to overcome this obstacle and provide a deeper insight into the proteome. Here we report a novel approach, which enables iTRAQ reagent quantitation of peptides fractionated based on presence of a cysteine residue (thus CysTRAQ). For the first time, we prove that iTRAQ quantitation is fully compatible with cysteinyl peptide enrichment and is not influenced by the fractionation process. Moreover, the employment of the method combined with high-resolution TripleTOF 5600 mass spectrometer for very fast MS/MS acquisition in human amniotic fluid analysis significantly increased the number of identified proteins, which were simultaneously quantified owing to the introduction of iTRAQ labeling. We herein show that CysTRAQ is a robust and straightforward method with potential application in quantitative proteomics experiments, i.e. as an alternative to the ICAT reagent approach.     

Stepwise identification of potent antimicrobial peptides from human genome

The increasing incidence of hospital acquired infections caused by antibiotic resistant pathogens has led to an increase in morbidity and mortality, finding alternative antibiotics unaffected by resistance mechanisms is fundamentally important for treating this problem. Naturally occurring proteins usually carry short peptide fragments that exhibit noticeable biological activity against a wide variety of microorganisms such as bacteria, fungi and protozoa. Traditional discovery of such antimicrobially active fragments (i.e. antimicrobial peptides, AMPs) from protein repertoire is either random or led by chance. Here, we report the use of a rational protocol that combines in silico prediction and in vitro assay to identify potential AMPs with high activity and low toxicity from the entire human genome. In the procedure, a three-step inference strategy is first proposed to perform genome-wide analysis to infer AMPs in a high-throughput manner. By employing this strategy we are able to screen more than one million peptide candidates generated from various human proteins, from which we identify four highly promising samples, and subsequently their antibacterial activity on five strains as well as cytotoxicity on human myoblasts are tested experimentally. As a consequence, two high-activity, low-toxicity peptides are discovered, which could be used as the structural basis to further develop new antibiotics. In addition, from 1491 known AMPs we also derive a quantitative measure called antibacterial propensity index (API) for 20 naturally occurring amino acids, which shows a significant allometric correlation with the theoretical minimal inhibitory concentration of putative peptides against Gram-positive and Gram-negative bacteria. This study may provide a proof-of-concept paradigm for the genome-wide discovery of novel antimicrobial peptides by using a combination of in silico and in vitro analyses.     

A quantitative analysis of spontaneous isoaspartate formation from N-terminal asparaginyl and aspartyl residues

The formation of isoaspartate (isoAsp) from asparaginyl or aspartyl residues is a spontaneous post-translational modification of peptides and proteins. Due to isopeptide bond formation, the structure and possibly function of peptides and proteins is altered. IsoAsp modifications within the peptide chain have been reported for many cytosolic proteins. Amyloid peptides (Aβ) deposited in Alzheimer’s disease may carry an N-terminal isoAsp-modification. Here, we describe a quantitative investigation of isoAsp-formation from N-terminal Asn and Asp using model peptides similar to the Aβ N-terminus. The study is based on a newly developed separation of peptides using capillary electrophoresis (CE). ¹H NMR was employed to validate the basic finding of N-terminal isoAsp-formation from Asp and Asn. Thereby, the isomerization of Asn at neutral pH (0.6 day^?1, peptide NGEF) is approximately six times faster than that within the peptide chain (AANGEF). The difference in velocity between Asn and Asp isomerization is approximately 50-fold. In contrast to N-terminal Asn, Asp isomerization is significantly accelerated at acidic pH. The kinetic solvent isotope (k _D2O/k _H2O) effect of 2.46 suggests a rate-limiting proton transfer in isoAsp-formation. The proton inventory is consistent with transfer of one proton in the transition state, supporting the previous notion of rate-limiting deprotonation of the peptide backbone amide during succinimide-intermediate formation. The study provides evidence for a spontaneous N-terminal isoAsp-formation within peptides and might explain the accumulation of N-terminal isoAsp in amyloid deposits.     

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

O-linked N-acetyl-β-d-glucosamine (O-GlcNAc) is a ubiquitous and dynamic post-translational modification known to modify over 3,000 nuclear, cytoplasmic, and mitochondrial eukaryotic proteins. Addition of O-GlcNAc to proteins is catalyzed by the O-GlcNAc transferase and is removed by a neutral-N-acetyl-β-glucosaminidase (O-GlcNAcase). O-GlcNAc is thought to regulate proteins in a manner analogous to protein phosphorylation, and the cycling of this carbohydrate modification regulates many cellular functions such as the cellular stress response. Diverse forms of cellular stress and tissue injury result in enhanced O-GlcNAc modification, or O-GlcNAcylation, of numerous intracellular proteins. Stress-induced O-GlcNAcylation appears to promote cell/tissue survival by regulating a multitude of biological processes including: the phosphoinositide 3-kinase/Akt pathway, heat shock protein expression, calcium homeostasis, levels of reactive oxygen species, ER stress, protein stability, mitochondrial dynamics, and inflammation. Here, we will discuss the regulation of these processes by O-GlcNAc and the impact of such regulation on survival in models of ischemia reperfusion injury and trauma hemorrhage. We will also discuss the misregulation of O-GlcNAc in diseases commonly associated with the stress response, namely Alzheimer’s and Parkinson’s diseases. Finally, we will highlight recent advancements in the tools and technologies used to study the O-GlcNAc modification.     

Identification of secreted proteins regulated by cAMP in glioblastoma cells using glycopeptide capture and label‐free quantification

Exposure of glioblastoma U87MG cells to a cAMP analog leads to a decrease in proliferation, invasion, and angiogenic potential. Here, we apply a label‐free MS‐based approach to identify formerly N‐linked glycopeptides that change in abundance upon cAMP treatment. Over 150 unique glycopeptides in three biological repetitions were quantified, leading to the identification of 14 upregulated proteins and 21 downregulated proteins due to cAMP treatment. Of these, eight have been validated, either through comparison with microarray data or by Western blot. We estimate our ability to identify differentially expressed peptides at greater than 85% in a single biological repetition, while the analysis of multiple biological repetitions lowers the false positive rate to ～2%. Many of the proteins identified in this study are involved in cell signaling and some, such as Tenascin C, Cathepsin L, Neuroblastoma suppressor of tumorigenicity, and AXL/UFO tyrosine–protein kinase receptor, have been previously shown to be involved in glioblastoma progression. We also identify several semitryptic peptides that increase in abundance upon cAMP treatment, suggesting that cAMP regulates protease activity in these cells. Overall, these results demonstrate the benefits of using a highly specific enrichment method for quantitative proteomic experiments.     

Isoform-specific O-Glycosylation of Osteopontin and Bone Sialoprotein by Polypeptide N-Acetylgalactosaminyltransferase-1

Mucin-type O-glycan biosynthesis is regulated by the family of UDP-GalNAc polypeptide:N-acetylgalactosaminlytransfersases (ppGalNAcTs) that catalyzes the first step in the pathway by transferring GalNAc to Ser or Thr residues in a protein from the sugar donor UDP-GalNAc. Because not all Ser/Thr residues are glycosylated, rules must exist that signal which hydroyxamino acids acquire sugar. To date, no universal consensus signal has emerged. Therefore, strategies to deduce the subset of proteins that will be glycosylated by distinct ppGalNAcTs must be developed. Mucin-type O-glycoproteins are present abundantly in bone, where we found multiple ppGalNAcT isoforms, including ppGalNAcT-1, to be highly expressed. Thus, we compared glycoproteins expressed in wild-type and Galnt1-null mice to identify bone-associated proteins that were glycosylated in a ppGalNAcT-1-dependent manner. A reduction in the apparent molecular masses of two SIBLINGs (small integrin binding ligand N-linked glycoproteins), osteopontin (OPN) and bone sialoprotein (BSP) in the Galnt1-null mice relative to those of the wild-type was observed. Several synthetic peptides derived from OPN and BSP sequences were designed to include either known or predicted (in silico) glycosylation sites. In vitro glycosylation assays of these peptides with recombinant ppGalNAcT-1, ppGalNAcT-2, or ppGalNAcT-3 demonstrated that both SIBLINGs contained Thr/Ser residues that were preferentially glycosylated by ppGalNAcT-1. In addition, lysates prepared from wild-type, but not those from Galnt1-null derived osteoblasts, could glycosylate these peptides efficiently, suggesting that OPN and BSP contain sites that are specific for ppGalNAcT-1. Our study presents a novel and systematic approach for identification of isoform-specific substrates of the ppGalNAcT family and suggests ppGalNAcT-1 to be indispensable for O-glycosylation at specific sites of the bone glycoproteins OPN and BSP.     

18O Labeling for a Quantitative Proteomic Analysis of Glycoproteins in Hepatocellular Carcinoma

Introduction

Quantitative proteomics using tandem mass spectrometry is an attractive approach for identification of potential cancer biomarkers. Fractionation of complex tissue samples into subproteomes prior to mass spectrometric analyses increases the likelihood of identifying cancer-specific proteins that might be present in low abundance. In this regard, glycosylated proteins are an interesting class of proteins that are already established as biomarkers for several cancers.

Materials and Methods

In this study, we carried out proteomic profiling of tumor and adjacent non-cancer liver tissues from hepatocellular carcinoma (HCC) patients. Glycoprotein enrichment from liver samples using lectin affinity chromatography and subsequent ¹⁸O/¹⁶O labeling of peptides allowed us to obtain relative abundance levels of lectin-bound proteins. As a complementary approach, we also examined the relative expression of proteins in HCC without glycoprotein enrichment. Lectin affinity enrichment was found to be advantageous to quantitate several interesting proteins, which were not detected in the whole proteome screening approach. We identified and quantitated over 200 proteins from the lectin-based approach. Interesting among these were fetuin, cysteine-rich protein 1, serpin peptidase inhibitor, leucine-rich alpha-2-glycoprotein 1, melanoma cell adhesion molecule, and heparan sulfate proteoglycan-2. Using lectin affinity followed by PNGase F digestion coupled to ¹⁸O labeling, we identified 34 glycosylation sites with consensus sequence N-X-T/S. Western blotting and immunohistochemical staining were carried out for several proteins to confirm mass spectrometry results.

Conclusion

This study indicates that quantitative proteomic profiling of tumor tissue versus non-cancerous tissue is a promising approach for the identification of potential biomarkers for HCC.