Benjamin L. Parker

Simultaneous Glycan-Peptide Characterization Using Hydrophilic Interaction Chromatography and Parallel Fragmentation by CID, Higher Energy Collisional Dissociation, and Electron Transfer Dissociation MS Applied to the N-Linked Glycoproteome of Campylobacter jejuni

Campylobacter jejuni is a gastrointestinal pathogen that is able to modify membrane and periplasmic proteins by the N-linked addition of a 7-residue glycan at the strict attachment motif (D/E)XNX(S/T). Strategies for a comprehensive analysis of the targets of glycosylation, however, are hampered by the resistance of the glycan-peptide bond to enzymatic digestion or β-elimination and have previously concentrated on soluble glycoproteins compatible with lectin affinity and gel-based approaches. We developed strategies for enriching C. jejuni HB93-13 glycopeptides using zwitterionic hydrophilic interaction chromatography and examined novel fragmentation, including collision-induced dissociation (CID) and higher energy collisional (C-trap) dissociation (HCD) as well as CID/electron transfer dissociation (ETD) mass spectrometry. CID/HCD enabled the identification of glycan structure and peptide backbone, allowing glycopeptide identification, whereas CID/ETD enabled the elucidation of glycosylation sites by maintaining the glycan-peptide linkage. A total of 130 glycopeptides, representing 75 glycosylation sites, were identified from LC-MS/MS using zwitterionic hydrophilic interaction chromatography coupled to CID/HCD and CID/ETD. CID/HCD provided the majority of the identifications (73 sites) compared with ETD (26 sites). We also examined soluble glycoproteins by soybean agglutinin affinity and two-dimensional electrophoresis and identified a further six glycosylation sites. This study more than doubles the number of confirmed N-linked glycosylation sites in C. jejuni and is the first to utilize HCD fragmentation for glycopeptide identification with intact glycan. We also show that hydrophobic integral membrane proteins are significant targets of glycosylation in this organism. Our data demonstrate that peptide-centric approaches coupled to novel mass spectrometric fragmentation techniques may be suitable for application to eukaryotic glycoproteins for simultaneous elucidation of glycan structures and peptide sequence.

Selective enrichment of sialic acid–containing glycopeptides using titanium dioxide chromatography with analysis by HILIC and mass spectrometry

The terminal monosaccharide of cell surface glycoconjugates is typically a sialic acid (SA), and aberrant sialylation is involved in several diseases. Several methodological approaches in sample preparation and subsequent analysis using mass spectrometry (MS) have enabled the identification of glycosylation sites and the characterization of glycan structures. In this paper, we describe a protocol for the selective enrichment of SA-containing glycopeptides using a combination of titanium dioxide (TiO2) and hydrophilic interaction liquid chromatography (HILIC). The selectivity of TiO2 toward SA-containing glycopeptides is achieved by using a low-pH buffer that contains a substituted acid such as glycolic acid to improve the binding efficiency and selectivity of SA-containing glycopeptides to the TiO2 resin. By combining TiO2 enrichment of sialylated glycopeptides with HILIC separation of deglycosylated peptides, a more comprehensive analysis of formerly sialylated glycopeptides by MS can be achieved. Here we illustrate the efficiency of the method by the identification of 1,632 unique formerly sialylated glycopeptides from 817 sialylated glycoproteins. The TiO2/HILIC protocol requires 2 d and the entire procedure from protein isolation can be performed in <5 d, depending on the time taken to analyze data.

Quantitative N-linked Glycoproteomics of Myocardial Ischemia and Reperfusion Injury Reveals Early Remodeling in the Extracellular Environment

Extracellular and cell surface proteins are generally modified with N-linked glycans and glycopeptide enrichment is an attractive tool to analyze these proteins. The role of N-linked glycoproteins in cardiovascular disease, particularly ischemia and reperfusion injury, is poorly understood. Observation of glycopeptides by mass spectrometry is challenging due to the presence of abundant, nonglycosylated analytes, and robust methods for purification are essential. We employed digestion with multiple proteases to increase glycoproteome coverage coupled with parallel glycopeptide enrichments using hydrazide capture, titanium dioxide, and hydrophilic interaction liquid chromatography with and without an ion-pairing agent. Glycosylated peptides were treated with PNGase F and analyzed by liquid chromatography-MS/MS. This allowed the identification of 1556 nonredundant N-linked glycosylation sites, representing 972 protein groups from ex vivo rat left ventricular myocardium. False positive “glycosylations” were observed on 44 peptides containing a deamidated Asn-Asp in the N-linked sequon by analysis of samples without PNGase F treatment. We used quantitation via isobaric tags for relative and absolute quantitation (iTRAQ) and validation with dimethyl labeling to analyze changes in glycoproteins from tissue following prolonged ischemia and reperfusion (40 mins ischemia and 20 mins reperfusion) indicative of myocardial infarction. The iTRAQ approach revealed 80 of 437 glycopeptides with altered abundance, while dimethyl labeling confirmed 46 of these and revealed an additional 62 significant changes. These were mainly from predicted extracellular matrix and basement membrane proteins that are implicated in cardiac remodeling. Analysis of N-glycans released from myocardial proteins suggest that the observed changes were not due to significant alterations in N-glycan structures. Altered proteins included the collagen-laminin-integrin complexes and collagen assembly enzymes, cadherins, mast cell proteases, proliferation-associated secreted protein acidic and rich in cysteine, and microfibril-associated proteins. The data suggest that cardiac remodeling is initiated earlier during reperfusion than previously hypothesized.

Site-specific glycan-peptide analysis for determination of N-glycoproteome heterogeneity.

A combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity from a complex mammalian protein mixture. Initially, global characterization of the N-glycome was performed using porous graphitized carbon liquid chromatography-tandem mass spectrometry (PGC-LC-MS/MS) and the data used to create an N-glycan modification database. In the next step, tryptic glycopeptides were enriched using zwitterionic hydrophilic interaction liquid chromatography (Zic-HILIC) and fractionated by reversed-phase liquid chromatography (RPLC; pH 7.9). The resulting fractions were each separated into two equal aliquots. The first set of aliquots were treated with peptide-N-glycosidase F (PNGase F) to remove N-glycans and the former N-glycopeptides analyzed by nano-RPLC-MS/MS (pH 2.7) and identified by Mascot database search. This enabled the creation of a glycopeptide-centric concatenated database for each fraction. The second set of aliquots was analyzed directly by nanoRPLC-MS/MS (pH 2.7), employing fragmentation by CID and HCD. The assignment of glycan compositions to peptide sequences was achieved by searching the N-glycopeptide HCD MS/MS spectra against the glycopeptide-centric concatenated databases employing the N-glycan modification database. CID spectra were used to assign glycan structures identified in the glycomic analysis to peptide sequences. This multidimensional approach allowed confident identification of 863 unique intact N-linked glycopeptides from 161 rat brain glycoproteins.

A Novel Method for the Simultaneous Enrichment, Identification, and Quantification of Phosphopeptides and Sialylated Glycopeptides Applied to a Temporal Profile of Mouse Brain Development

We describe a method that combines an optimized titanium dioxide protocol and hydrophilic interaction liquid chromatography to simultaneously enrich, identify and quantify phosphopeptides and formerly N-linked sialylated glycopeptides to monitor changes associated with cell signaling during mouse brain development. We initially applied the method to enriched membrane fractions from HeLa cells, which allowed the identification of 4468 unique phosphopeptides and 1809 formerly N-linked sialylated glycopeptides. We subsequently combined the method with isobaric tagging for relative quantification to compare changes in phosphopeptide and formerly N-linked sialylated glycopeptide abundance in the developing mouse brain. A total of 7682 unique phosphopeptide sequences and 3246 unique formerly sialylated glycopeptides were identified. Moreover 669 phosphopeptides and 300 formerly N-sialylated glycopeptides differentially regulated during mouse brain development were detected. This strategy allowed us to reveal extensive changes in post-translational modifications from postnatal mice from day 0 until maturity at day 80. The results of this study confirm the role of sialylation in organ development and provide the first extensive global view of dynamic changes between N-linked sialylation and phosphorylation.

Purification and identification of O-GlcNAc-modified peptides using phosphate-based alkyne CLICK chemistry in combination with titanium dioxide chromatography and mass spectrometry

A selective method for the enrichment of O-GlcNAcylated peptides using a novel CLICK chemistry reagent is described. Peptides modified by O-GlcNAc were enzymatically labeled with N-azidoacetylgalactosamine. The azide was then reacted with a phospho-alkyne using CLICK chemistry and O-GlcNAcGalNAzPO(4)-containing peptides were enriched using titanium dioxide chromatography. Modified peptides were analyzed using a combination of higher energy collision dissociation for identification and electron transfer dissociation to localize the site of O-GlcNAc attachment. The enrichment method was developed and optimized using an alpha-crystallin standard protein and then applied to a soluble protein preparation of mouse brain tissue and a nuclear preparation generated from HeLa cells. A total of 42 unique O-GlcNAcylated peptides were identified, including 7 novel O-GlcNAc sites.

Terminal Galactosylation and Sialylation Switching on Membrane Glycoproteins upon TNF-Alpha-Induced Insulin Resistance in Adipocytes

Insulin resistance (IR) is a complex pathophysiological state that arises from both environmental and genetic perturbations and leads to a variety of diseases, including type-2 diabetes (T2D). Obesity is associated with enhanced adipose tissue inflammation, which may play a role in disease progression. Inflammation modulates protein glycosylation in a variety of cell types, and this has been associated with biological dysregulation. Here, we have examined the effects of an inflammatory insult on protein glycosylation in adipocytes. We performed quantitative N-glycome profiling of membrane proteins derived from mouse 3T3-L1 adipocytes that had been incubated with or without the proinflammatory cytokine TNF-alpha to induce IR. We identified the regulation of specific terminal N-glycan epitopes, including an increase in terminal di-galactose- and a decrease in biantennary alpha-2,3-sialoglycans. The altered N-glycosylation of TNF-alpha-treated adipocytes correlated with the regulation of specific glycosyltransferases, including the up-regulation of B4GalT5 and Ggta1 galactosyltransferases and down-regulation of ST3Gal6 sialyltransferase. Knockdown of B4GalT5 down-regulated the terminal di-galactose N-glycans, confirming the involvement of this enzyme in the TNF-alpha-regulated N-glycome. SILAC-based quantitative glycoproteomics of enriched N-glycopeptides with and without deglycosylation were used to identify the protein and glycosylation sites modified with these regulated N-glycans. The combined proteome and glycoproteome workflow provided a relative quantification of changes in protein abundance versus N-glycosylation occupancy versus site-specific N-glycans on a proteome-wide level. This revealed the modulation of N-glycosylation on specific proteins in IR, including those previously associated with insulin-stimulated GLUT4 trafficking to the plasma membrane.

Human neutrophils secrete bioactive paucimannosidic proteins from azurophilic granules into pathogen-infected sputum

Background: Protein paucimannosylation is considered an important invertebrate- and plant-specific glycoepitope. Results: Azurophilic granule-specific human neutrophil proteins from pathogen-infected sputum displayed significant core-fucosylated paucimannosylation generated by maturation- and granule-specific β-hexosaminidase A and were preferentially secreted from non-lysosomal origins into sputum upon P. aeruginosa stimulation. Conclusion: Human neutrophils produce, store, and selectively secrete bioactive paucimannosidic proteins. Significance: This work will aid in understanding the function(s) of human paucimannosylation in glycoimmunology. Unlike plants and invertebrates, mammals reportedly lack proteins displaying asparagine (N)-linked paucimannosylation (mannose1–3fucose0–1N-acetylglucosamine2Asn). Enabled by technology advancements in system-wide biomolecular characterization, we document that protein paucimannosylation is a significant host-derived molecular signature of neutrophil-rich sputum from pathogen-infected human lungs and is negligible in pathogen-free sputum. Five types of paucimannosidic N-glycans were carried by compartment-specific and inflammation-associated proteins of the azurophilic granules of human neutrophils including myeloperoxidase (MPO), azurocidin, and neutrophil elastase. The timely expressed human azurophilic granule-resident β-hexosaminidase A displayed the capacity to generate paucimannosidic N-glycans by trimming hybrid/complex type N-glycan intermediates with relative broad substrate specificity. Paucimannosidic N-glycoepitopes showed significant co-localization with β-hexosaminidase A and the azurophilic marker MPO in human neutrophils using immunocytochemistry. Furthermore, promyelocyte stage-specific expression of genes coding for paucimannosidic proteins and biosynthetic enzymes indicated a novel spatio-temporal biosynthetic route in early neutrophil maturation. The absence of bacterial exoglycosidase activities and paucimannosidic N-glycans excluded exogenous origins of paucimannosylation. Paucimannosidic proteins from isolated and sputum neutrophils were preferentially secreted upon inoculation with virulent Pseudomonas aeruginosa. Finally, paucimannosidic proteins displayed affinities to mannose-binding lectin, suggesting immune-related functions of paucimannosylation in activated human neutrophils. In conclusion, we are the first to document that human neutrophils produce, store and, upon activation, selectively secrete bioactive paucimannosidic proteins into sputum of lungs undergoing pathogen-based inflammation.

Targeted phosphoproteomics of insulin signaling using data-independent acquisition mass spectrometry.

A mass spectrometry method may enable the simultaneous and rapid quantification of multiple phosphorylation events in multiple samples. Cross sample proteomics Nontargeted proteomic approaches are not well suited for comparisons of different samples and have detection limits, meaning that many biologically important but low abundance proteins are not detected. By applying a proteomic approach called DIA-MS (data-independent acquisition mass spectrometry) to an adipocyte cell line, Parker et al. showed that this method successfully mapped the phosphorylation events that occurred in response to insulin in a quantitative manner. Furthermore, by comparing the quantification of phosphorylation in cells exposed to various kinase inhibitors, the authors assigned specific kinases to many of phosphorylation events, which identified points of crosstalk between signaling pathways. This study provides proof of principle that this approach enables simultaneous analysis of phosphorylation events in multiple samples from different time points, doses, or patients. A major goal in signaling biology is the establishment of high-throughput quantitative methods for measuring changes in protein phosphorylation of entire signal transduction pathways across many different samples comprising temporal or dose data or patient samples. Data-independent acquisition (DIA) mass spectrometry (MS) methods, which involve tandem MS scans that are collected independently of precursor ion information and then are followed by targeted searching for known peptides, may achieve this goal. We applied DIA-MS to systematically quantify phosphorylation of components in the insulin signaling network in response to insulin as well as in stimulated cells exposed to a panel of kinase inhibitors targeting key downstream effectors in the network. We accurately quantified the effect of insulin on phosphorylation of 86 protein targets in the insulin signaling network using either stable isotope standards (SIS) or label-free quantification (LFQ) and mapped signal transmission through this network. By matching kinases to specific phosphorylation events (based on linear consensus motifs and temporal phosphorylation) to the quantitative phosphoproteomic data from cells exposed to inhibitors, we investigated predicted kinase-substrate relationships of AKT and mTOR in a targeted fashion. Furthermore, we applied this approach to show that AKT2-dependent phosphorylation of GAB2 promoted insulin signaling but inhibited epidermal growth factor (EGF) signaling in a manner dependent on 14-3-3 binding. Because DIA-MS can increase throughput and improve the reproducibility of peptide detection across multiple samples, this approach should facilitate more accurate, comprehensive, and quantitative assessment of signaling networks under various experimental conditions than are possible using other MS proteomic methods.

Global Phosphoproteomic Mapping of Early Mitotic Exit in Human Cells Identifies Novel Substrate Dephosphorylation Motifs

Entry into mitosis is driven by the coordinated phosphorylation of thousands of proteins. For the cell to complete mitosis and divide into two identical daughter cells it must regulate dephosphorylation of these proteins in a highly ordered, temporal manner. There is currently a lack of a complete understanding of the phosphorylation changes that occur during the initial stages of mitotic exit in human cells. Therefore, we performed a large unbiased, global analysis to map the very first dephosphorylation events that occur as cells exit mitosis. We identified and quantified the modification of >16,000 phosphosites on >3300 unique proteins during early mitotic exit, providing up to eightfold greater resolution than previous studies. The data have been deposited to the ProteomeXchange with identifier PXD001559. Only a small fraction (∼10%) of phosphorylation sites were dephosphorylated during early mitotic exit and these occurred on proteins involved in critical early exit events, including organization of the mitotic spindle, the spindle assembly checkpoint, and reformation of the nuclear envelope. Surprisingly this enrichment was observed across all kinase consensus motifs, indicating that it is independent of the upstream phosphorylating kinase. Therefore, dephosphorylation of these sites is likely determined by the specificity of phosphatase/s rather than the activity of kinase/s. Dephosphorylation was significantly affected by the amino acids at and surrounding the phosphorylation site, with several unique evolutionarily conserved amino acids correlating strongly with phosphorylation status. These data provide a potential mechanism for the specificity of phosphatases, and how they co-ordinate the ordered events of mitotic exit. In summary, our results provide a global overview of the phosphorylation changes that occur during the very first stages of mitotic exit, providing novel mechanistic insight into how phosphatase/s specifically regulate this critical transition.