The selective capture of target peptides poses a great challenge to modern chemists and biologists, especially when enriching them from proteome samples possessing extremes in concentration dynamic range and sequence diversity. While approaches based on traditional techniques such as biotin-avidin pairing offer versatile tools to design strategies for selective enrichment, problems are still encountered due to sample loss or poor selectivity of enrichment. Here we show that the recently introduced fluorous chemistry approach has attractive properties as an alternative method for selective enrichment. Through appending a perfluorine group to the target peptide, it is possible to dramatically increase the peptide's hydrophobicity and thus enable facile separation of labeled from non-labeled peptides. Use of reversed-phase chromatography allowed for improved peptide recovery in comparison with results obtained using the formerly reported fluorous bonded phase methods. Furthermore, this approach also allowed for on-line separation and identification of both labeled and unlabeled peptides in a single experiment. The net result is an increase in the confidence of protein identification by tandem mass spectrometry (MS2) as all peptides and subsequent information are retained. Successful off-line and on-line enrichment of cysteine-containing peptides was obtained, and high quality MS2 spectra were obtained by tandem mass spectrometry due to the stability of the tag, allowing for facile identification via standard database searching. We believe that this strategy holds great promise for selective enrichment and identification of low abundance target proteins or peptides.
Oral sodium phenyl butyrate (SPB) is currently under investigation as a histone deacetylation (HDAC) inhibitor in Huntington disease (HD). Ongoing studies indicate that symptoms related to HD genetic abnormalities decrease with SPB therapy. In a recently reported safety and tolerability study of SPB in HD, we analyzed overall chromatographic patterns from a method that employs gradient Liquid Chromatography with series Electrochemical array, UV and Fluorescence (LCECA/UV/F) for measuring SPB and its metabolite phenylacetate (PA). We found that plasma and urine from SPB-treated patients yielded individual-specific patterns of ca. 20 metabolites which may provide a means for the selection of subjects for extended trials of SPB. The structural identification of these metabolites is of critical importance, since their characterization will facilitate understanding the mechanisms of drug action and possible side effects. We have now developed an iterative process with LCECA, parallel LCECA/LCMS, and high performance tandem MS, for metabolite characterization. We report here the details of this method and its use for identification of 10 plasma and urinary metabolites in treated subjects, including indole species in urine that are not themselves metabolites of SPB. This approach thus contributes to understanding metabolic pathways that differ among HD individuals being treated with SPB.
Sodium phenyl butyrate; Huntington disease; LCECA; metabolites; parallel LC-EC-array/LC-MS; tandem mass spectrometry
Transthyretin (TTR) amyloidosis and hemoglobinopathies are the archetypes of molecular diseases where point mutation characterization is diagnostically critical. We have developed a Top-down analytical platform for variant and/or modified protein sequencing and are examining the feasibility of using this platform for the analysis of hemoglobin/TTR patient samples and evaluating the potential clinical applications. The platform is based on a commercial high resolution hybrid orbitrap mass spectrometer (LTQ-Orbitrap™) with automated sample introduction; automated data analysis is performed by our own software algorithm (BUPID topdown).
The analytical strategy consists of iterative data capture, first recording a mass profile of the protein(s). The presence of a variant is revealed by a mass shift consistent with the amino acid substitution. Nozzle-skimmer dissociation (NSD) of the protein(s) yields a wide variety of sequence-defining fragment ions. The fragment ion containing the amino acid substitution or modification can be identified by searching for a peak exhibiting the mass shift observed in the protein mass profile. This fragment ion can then be selected for MS/MS analysis in the ion trap to yield sequence information permitting the identification of the variant. Substantial sequence coverage has been obtained in this manner. This strategy allows for a stepwise MS/MS analysis of the protein structure. The sequence information obtained can be supplemented with whole protein NSD fragmentation and MS/MS analysis of specific protein charge states. The analyses of variant forms of TTR and hemoglobin are presented to illustrate the potential of the method.
Top-down; proteins; variants; transthyretin; hemoglobin; post-translational modifications
Tularemia is a severe infectious disease in humans caused by the gram-negative bacterium Francisella tularensis (Ft). Due to its low infectious dose, high mortality rate, and the threat of its large-scale dissemination in weaponized form, development of vaccines and immunotherapeutics against Ft is essential. Ft lipopolysaccharide (LPS), which contains the linear graded-length saccharide component O-antigen (OAg) attached to a core oligosaccharide, has been reported as a protective antigen. Purification of LPS saccharides of defined length and composition is necessary to reveal the epitopes targeted by protective antibodies. In this study, we purified saccharides from LPS preparations from both the Ft subspecies holarctica live vaccine strain (LVS) and the virulent Ft subspecies tularensis SchuS4 strain using liquid chromatography. We then characterized the fractions using high resolution mass spectrometry and tandem mass spectrometry. Three types of saccharides were observed in both the LVS and SchuS4 preparations: two consisting of OAg tetrasaccharide repeats attached to one of two core-oligosaccharide variants, and one consisting of tetrasaccharide repeats only (coreless). The coreless OAg oligosaccharides were shown to contain Qui4NFm (4,6-dideoxy-4-formamido-D-glucose) at the non-reducing end and QuiNAc (2-acetamido-2,6-dideoxy-O-D-glucose) at the reducing end. Purified homogeneous preparations of saccharides of each type will allow mapping of protective epitopes in Ft LPS.
Little is known about the antigen specificity of CD1d-restricted T cells, except that they frequently recognize CD1d-expressing antigen-presenting cells in the absence of exogenous antigen. We previously demonstrated that the 24.8.A iNKT cell hybridoma was broadly reactive with CD1d-transfected cell lines and recognized the polar lipid fraction of a tumor cell extract. In the present study, the antigen recognized by the 24.8.A iNKT cell hybridoma was purified to homogeneity and identified as palmitoyl-oleoyl-sn-glycero-3-phosphoethanolamine (16:0–18:1 PE). The 24.8.A iNKT cell hybridoma recognized synthetic 16:0-18:1[cis] PE, confirming that this phospholipid is antigenic. Recognition correlated with the degree of unsaturation of the acyl chains. Using a panel of synthetic PEs, the 24.8.A iNKT cell hybridoma was shown to be activated by PEs that contained at least one unsaturated acyl chain. The configuration of the double bonds was important, as the 24.8.A iNKT cell hybridoma recognized unsaturated acyl chains in the cis, but not the trans, configuration. PEs with multiple double bonds were recognized better than those with a single double bond, and increasing acyl chain unsaturation correlated with increased binding of PE to CD1d. These data illustrate the potential importance of the acyl chain structure for phospholipid antigen binding to CD1d.
PMID: 12963715 CAMSID: cams2348
The walls of infectious pathogens, which are essential for transmission, pathogenesis, and diagnosis, contain sugar polymers that are defining structural features, e.g., β-1,3-glucan and chitin in fungi, chitin in Entamoeba cysts, β-1,3-GalNAc in Giardia cysts, and peptidoglycans in bacteria. The goal here was to determine in which of three walled forms of Toxoplasma gondii (oocyst, sporocyst, or tissue cyst) is β-1,3-glucan, the product of glucan synthases and glucan hydrolases predicted by whole-genome sequences of the parasite. The three most important discoveries were as follows. (i) β-1,3-glucan is present in oocyst walls of Toxoplasma and Eimeria (a chicken parasite that is a model for intestinal stages of Toxoplasma) but is absent from sporocyst and tissue cyst walls. (ii) Fibrils of β-1,3-glucan are part of a trabecular scaffold in the inner layer of the oocyst wall, which also includes a glucan hydrolase that has a novel glucan-binding domain. (iii) Echinocandins, which target the glucan synthase and kill fungi, arrest development of the Eimeria oocyst wall and prevent release of the parasites into the intestinal lumen. In summary, β-1,3-glucan, which can be targeted by drugs, is an important component of oocyst walls of Toxoplasma but is not a component of sporocyst and tissue cyst walls.
We show here that walls of Toxoplasma oocysts, the infectious stage shed by cats, contain β-1,3-glucan, a sugar polymer that is a major component of fungal walls. In contrast to fungi, β-1,3-glucan is part of a trabecular scaffold in the inner layer of the oocyst wall that is independent of the permeability barrier formed by the outer layer of the wall. While glucan synthase inhibitors kill fungi, these inhibitors arrest the development of the oocyst walls of Eimeria (an important chicken pathogen that is a surrogate for Toxoplasma) and block release of oocysts into the intestinal lumen. The absence of β-1,3-glucan in tissue cysts of Toxoplasma suggests that drugs targeted at the glucan synthase might be used to treat Eimeria in chickens but not to treat Toxoplasma in people.
Myelin protein zero (P0), a glycosylated single-pass transmembrane protein, is essential in the formation and maintenance of peripheral nervous system (PNS) compact myelin. P0 in Xenopus (xP0) exists primarily as a dimeric form that remains stable after various physical and chemical treatments. In exploring the nature of the interactions underlying the dimer stability, we found that xP0 dimer dissociated into monomer during continuous elution gel electrophoresis and conventional SDS-PAGE, indicating that the dimer is stabilized by non-covalent interactions. Furthermore, as some of the gel-purified monomer re-associated into dimer on SDS-PAGE gels, there is likely a dynamic equilibrium between xP0 dimer and monomer in vivo. Because the carbohydrate and fatty acyl moieties may be crucial for the adhesion role of P0, we used sensitive mass spectrometry approaches to elucidate the detailed N-glycosylation and S-acylation profiles of xP0. Asn92 was determined to be the single, fully-occupied glycosylation site of xP0, and a total of 12 glycans was detected that exhibited new structural features compared with those observed from P0 in other species: (1) the neutral glycans were composed mainly of high mannose and hybrid types; (2) five of twelve were acidic glycans, among which three were sialylated and the other two were sulfated; (3) none of the glycans had core fucosylation; and (4) no glucuronic acid, hence no HNK-1 epitope, was detected. The drastically different carbohydrate structures observed here support the concept of the species-specific variation in N-glycosylation of P0. Cys152 was found to be acylated with stearoyl (C18:0), whereas palmitoyl (C16:0) is the corresponding predominant fatty acyl group on P0 from higher vertebrates. We propose that the unique glycosylation and acylation patterns of Xenopus P0 may underlie its unusual dimerization behaviour. Our results should shed light on the understanding of the phylogenetic development of P0’s adhesion role in PNS compact myelin.
De novo N-linked glycan sequencing; mass spectrometry; species-specific N-glycosylation
Heparan Sulfate (HS) and heparin are linear, heterogeneous carbohydrates of the glycosaminoglycan (GAG) family that are modified by N-acetylation, N-sulfation, O-sulfation, and uronic acid epimerization. HS interacts with growth factors in the extracellular matrix, thereby modulating signaling pathways that govern cell growth, development, differentiation, proliferation, and adhesion. HPLC-chip-based hydrophilic interaction liquid chromatography/mass spectrometry has emerged as a method for analyzing the domain structure of GAGs. However, analysis of highly sulfated GAG structures decasaccharide or larger in size has been limited by spray instability in the negative-ion mode. This report demonstrates that addition of post-column make-up flow to the amide-HPLC-chip configuration permits robust and reproducible analysis of extended GAG domains (up to degree of polymerization 18) from HS and heparin. This platform provides quantitative information regarding oligosaccharide profile, degree of sulfation, and non-reducing chain termini. It is expected that this technology will enable quantitative, comparative glycomics profiling of extended GAG oligosaccharide domains of functional interest.
Sirtuin-1 (SIRT1) is an NAD+-dependent protein deacetylase that is sensitive to oxidative signals. Our purpose was to determine whether SIRT1 activity is sensitive to the low molecular weight nitrosothiol, S-nitrosoglutathione (GSNO), which can transduce oxidative signals into physiological responses. SIRT1 formed mixed disulfides with GSNO-Sepharose, and mass spectrometry identified several cysteines that are modified by GSNO, including Cys-67 which was S-glutathiolated. GSNO had no effect on basal SIRT1deacetylase activity, but inhibited stimulation of activity by resveratrol (RSV) with an IC50 of 69 μM. These observations indicate that S-glutathiolation of SIRT1 by low concentrations of reactive glutathione can modulate its enzymatic activity.
Antioxid. Redox Signal. 13, 1023–1032.
Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, has a truncated Asn-linked glycan (N-glycan) precursor composed of seven sugars (Man5GlcNAc2). Here, we show that glycoproteins with unmodified N-glycans are aggregated and capped on the surface of E. histolytica trophozoites by the antiretroviral lectin cyanovirin-N and then replenished from large intracellular pools. Cyanovirin-N cocaps the Gal/GalNAc adherence lectin, as well as glycoproteins containing O-phosphodiester-linked glycans recognized by an anti-proteophosphoglycan monoclonal antibody. Cyanovirin-N inhibits phagocytosis by E. histolytica trophozoites of mucin-coated beads, a surrogate assay for amebic virulence. For technical reasons, we used the plant lectin concanavalin A rather than cyanovirin-N to enrich secreted and membrane proteins for mass spectrometric identification. E. histolytica glycoproteins with occupied N-glycan sites include Gal/GalNAc lectins, proteases, and 17 previously hypothetical proteins. The latter glycoproteins, as well as 50 previously hypothetical proteins enriched by concanavalin A, may be vaccine targets as they are abundant and unique. In summary, the antiretroviral lectin cyanovirin-N binds to well-known and novel targets on the surface of E. histolytica that are rapidly replenished from large intracellular pools.
Secondary fragmentations of three synthetic peptides (human αA crystallin peptide 1-11, the deamidated form of human βB2 crystallin peptide 4-14, and amyloid β peptide 25-35) were studied in both electron capture dissociation (ECD) and electron transfer dissociation (ETD) mode. In ECD, in addition to c and z• ion formations, charge remote fragmentations (CRF) of z• ions were abundant, resulting in internal fragment formation or partial/entire side chain losses from amino acids, sometimes several residues away from the backbone cleavage site, and to some extent multiple side chain losses. The internal fragments were observed in peptides with basic residues located in the middle of the sequences, which was different from most tryptic peptides with basic residues located at the C-terminus. These secondary cleavages were initiated by hydrogen abstraction at the α-, β-, or γ-position of the amino acid side chain. In comparison, ETD generates fewer CRF fragments than ECD. This secondary cleavage study will facilitate ECD/ETD spectra interpretation, and help de novo sequencing and database searching.
In order that biological meaning may be derived and testable hypotheses may be built from proteomics experiments, assignments of proteins identified by mass spectrometry or other techniques must be supplemented with additional notation, such as information on known protein functions, protein-protein interactions, or biological pathway associations. Collecting, organizing, and interpreting this data often requires the input of experts in the biological field of study, in addition to the time-consuming search for and compilation of information from online protein databases. Furthermore, visualizing this bulk of information can be challenging due to the limited availability of easy-to-use and freely available tools for this process. In response to these constraints, we have undertaken the design of software to automate annotation and visualization of proteomics data in order to accelerate the pace of research. Here we present the Software Tool for Researching Annotations of Proteins (STRAP) – a user-friendly, open-source C# application. STRAP automatically obtains gene ontology (GO) terms associated with proteins in a proteomics results ID list using the freely accessible UniProtKB and EBI GOA databases. Summarized in an easy-to-navigate tabular format, STRAP includes meta-information on the protein in addition to complimentary GO terminology. Additionally, this information can be edited by the user so that in-house expertise on particular proteins may be integrated into the larger dataset. STRAP provides a sortable tabular view for all terms, as well as graphical representations of GO-term association data in pie (biological process, cellular component and molecular function) and bar charts (cross comparison of sample sets) to aid in the interpretation of large datasets and differential analyses experiments. Furthermore, proteins of interest may be exported as a unique FASTA-formatted file to allow for customizable re-searching of mass spectrometry data, and gene names corresponding to the proteins in the lists may be encoded in the Gaggle microformat for further characterization, including pathway analysis. STRAP, a tutorial, and the C# source code are freely available from http://cpctools.sourceforge.net.
proteomics; annotation; software; mass spectrometry; data analysis
NSP protein family members associate with p130Cas, a focal adhesion adapter protein best known as a Src substrate that integrates adhesion-related signaling. Over-expression of AND-34/BCAR3/NSP2 (BCAR3), but not NSP1 or NSP3, induces anti-estrogen resistance in human breast cancer cell lines. BCAR3 over-expression in epithelial MCF-7 cells augments levels of a phosphorylated p130Cas species that migrates more slowly on SDS PAGE while NSP-1 and NSP3 induce modest or no phosphorylation, respectively. Conversely, reduction in BCAR3 expression in mesenchymal MDA-231 cells by inducible shRNA results in loss of such p130Cas phosphorylation. Replacement of NSP3's serine/proline-rich domain with that of AND-34/BCAR3 instills the ability to induce p130Cas phosphorylation. Phospho-amino acid analysis demonstrates that BCAR3 induces p130Cas serine phosphorylation. Mass spectrometry identified phosphorylation at p130Cas serines 139, 437 and 639. p130Cas serine phosphorylation accumulates for several hours after adhesion of MDA-231 cells to fibronectin and is dependent upon BCAR3 expression. BCAR3 knockdown alters p130Cas localization and converts MDA-231 growth to an epithelioid pattern characterized by striking cohesiveness and lack of cellular projections at colony borders. These studies demonstrate that BCAR3 regulates p130Cas serine phosphorylation that is adhesion-dependent, temporally distinct from previously well-characterized rapid Fak and Src kinase-mediated p130Cas tyrosine phosphorylation and that correlates with invasive phenotype.
BCAR3; p130Cas; breast cancer; adhesion; phosphorylation; anti-estrogen resistance
The infectious and diagnostic stage of Giardia lamblia (also known as G. intestinalis or G. duodenalis) is the cyst. The Giardia cyst wall contains fibrils of a unique β-1,3-linked N-acetylgalactosamine (GalNAc) homopolymer and at least three cyst wall proteins (CWPs) composed of Leu-rich repeats (CWPLRR) and a C-terminal conserved Cys-rich region (CWPCRR). Our goals were to dissect the structure of the cyst wall and determine how it is disrupted during excystation. The intact Giardia cyst wall is thin (∼400 nm), easily fractured by sonication, and impermeable to small molecules. Curled fibrils of the GalNAc homopolymer are restricted to a narrow plane and are coated with linear arrays of oval-shaped protein complex. In contrast, cyst walls of Giardia treated with hot alkali to deproteinate fibrils of the GalNAc homopolymer are thick (∼1.2 µm), resistant to sonication, and permeable. The deproteinated GalNAc homopolymer, which forms a loose lattice of curled fibrils, is bound by native CWP1 and CWP2, as well as by maltose-binding protein (MBP)-fusions containing the full-length CWP1 or CWP1LRR. In contrast, neither MBP alone nor MBP fused to CWP1CRR bind to the GalNAc homopolymer. Recombinant CWP1 binds to the GalNAc homopolymer within secretory vesicles of Giardia encysting in vitro. Fibrils of the GalNAc homopolymer are exposed during excystation or by treatment of heat-killed cysts with chymotrypsin, while deproteinated fibrils of the GalNAc homopolymer are degraded by extracts of Giardia cysts but not trophozoites. These results show the Leu-rich repeat domain of CWP1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. During excystation, host and Giardia proteases appear to degrade bound CWPs, exposing fibrils of the GalNAc homopolymer that are digested by a stage-specific glycohydrolase.
While the walls of plants and fungi contain numerous sugar homopolymers (cellulose, chitin, and β-1,3-glucans) and dozens of proteins, the cyst wall of Giardia is relatively simple. The Giardia wall contains a unique homopolymer of β-1,3-linked N-acetylgalactosamine (GalNAc) and at least three cyst wall proteins (CWPs), each of which is composed of Leu-rich repeats and a C-terminal Cys-rich region. The three major discoveries here are: 1) Fibrils of the GalNAc homopolymer are curled and form a lattice that is compressed into a narrow plane by bound protein in intact cyst walls. 2) Leu-rich repeats of CWP1 form a novel lectin domain that is specific for fibrils of the GalNAc homopolymer, which can be isolated by methods used to deproteinate fungal walls. 3) A cyst-specific glycohydrolase is able to degrade deproteinated fibrils of the GalNAc homopolymer. We incorporate these findings into a new curled fiber and lectin model of the intact Giardia cyst wall and a protease and glycohydrolase model of excystation.
Immunoglobulin light chain amyloidosis is a protein misfolding disease in which a monoclonal immunoglobulin (Ig) light chain (LC) with a critically folded β-conformation self-aggregates to form highly ordered, nonbranching amyloid fibrils. The insoluble nature of amyloid fibrils ultimately results in the extracellular deposition of the LC in tissues and organs throughout the body. Structural features that confer amyloidogenic properties on an Ig LC likely include amino acid sequence variations and post-translational modifications, but the specific natures of these changes remain to be defined. As part of an exploration of the effective range of amyloidogenic modifications, this study details the structural and genetic analyses of nine κ1 LC proteins. Urinary LCs were purified by size exclusion chromatography using FPLC, and structural analyses were performed by electrospray ionization, matrix-assisted laser desorption/ionization, and tandem mass spectrometry. RT-PCR amplification, cloning, and sequencing of the monoclonal LC genes were accomplished using bone marrow-derived mRNA. Clinical data were reviewed retrospectively. Characterization of the urinary κ1 LCs revealed extensive post-translational modification in all proteins, in addition to somatic mutations expected on the basis of results from genetic analyses. Post-translational modifications included disulfide-linked dimerization, S-cysteinylation, glycosylation, fragmentation, S-sulfonation, and 3-chlorotyrosine formation. Genetic analyses showed that several LC variable region germline gene donors were represented including O18/O8, O12/O2, L15, and L5. Clinical features included soft tissue, cardiac, renal, and hepatic involvement. This study demonstrated the extensive heterogeneity in primary structure, post-translational modifications, and germline gene usage that occurred in nine amyloidogenic κ1 LC proteins.
In this study, the membrane lipids of B. burgdorferi were separated into 16 fractions; the components in each fraction were identified, and the immunogenicity of each fraction was determined by ELISA using sera from Lyme disease patients. Only the 2 glycolipids, acylated cholesteryl galactoside (ACG, BbGL-I) and monogalactosyl diacylglycerol (MgalD, BbGL-II), were immunogenic. Early in the infection, 24 of 84 patients (29%) who were convalescent from erythema migrans and 19 of the 35 patients (54%) with neuroborreliosis had weak IgG responses to purified MgalD, and a smaller percentage of patients had early responses to synthetic ACG. However, almost all of 75 patients with Lyme arthritis, a late disease manifestation, had strong IgG reactivity with both glycolipids. Thus, almost all patients with Lyme arthritis have strong IgG antibody responses to B. burgdorferi glycolipid antigens.
Lyme disease; Bacterial infection; Borrelia burgdorferi; Antibodies; Glycolipid antigens
We use affinity purification of the double bromodomain protein Brd2 to isolate a multicomponent nuclear complex from cultured cells, and apply mass spectrometry/proteomics methods to identify the participants. We then confirm by immunoblot several transcription co-activators and co-repressors, proteins of the Swi/Snf chromatin remodeling complex, which regulate transcription control of cyclin A. This multiprotein complex is likely to contribute to cell cycle control and play a role in proliferation and cancer.
bromodomain; multiprotein complexes; affinity chromatography; high pressure liquid chromatography; mass spectral analysis; transcription factors; TATA binding protein-associated factors; chromatin assembly and disassembly; cyclin A
The recent discovery of dideoxymycobactin (DDM) as a ligand for CD1a demonstrates how a nonribosomal lipopeptide antigen is presented to T cells. DDM contains an unusual acylation motif and a peptide sequence present only in mycobacteria, but its discovery raises the possibility that ribosomally produced viral or mammalian proteins that commonly undergo lipidation might also function as antigens. To test this, we measured T cell responses to synthetic acylpeptides that mimic lipoproteins produced by cells and viruses. CD1c presented an N-acyl glycine dodecamer peptide (lipo-12) to human T cells, and the response was specific for the acyl linkage as well as the peptide length and sequence. Thus, CD1c represents the second member of the CD1 family to present lipopeptides. lipo-12 was efficiently recognized when presented by intact cells, and unlike DDM, it was inactivated by proteases and augmented by protease inhibitors. Although lysosomes often promote antigen presentation by CD1, rerouting CD1c to lysosomes by mutating CD1 tail sequences caused reduction in lipo-12 presentation. Thus, although certain antigens require antigen processing in lysosomes, others are destroyed there, providing a hypothesis for the evolutionary conservation of large CD1 families containing isoforms that survey early endosomal pathways.
In addition to hemostasis, platelets mediate inflammation and clearance of bacteria from the bloodstream. As with platelet-platelet interactions, platelet-bacteria interactions involve cytoskeletal rearrangements and release of granular content. Stimulation of the immune Toll-like receptor 2 (TLR2) on the platelet surface, activates phosphoinositide-3-kinase (PI3K) and causes platelet activation and platelet-dependent thrombosis. It remains unknown if platelet activation by immune vs. thrombotic pathways leads to the differential regulation of signal transduction, protein-protein interactions, and α-granule release, and the physiological relevance of these potential differences. We investigated these processes after immune vs. thrombotic platelet stimulation. We examined selected signaling pathways and found that phosphorylation kinetics of Akt, ERK1/2 and p38 differed dramatically between agonists. Next, we investigated platelet protein-protein interactions by mass spectrometry (MS)-based proteomics specifically targeting cytosolic factor XIIIa (FXIIIa) because of its function as a cytoskeleton-crosslinking protein whose binding partners have limited characterization. Four FXIIIa-binding proteins were identified, two of which are novel interactions: FXIIIa-focal adhesion kinase (FAK) and FXIIIagelsolin. The binding of FAK to FXIIIa was found to be altered differentially by immune vs. thrombotic stimulation. Lastly, we studied the effect of thrombin- vs. Pam3CSK4-stimulation on α-granule release and observed differential release patterns for selected granule proteins and decreased fibrin clot formation compared with thrombin. The inhibition of PI3K caused a decrease in protein release after Pam3CSK4- but not after thrombin-stimulation. In summary, stimulation of plates by either thrombotic or immune receptors leads to markedly different signaling responses and granular protein release consistent with differential contribution to coagulation and thrombosis.
Toll-like receptor 2; thrombin; signal transduction; mass spectrometry-based proteomics; factor XIIIA
A key challenge to investigations into the functional roles of glycosaminoglycans (GAGs) in biological systems is the difficulty in achieving sensitive, stable and reproducible mass spectrometric analysis. GAGs are linear carbohydrates with domains that vary in the extent of sulfation, acetylation and uronic acid epimerization. It is of particular importance to determine spatial and temporal variations of GAG domain structures in biological tissues. In order to analyze GAGs from tissue, it is useful to couple mass spectrometry with an on-line separation system. The purposes of the separation system are both to remove components that inhibit GAG ionization and to enable the analysis of very complex mixtures. This contribution presents amide-silica hydrophilic interaction chromatography (HILIC) in a chip-based format for LC/MS of heparin, heparan sulfate and chondroitin/dermatan sulfate GAGs. The chip interface yields robust performance in the negative ion mode that is essential for GAGs and other acidic glycan classes while the built-in trapping cartridge reduces background from the biological tissue matrix. The HILIC chromatographic separation is based on a combination of the glycan chain lengths and the numbers of hydrophobic acetate groups and acidic sulfate groups. In summary, chip based amide-HILIC LC/MS is an enabling technology for GAG glycomics profiling.
Heparan sulfate (HS) is a sulfated glycosaminoglycan attached to a core protein on the cell surface. Protein binding to cell surface Heparan sulfate (HS) is a key regulatory event for many cellular processes. The concept whereby protein binding to HS is not random but requires a limited number of sulfation patterns is becoming clear. Here we describe a hydrophobic trapping assay to screen a library of heparin hexasaccharides for binders to Antithrombin III (ATIII). Out of five initial hexasaccharide compositions present in the library (1:2:3:6:1), (1:2:3:7:1), (1:2:3:7:0), (1:2:3:8:0), (1:2:3:9:0) only two are shown to be able to bind ATIII, namely (1:2:3:8:0) and (1:2:3:9:0). The use of an amide hydrophilic interaction (HILIC) LC/MS permitted reproducible quantitative analysis of the composition of the initial library as well as that of the binding fraction. This type of LC/MS has never been applied to heparinoids. The specificity of the hexasaccharides binding ATIII was confirmed by assaying their ability to enhance ATIII mediated inhibition of Factor Xa in vitro.
Transthyretin (TTR) is a serum protein that is also a prominent component of deposits in two different types of systemic amyloid disease, senile systemic and familial TTR amyloidoses. Studies of recombinant TTR (rTTR) have provided many insights into the relationship between protein structure and amyloidogenicity. Yet, there is no existing recombinant system that results in high yield production of a protein that is identical in primary structure to human TTR. To date, most published studies have generated rTTR using the human gene sequence, which is poorly expressed in E. coli. In addition, the gene sequence has been flanked by a 3′ AUG start codon to initiate translation, resulting in the expression of a protein containing an N-terminal methionine residue not present in the human protein. We present an improved technique which can be used to generate large quantities of human native sequence TTR. Our recombinant system utilizes a gene containing codons altered for efficient expression in E. coli and an N-terminal polyhistidine tag for simplified purification. Optimization of this system was accomplished by generating a modified polyhistidine tag that was efficiently removed by dipeptidyl aminopeptidase I (DAPase). This is the first report detailing an effective and useful method for producing rTTR containing an amino acid sequence identical to human TTR. Furthermore, we describe the thiol modification of the recombinant protein to achieve exact replication of the several prominent post-translationally modified forms of TTR that have been identified in human serum.
P0 glycoprotein is the major structural protein of peripheral nerve myelin where it is thought to modulate inter-membrane adhesion at both the extracellular apposition, which is labile upon changes in pH and ionic strength, and the cytoplasmic apposition, which is resistant to such changes. Most studies on P0 have focused on structure-function correlates in higher vertebrates. Here, we focused on its role in the structure and interactions of frog (Xenopus laevis) myelin, where it exists primarily in a dimeric form. As part of our study, we deduced the full sequence of Xenopus laevis P0 (xP0) from its cDNA. The xP0 sequence was found to be similar to P0 sequences of higher vertebrates, suggesting that a common mechanism of PNS myelin compaction via P0 interaction might have emerged through evolution. As previously reported for mouse PNS myelin, a similar change of extracellular apposition in frog PNS myelin as a function of pH and ionic strength was observed, which can be explained by a conformational change of P0 due to protonation-deprotonation of His52 at P0’s putative adhesive interface. On the other hand, the cytoplasmic apposition in frog PNS myelin, like that in the mouse, remained unchanged at different pH and ionic strength. The contribution of hydrophobic interactions to stabilizing the cytoplasmic apposition was tested by incubating sciatic nerves with detergents. Dramatic expansion at the cytoplasmic apposition was observed for both frog and mouse, indicating a common hydrophobic nature at this apposition. Urea also expanded the cytoplasmic apposition of frog myelin likely owing to denaturation of P0. Removal of the fatty acids that attached to the single Cys residue in the cytoplasmic domain of P0 did not change PNS myelin structure of either frog or mouse, suggesting that the P0-attached fatty acyl chain does not play a significant role in PNS myelin compaction and stability. These results help clarify the present understanding of P0’s adhesion role and the role of its acylation in compact PNS myelin.
x-ray diffraction; membrane-membrane interactions; protein zero cDNA; PNS; detergents; hydroxylamine; adhesion protein; frog; mass spectrometry
Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix, consisting of an integrated assembly of collagens, glycoproteins, proteoglycans and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed phase and strong anion exchange solid phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid- d4 and subjected to size exclusion chromatography with on-line electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.
cartilage; glycomics; glycosaminoglycan; mass spectrometry