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PubMed2006

Molecular characterization and comparison of the components and multiprotein complexes in the postsynaptic proteome.

Characterization of the composition of the postsynaptic proteome (PSP) provides a framework for understanding the overall organization and function of the synapse in normal and pathological conditions. We have identified 698 proteins from the postsynaptic terminal of mouse CNS synapses using a series of purification strategies and analysis by liquid chromatography tandem mass spectrometry and large-scale immunoblotting. Some 620 proteins were found in purified postsynaptic densities (PSDs), nine in AMPA-receptor immuno-purifications, 100 in isolates using an antibody against the NMDA receptor subunit NR1, and 170 by peptide-affinity purification of complexes with the C-terminus of NR2B. Together, the NR1 and NR2B complexes contain 186 proteins, collectively referred to as membrane-associated guanylate kinase-associated signalling complexes. We extracted data from six other synapse proteome experiments and combined these with our data to provide a consensus on the composition of the PSP. In total, 1124 proteins are present in the PSP, of which 466 were validated by their detection in two or more studies, forming what we have designated the Consensus PSD. These synapse proteome data sets offer a basis for future research in synaptic biology and will provide useful information in brain disease and mental disorder studies.

Animals↗
PubMed2002

Global proteome analysis of a human gastric carcinoma.

An approach that combines analysis of global protein digests (GPDs) of various subcellular fractions with a novel chromatographic-based method to map protein expression profiles is described. The KATO III gastric carcinoma cell line was fractionated into membrane and cytosol fractions. Each subcellular fraction was digested with trypsin to yield complex mixtures of global protein tags (GPTs). These mixtures were fractionated by two dimensions of chromatography, and GPTs were sequenced by microcapillary liquid chromatography-tandem mass spectrometry (LC-MS/MS), using two further complementary dimensions of chromatography. Additionally, a novel method of protein expression profiling was used to map the KATO III human gastric carcinoma cell line. This method uses the cells' natural proteolytic processes to derive in vivo peptide tags that represent proteins of every functional class and from all subcellular compartments. In one example, expressed protein tags (EPTs) are naturally displayed on the surface of cells by multiligand receptors. Isolation and sequence identification of EPTs is an efficient approach for protein profiling that is complementary to GPT analysis. The EPT approach also provides a further unique subcellular fraction of the biological starting material. Isolation of the multiligand receptors was by immunoaffinity chromatography (IAC). In the current study, five individual peptide maps (two EPTs and three GPTs) of the KATO III cell line were fractionated by multimodal chromatography, and sequenced by on-line multimodal microcapillary LC-MS/MS. This analysis led to the identification of 4291 individual peptide sequences, which defined 1966 unique proteins expressed by this human carcinoma cell line.

Chromatography, Affinity↗
PubMed2001

Similarity between condensed phase and gas phase chemistry: fragmentation of peptides containing oxidized cysteine residues and its implications for proteomics.

Amino acid residues containing thioethers are easily oxidized during protein purification, derivatization, and/or digestion. For instance, oxidation of methionine residues in proteins during SDS-PAGE is commonly observed. Under low energy collision induced dissociation this gives rise to a second series of fragment ion of lower abundance that are shifted by -64 Da when compared to the oxidized methionine-containing fragments. We report here that alkylated cysteine residues can be found in their oxidized form too, indicating that the oxidation of thioethers can occur during and following protein digestion and not only during SDS-PAGE or reduction and alkylation. Collision induced dissociation experiments on the singly- and multiply-charged species reveals that these peptides preferentially undergo elimination reactions that forms a dehydroalanine from the oxidized, alkylated cysteine residue. This contrasts to the less abundant elimination reaction of peptides containing oxidized methionines which cannot form an alpha,beta-unsaturated compound, but parallels the condensed phased chemistry of sulfoxides. The masses of both precursor and product ions are shifted such that these peptides cannot be identified in database searches with current algorithms. Incorporation of this fragmentation pattern is important for the isotope-coded affinity tag approach since this method is based on peptides containing cysteine residues.

Cysteine↗
PubMed2005

[Proteomics and diagnostic application].

Proteomics research possesses even greater potential than the achievements of genomics research. Clinical tests which more accurately identify pathology are needed. Clinical tests are also classified in terms of the metabolome. On the other hand, the techniques of proteomics research appear to be useful for developing diagnostic systems, not just searching for biomarkers. 2DG and the mass spectrometer are representative proteomics tools, and are now being used in blood tests. It is possible that systemic manifestations will come to be understood on the basis of the data obtained from them, and that they will be used for a variety of screening tests. Use of one of them, SELDI, is particularly interesting. High-throughput protein expression techniques, for example, phage display and the IVV method, are becoming useful for producing innovative high-affinity diagnostic antibodies. Protein chips that use ELISA and fluorescence for detection can be regarded as simultaneous multi-item tests. Up until now there have been chips with several spots, but it should soon become possible to make high-density chips in addition. Array devices for SPR, a new principle for viewing protein-protein interactions, have already been developed. Although they may not be very sensitive, they are useful in antibody tests because they make it possible to measure affinity. Simultaneously using different types of detection systems on the thin gold film of the SPR array would make possible a wider range of tests than ever before. Moreover, enormous chip data processing has the potential to create a new concept in profiling test methodology. Proteomics research such as that described above has the potential to give rise to new concepts in the field of diagnosis.

Biomarkers↗
PubMed2003

HysTag--a novel proteomic quantification tool applied to differential display analysis of membrane proteins from distinct areas of mouse brain.

A novel isotopically labeled cysteine-tagging and complexity-reducing reagent, called HysTag, has been synthesized and used for quantitative proteomics of proteins from enriched plasma membrane preparations from mouse fore- and hindbrain. The reagent is a 10-mer derivatized peptide, H(2)N-(His)(6)-Ala-Arg-Ala-Cys(2-thiopyridyl disulfide)-CO(2)H, which consists of four functional elements: i) an affinity ligand (His(6)-tag), ii) a tryptic cleavage site (-Arg-Ala-), iii) Ala-9 residue that contains four (d(4)) or no (d(0)) deuterium atoms, and iv) a thiol-reactive group (2-thiopyridyl disulfide). For differential analysis cysteine residues in the compared samples are modified using either (d(4)) or (d(0)) reagent. The HysTag peptide is preserved in Lys-C digestion of proteins and allows charge-based selection of cysteine-containing peptides, whereas subsequent tryptic digestion reduces the labeling group to a di-peptide, which does not hinder effective fragmentation. Furthermore, we found that tagged peptides containing Ala-d(4) co-elute with their d(0)-labeled counterparts. To demonstrate effectiveness of the reagent, a differential analysis of mouse forebrain versus hindbrain plasma membranes was performed. Enriched plasma membrane fractions were partially denatured, reduced, and reacted with the reagent. Digestion with endoproteinase Lys-C was carried out on nonsolubilized membranes. The membranes were sedimented by ultra centrifugation, and the tagged peptides were isolated by Ni(2+) affinity or cation-exchange chromatography. Finally, the tagged peptides were cleaved with trypsin to release the histidine tag (residues 1-8 of the reagent) followed by liquid chromatography tandem mass spectroscopy for relative protein quantification and identification. A total of 355 unique proteins were identified, among which 281 could be quantified. Among a large majority of proteins with ratios close to one, a few proteins with significant quantitative changes were retrieved. The HysTag offers advantages compared with the isotope-coded affinity tag reagent, because the HysTag reagent is easy to synthesize, economical due to use of deuterium instead of (13)C isotope label, and allows robust purification and flexibility through the affinity tag, which can be extended to different peptide functionalities.

Amino Acid Sequence↗
PubMed2005

Lessons from nature: On the molecular recognition elements of the phosphoprotein binding-domains.

The reversible phosphorylation of proteins regulates many biological processes. Despite the technological advances in the enrichment and detection of phosphorylated proteins, the currently available techniques still struggle with the complexity of the human proteome. The aim of this review is to highlight the molecular recognition elements of the interaction between phosphorylated proteins and peptides and pTyr or pSer/Thr-binding domains. The identification of the recognition features of the naturally occurring pTyr- and pSer/Thr-binding domains can contribute to an understanding of the molecular aspects of the affinity and specificity for phosphorylated residues. This might inspire the design of small "biomimetic" molecules with potential applications in assessing the extent of the phosphoproteome using affinity-based strategies.

Binding Sites↗
PubMed2001

In vitro display technologies: novel developments and applications.

In vitro display techniques are powerful tools to select polypeptide binders against various target molecules. Novel applications include maturation of protein affinity and stability, selection for enzymatic activity, and the display of cDNA and random polypeptide libraries. Taken together, these display techniques have great potential for biotechnological, medical and proteomic applications.

Binding Sites↗
PubMed2005

Use of multidimensional lectin affinity chromatography in differential glycoproteomics.

This paper reports studies comparing the relative degree of sialylation among human serum glycoproteins carrying complex biantennary N-linked, hybrid, and high-mannose oligosaccharides. Comparisons were made by coupling lectin affinity selection with stable isotope coding of peptides from tryptic digests of serum. After proteolysis, samples were split and differentially acetylated with stable isotope coding agents according to either origin or the separation method by which they would be fractionated. A lectin column prepared from Sambucus nigra agglutinin (SNA) was used to select and compare the concentration of sialic acid containing glycopeptides. The relative standard deviation in quantification using this method was 4%. Using this method the concentration of sialic acid containing glycoproteins from a normal individual were compared to those in a pooled serum sample from a large number of normal individuals. It was found that sialylation varied less than 2-fold in all but four or five glycoproteins. Further studies were done on the degree of sialylation within glycoproteins. Samples labeled with the light isoform of the coding agent were applied to a set of serial lectin columns consisting of a concanavalin A (Con A) column coupled to an SNA column for selecting sialic acid appended to glycopeptides with complex biantennary N-linked, hybrid, and high-mannose glycans. In contrast, samples labeled with the heavy isoform of the coding agent were applied to a Con A lectin column alone to select glycopeptides containing complex biantennary N-linked, hybrid, and high-mannose glycans, without regard to sialylation. Glycopeptides thus selected were mixed, deglycosylated by PNGase F, and fractionated by reversed-phase chromatography (RPC). The RPC fractions were then analyzed by ESI-MS. The relative standard deviation of the method was 4%. All glycopeptides identified contained sialic acid except one. Peptides in which the relative abundance of isotopic isoforms was equal were considered to indicate that the protein parent was fully sialylated at that specific glycosylation site.

Amino Acid Sequence↗
PubMed2006

Phosphoproteomic analysis of rat liver by high capacity IMAC and LC-MS/MS.

Proper liver function is crucial for metabolism control and to clear toxic substances from the bloodstream. Many small-molecule therapeutics accumulate in the liver, negatively impacting liver function and often resulting in hepatotoxicity and cell death. Several analytical methods are currently utilized to evaluate hepatotoxicity and monitor liver function. To date, none of these methods have specifically targeted protein phosphorylation-mediated signal transduction pathways which should be altered in response to toxic effects of small molecule therapeutics. To develop novel assays to probe specific signaling pathways in the liver, identification and quantification of specific protein phosphorylation sites in this complex organ is necessary. Here, we have utilized an optimized immobilized metal affinity chromatography (IMAC) protocol to enrich phosphorylated peptides from a tryptic digest of proteins isolated from whole liver lysate. LC-MS/MS analysis of IMAC-enriched peptides resulted in the identification of more than 300 phosphorylation sites from over 200 proteins in rat liver, a significant advance over previously published analyses of the liver phosphoproteome. Previously characterized phosphorylation sites and potentially novel sites were identified in the current study, including sites on proteins implicated in metabolism regulation, transcription, translation, and canonical signaling pathways. Moreover, protein phosphorylation analysis was performed without prior fractionation of the sample, enabling analysis of small sample amounts while minimizing analysis time, potentially allowing for high-throughput assays to be performed with this methodology. From these data, it appears that this methodology can be used to identify new phosphorylation sites and, in combination with a stable isotope-labeling step, to investigate the effects of liver diseases, cancer and evaluate potential toxicology of new drug substances.

Amino Acid Motifs↗
PubMed2005

Construction and application of a yeast surface-displayed human cDNA library to identify post-translational modification-dependent protein-protein interactions.

Although post-translational modifications such as phosphorylation mediate fundamental biological processes within the cell, relatively few methods exist that allow proteome-wide identification of proteins that interact with these modifications. We constructed a yeast surface-displayed human cDNA library and utilized it to identify protein fragments with affinity for phosphorylated peptides derived from the major tyrosine autophosphorylation sites of the epidermal growth factor receptor or focal adhesion kinase. We identified cDNAs encoding the Src homology 2 domains from adapter protein APS, phosphoinositide 3-kinase regulatory subunit 3, SH2B, and tensin, demonstrating the effectiveness of this approach. Our results suggest that large libraries of functional human protein fragments can be efficiently displayed on the yeast surface. In addition to the analysis of post-translational modifications, yeast surface-displayed human cDNA libraries have many potential applications, including identifying targets and defining potential cross-reactive proteins for small molecules or drugs.

Adult↗
PubMed2006

Identification and validation of mannose 6-phosphate glycoproteins in human plasma reveal a wide range of lysosomal and non-lysosomal proteins.

Acid hydrolase activities are normally confined within the cell to the lysosome, a membrane-delimited cytoplasmic organelle primarily responsible for the degradation of macromolecules. However, lysosomal proteins are also present in human plasma, and a proportion of these retain mannose 6-phosphate (Man-6-P), a modification on N-linked glycans that is recognized by Man-6-P receptors (MPRs) that normally direct the targeting of these proteins to the lysosome. In this study, we purified the Man-6-P glycoforms of proteins from human plasma by affinity chromatography on immobilized MPRs and characterized this subproteome by two-dimensional gel electrophoresis and by tandem mass spectrometry. As expected, we identified many known and potential candidate lysosomal proteins. In addition, we also identified a number of abundant classical plasma proteins that were retained even after two consecutive rounds of affinity purification. Given their abundance in plasma, we initially considered these proteins to be likely contaminants, but a mass spectrometric study of Man-6-phosphorylation sites using MPR-purified glycopeptides revealed that some proportion of these classical plasma proteins contained the Man-6-P modification. We propose that these glycoproteins are phosphorylated at low levels by the lysosomal enzyme phosphotransferase, but their high abundance results in detection of Man-6-P glycoforms in plasma. These results may provide useful insights into the molecular processes underlying Man-6-phosphorylation and highlight circumstances under which the presence of Man-6-P may not be indicative of lysosomal function. In addition, characterization of the plasma Man-6-P glycoproteome should facilitate development of mass spectrometry-based tools for the diagnosis of lysosomal storage diseases and for investigating the involvement of Man-6-P-containing glycoproteins in more widespread human diseases and their potential utility as biomarkers.

Blood Proteins↗
PubMed2007

Rapid and individual-specific glycoprofiling of the low abundance N-glycosylated protein tissue inhibitor of metalloproteinases-1.

A gel-based method for a mass spectrometric site-specific glycoanalysis was developed using a recombinant glycoprotein expressed in two different cell lines. Hydrophilic interaction liquid chromatography at nanoscale level was used to enrich for glycopeptides prior to MS. The glycoprofiling was performed using matrix-assisted laser desorption/ionization MS and MS/MS. The method proved to be fast and sensitive and furthermore yielded a comprehensive site-specific glycan analysis, allowing a differentiation of the glycoprofiles of the two sources of recombinant protein, both comprising N-glycans of a highly heterogeneous nature. To test the potential of the method, tissue inhibitor of metalloproteinases-1 (TIMP-1), a secreted low abundance N-glycosylated protein and a cancer marker, was purified in an individual-specific manner from plasma of five healthy individuals using IgG depletion and immunoaffinity chromatography. The corresponding TIMP-1 glycoprofiles were determined to be highly similar, comprising mainly bi- and triantennary complex oligosaccharides. Additionally it was shown that platelet-derived TIMP-1 displayed a similar glycoprofile. This is the first study to investigate the glycosylation of naturally occurring human TIMP-1, and the high similarity of the glycoprofiles showed that individual-specific glycosylation variations of TIMP-1 are minimal. In addition, the results showed that TIMP-1 derived from platelets and plasma is similarly glycosylated. This comprehensive and rapid glycoprofiling of a low abundance glycoprotein performed in an individual-specific manner allows for future studies of glycosylated biomarkers for person-specific detection of altered glycosylation and may thus allow early detection and monitoring of diseases.

Amino Acid Sequence↗
PubMed2006

Multilectin affinity chromatography for characterization of multiple glycoprotein biomarker candidates in serum from breast cancer patients.

BACKGROUND: Glycoproteins are often associated with cancer and are important in serum studies, for which glycosylation is a common posttranslational modification. METHODS: We used multilectin affinity chromatography (M-LAC) to isolate glycoproteins from the sera of breast cancer patients and controls. The proteins were identified by HPLC-tandem mass spectrometry (MS/MS) analysis of the corresponding tryptic digests. We used the FuncAssociate Gene Ontology program for association analysis of the identified proteins. Biomarker candidates in these groups were comparatively quantitated by use of peak area measurements, with inclusion of an internal standard. We analyzed data for concordance within the ontology association groups for vector of change with the development of breast cancer. RESULTS: Detection of the known low-concentration biomarker HER-2 (8-24 mug/L) enabled us to establish a dynamic range of 10(6), relative to the amount of albumin, for the depletion step. We then used ELISA to confirm this range. Proteins associated with lipid transport and metabolism, cell growth and maintenance, ion homeostasis, and protease inhibition were found to be differentially regulated in serum from women with breast cancer compared with serum from women without breast cancer. CONCLUSIONS: M-LAC for isolation of the serum glycoproteome, coupled with liquid chromatography-MS/MS and the use of gene ontology associations, can be used to characterize large panels of candidate markers, which can then be evaluated in a particular patient population.

Biomarkers, Tumor↗
PubMed2003

Phosphoproteomic fingerprinting of epidermal growth factor signaling and anticancer drug action in human tumor cells.

Many proteins regulating cancer cell growth are tyrosine phosphorylated. Using antiphosphotyrosine affinity chromatography, thiourea protein solubilization, two-dimensional PAGE, and mass spectrometry, we report here the characterization of the epidermal growth factor (EGF)-induced phosphoproteome in A431 human epidermoid carcinoma cells. Using this approach, more than 50 distinct tyrosine phosphoproteins are identifiable within five main clusters-cytoskeletal proteins, signaling enzymes, SH2-containing adaptors, chaperones, and focal adhesion proteins. Comparison of the phosphoproteomes induced in vitro by transforming growth factor-alpha and platelet-derived growth factor demonstrates the pathway- and cell-specific nature of the phosphoproteomes induced. Elimination of both basal and ligand-dependent phosphoproteins by cell exposure to the EGF receptor catalytic inhibitor gefitinib (Iressa, ZD1839) suggests either an autocrine growth loop or the presence of a second inhibited kinase in A431 cells. By identifying distinct patterns of phosphorylation involving novel signaling substrates, and by clarifying the mechanism of action of anticancer drugs, these findings illustrate the potential of immunoaffinity-based phosphoproteomics for guiding the discovery of new drug targets and the rational utilization of pathway-specific chemotherapies.

Antineoplastic Agents↗
PubMedDate not supplied

Development of mammalian serum albumin affinity purification media by peptide phage display.

Several phage isolates that bind specifically to human serum albumin (HSA) were isolated from disulfide-constrained cyclic peptide phage-display libraries. The majority of corresponding synthetic peptides bind with micromolar affinity to HSA in low salt at pH 6.2, as determined by fluorescence anisotropy. One of the highest affinity peptides, DX-236, also bound well to several mammalian serum albumins (SA). Immobilized DX-236 quantitatively captures HSA from human serum; mild conditions (100 mM Tris, pH 9.1) allow release of HSA. The DX-236 affinity column bound HSA from human serum with a greater specificity than does Cibacron Blue agarose beads. In addition to its likely utility in HSA and other mammalian SA purifications, this peptide media may be useful in the proteomics and medical research markets for selective removal of mammalian albumin from serum prior to mass spectrometric and other analyses.

Amino Acid Sequence↗
PubMed2004

Proteomic characterization of messenger ribonucleoprotein complexes bound to nontranslated or translated poly(A) mRNAs in the rat cerebral cortex.

Receptor-triggered control of local postsynaptic protein synthesis plays a crucial role for enabling long lasting changes in synaptic functions, but signaling pathways that link receptor stimulation with translational control remain poorly known. Among the putative regulatory factors are mRNA-binding proteins (messenger ribonucleoprotein, mRNP), which control the fate of cytosolic localized mRNAs. Based on the assumption that a subset of mRNA is maintained in an inactive state, mRNP-mRNA complexes were separated into polysome-bound (translated) and polysome-free (nontranslated) fractions by sucrose density centrifugation. Poly(A) mRNA-mRNP complexes were purified from a postmitochondrial extract of rat cerebral cortex by oligo(dT)-cellulose affinity chromatography. The mRNA processing proteins were characterized, from solution, by a nanoflow reverse phase-high pressure liquid chromatography-mu-electrospray ionization mass spectrometry. The majority of detected mRNA-binding proteins was found in both fractions. However, a small number of proteins appeared to be fraction-specific. This subset of proteins is by far the most interesting because the proteins are potentially involved in controlling an activity-dependent onset of translation. They include transducer proteins, kinases, and anchor proteins. This study of the mRNP proteome is the first step in allowing future experimentation to characterize individual proteins responsible for mRNA processing and translation in dendrites.

Animals↗
PubMed2002

High-throughput protein characterization using mass spectrometric immunoassay.

A high-throughput mass spectrometric immunoassay system for the analysis of proteins directly from plasma is reported. A 96-well format robotic workstation was used to prepare antibody-derivatized affinity pipette tips for subsequent use in the extraction of specific proteins from plasma and deposition onto 96-well format matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) targets. Samples from multiple individuals were screened with regard to the plasma protein transthyretin (TTR), followed by analysis of the same plasma samples for the transthyretin-associated transport protein, retinol-binding protein (RBP). Analyses were able to detect the presence of posttranslationally modified TTR and RBP, as well as a mutation present in the TTR of one individual. Subsequent analyses of wild-type and mutated TTR using enzymatically active MALDI-TOF MS targets were able to identify the site and nature of the point mutation. The approach represents a rapid (approximately 100 samples/2 h, reagent preparation-to-data) and accurate means of characterizing specific proteins present in large numbers of individuals for proteomic and clinical/diagnostic purposes.

Antibody Affinity↗
PubMed2004

Evidence of glutathione transferase complexing and signaling in the model nematode Caenorhabditis elegans using a pull-down proteomic assay.

Phage display techniques using random peptide interactions have supported the role of mammalian glutathione transferase (GST) as part of a signalling pathway for both oxidative stress and an apoptosis pathway. Little is known about the interaction of nonmammalian GST with other proteins. GSTs have been implicated in the development of chronic nematode infections by neutralising cytotoxic products arising from host immune initiated reactive oxygen species (ROS) assault. In this study we attached one of the key GSTs expressed in the model nematode Caenorhabditis elegans to an affinity support matrix and directly identified major interacting proteins by two-dimensional electrophoresis and peptide mass fingerprinting before and following oxidative stress. Nematode GST does not appear to be a stand-alone enzyme and interacts with many types of proteins in both normal and ROS stress conditions. Pull-down proteomic presents a flexible, label free, rapid and economical assay without specialised ligand fishing equipment to identify protein binding partners.

Amino Acid Sequence↗