Immunoaffinity enrichment based on antipeptide antibodies coupled to mass spectrometry-based iden... more Immunoaffinity enrichment based on antipeptide antibodies coupled to mass spectrometry-based identification and quantification (immuno-MS) is a promising approach to translate proteomics to clinical assays with diagnostic value. This is linked to precision cancer medicine, where immuno-MS based studies of protein phosphorylation dependent-signalling states of cells enable pathway targeted therapies and response monitoring to be developed. Clinically robust methods depend on the availability of high quality phosphopeptide antibodies but these rarely meet the stringent demands on reproducibility, robustness, availability and peptide specificity. We here show that polymer-based “plastic antibodies” prepared by molecular imprinting could play this role. Focusing on the Tyr-492 and Tyr-493 kinase regulatory motif of the SH2 domain in ZAP-70, a critical mediator in T-cell receptor signalling, we show that MIPs can be easily generated to recognize corresponding mono- or di- phosphorylated ...
Proceedings of the National Academy of Sciences, 1999
Gα o , the most abundant G protein in mammalian brain, occurs at least in two subforms, i.e., Gα ... more Gα o , the most abundant G protein in mammalian brain, occurs at least in two subforms, i.e., Gα o1 and Gα o2 , derived by alternative splicing of the mRNA. A third Gα o1 -related isoform, Gα o3 , has been purified, representing about 30% of total G o in brain. Initial studies revealed distinct biochemical properties of Gα o3 as compared with other Gα o isoforms. In matrix-assisted laser desorption/ionization peptide mass mapping of gel-isolated Gα o1 and Gα o3 , C-terminal peptides showed a difference of +1 Da for Gα o3 . Nanoelectrospray tandem mass spectrometry sequencing revealed an Asp instead of an Asn at position 346 of Gα o3 . Gel electrophoretic analysis of recombinant Gα o3 showed the same mobility as native Gα o3 but distinct to Gα o1 . The conversion of 346 Asn→Asp changed the signaling properties, including the velocity of the basal guanine nucleotide-exchange reaction, which points to the involvement of the C terminus in basal guanosine 5′-[γ-thio]triphosphate binding....
Vaccinia virus assembly has been well studied at the ultrastructural level, but little is known a... more Vaccinia virus assembly has been well studied at the ultrastructural level, but little is known about the molecular events that occur during that process. Towards this goal, we have identified the major membrane and core proteins of the intracellular mature virus (IMV). Pure IMV preparations were subjected to Nonidet P-40 (NP-40) and dithiothreitol (DTT) treatment to separate the core proteins from the membrane proteins. These proteins were subsequently separated by two-dimensional (2D) gel electrophoresis, and the major polypeptide spots, as detected by silver staining and 35S labeling, were identified by either matrix-assisted laser desorption/ionization mass spectrometry, N-terminal amino acid sequencing, or immunoprecipitation with defined antibodies. Sixteen major spots that partitioned into the NP-40-DTT-soluble fraction were identified; 11 of these were previously described virally encoded proteins and 5 were cellular proteins, mostly of mitochondrial origin. The core fractio...
Publisher Summary This chapter describes the mass spectrometric protocol for the analysis of UV-c... more Publisher Summary This chapter describes the mass spectrometric protocol for the analysis of UV-crosslinked protein–nucleic acid complexes. It presents a protocol that combines developed methods for UV-light-induced photochemical crosslinking of proteins to nucleic acids with MALDI, and ESI mass spectrometry to locate and identify the particular amino acid and nucleotide residues that covalently bind to each other. The chapter explains the application of some of the stages in this analytical scheme to two different systems of protein–nucleic acid complexes, the phage T4 gene 32 protein UV-laser cross-linked to the oligonucleotide photoaffinity-labeled with 4-thiouridinediphosphate. In the first stage, the nucleic acid binding protein and its nucleic acid substrate or a photoactivatable analogue thereof are incubated under proper conditions to form the protein–nucleic acid complex. The sample is subsequently irradiated with UV light for a given period of time to create photochemically crosslinked protein–nucleic acid complexes.
Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a mult... more Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a multisubunit “cis” complex on isolated organelles. We now identify the v-SNAREs Vti1p and Ykt6p by mass spectrometry as additional components of the immunoisolated vacuolar SNARE complex. Immunodepletion of detergent extracts with anti-Vti1p removes all the Ykt6p that is in a complex with Vam3p, immunodepletion with anti-Ykt6p removes all the Vti1p that is complexed with Vam3p, and immunodepletion with anti-Nyv1p removes all the Ykt6p in complex with other SNAREs, demonstrating that they are all together in the same cis multi-SNARE complex. After priming, which disassembles the cis-SNARE complex, antibodies to any of the five SNARE proteins still inhibit the fusion assay until the docking stage is completed, suggesting that each SNARE plays a role in docking. Furthermore, vti1 temperature-sensitive alleles cause a synthetic fusion-defective phenotype in our reaction. Our data show that vacuo...
Proceedings of the National Academy of Sciences, 1996
The function of many of the uncharacterized open reading frames discovered by genomic sequencing ... more The function of many of the uncharacterized open reading frames discovered by genomic sequencing can be determined at the level of expressed gene products, the proteome. However, identifying the cognate gene from minute amounts of protein has been one of the major problems in molecular biology. Using yeast as an example, we demonstrate here that mass spectrometric protein identification is a general solution to this problem given a completely sequenced genome. As a first screen, our strategy uses automated laser desorption ionization mass spectrometry of the peptide mixtures produced by in-gel tryptic digestion of a protein. Up to 90% of proteins are identified by searching sequence data bases by lists of peptide masses obtained with high accuracy. The remaining proteins are identified by partially sequencing several peptides of the unseparated mixture by nanoelectrospray tandem mass spectrometry followed by data base searching with multiple peptide sequence tags. In blind trials, t...
Proceedings of the National Academy of Sciences, 2009
D-serine is a physiological coagonist of N-methyl D-aspartate receptors (NMDARs) that plays a maj... more D-serine is a physiological coagonist of N-methyl D-aspartate receptors (NMDARs) that plays a major role in several NMDAR-dependent events. In this study we investigate mechanisms regulating D-serine production by the enzyme serine racemase (SR). We now report that NMDAR activation promotes translocation of SR to the plasma membrane, which dramatically reduces the enzyme activity. Membrane-bound SR isolated from rat brain is not extracted from the membrane by high detergent and salt concentration, indicating a strong association. Colocalization studies indicate that most membrane-bound SR is located at the plasma membrane and dendrites, with much less SR observed in other types of membrane. NMDAR activation promotes translocation of the cytosolic SR to the membrane, resulting in reduced D-serine synthesis, and this effect is averted by blockade of NMDARs. In primary neuronal cultures, SR translocation to the membrane is blocked by a palmitoylation inhibitor, indicating that membrane...
Immunoaffinity enrichment based on antipeptide antibodies coupled to mass spectrometry-based iden... more Immunoaffinity enrichment based on antipeptide antibodies coupled to mass spectrometry-based identification and quantification (immuno-MS) is a promising approach to translate proteomics to clinical assays with diagnostic value. This is linked to precision cancer medicine, where immuno-MS based studies of protein phosphorylation dependent-signalling states of cells enable pathway targeted therapies and response monitoring to be developed. Clinically robust methods depend on the availability of high quality phosphopeptide antibodies but these rarely meet the stringent demands on reproducibility, robustness, availability and peptide specificity. We here show that polymer-based “plastic antibodies” prepared by molecular imprinting could play this role. Focusing on the Tyr-492 and Tyr-493 kinase regulatory motif of the SH2 domain in ZAP-70, a critical mediator in T-cell receptor signalling, we show that MIPs can be easily generated to recognize corresponding mono- or di- phosphorylated ...
Proceedings of the National Academy of Sciences, 1999
Gα o , the most abundant G protein in mammalian brain, occurs at least in two subforms, i.e., Gα ... more Gα o , the most abundant G protein in mammalian brain, occurs at least in two subforms, i.e., Gα o1 and Gα o2 , derived by alternative splicing of the mRNA. A third Gα o1 -related isoform, Gα o3 , has been purified, representing about 30% of total G o in brain. Initial studies revealed distinct biochemical properties of Gα o3 as compared with other Gα o isoforms. In matrix-assisted laser desorption/ionization peptide mass mapping of gel-isolated Gα o1 and Gα o3 , C-terminal peptides showed a difference of +1 Da for Gα o3 . Nanoelectrospray tandem mass spectrometry sequencing revealed an Asp instead of an Asn at position 346 of Gα o3 . Gel electrophoretic analysis of recombinant Gα o3 showed the same mobility as native Gα o3 but distinct to Gα o1 . The conversion of 346 Asn→Asp changed the signaling properties, including the velocity of the basal guanine nucleotide-exchange reaction, which points to the involvement of the C terminus in basal guanosine 5′-[γ-thio]triphosphate binding....
Vaccinia virus assembly has been well studied at the ultrastructural level, but little is known a... more Vaccinia virus assembly has been well studied at the ultrastructural level, but little is known about the molecular events that occur during that process. Towards this goal, we have identified the major membrane and core proteins of the intracellular mature virus (IMV). Pure IMV preparations were subjected to Nonidet P-40 (NP-40) and dithiothreitol (DTT) treatment to separate the core proteins from the membrane proteins. These proteins were subsequently separated by two-dimensional (2D) gel electrophoresis, and the major polypeptide spots, as detected by silver staining and 35S labeling, were identified by either matrix-assisted laser desorption/ionization mass spectrometry, N-terminal amino acid sequencing, or immunoprecipitation with defined antibodies. Sixteen major spots that partitioned into the NP-40-DTT-soluble fraction were identified; 11 of these were previously described virally encoded proteins and 5 were cellular proteins, mostly of mitochondrial origin. The core fractio...
Publisher Summary This chapter describes the mass spectrometric protocol for the analysis of UV-c... more Publisher Summary This chapter describes the mass spectrometric protocol for the analysis of UV-crosslinked protein–nucleic acid complexes. It presents a protocol that combines developed methods for UV-light-induced photochemical crosslinking of proteins to nucleic acids with MALDI, and ESI mass spectrometry to locate and identify the particular amino acid and nucleotide residues that covalently bind to each other. The chapter explains the application of some of the stages in this analytical scheme to two different systems of protein–nucleic acid complexes, the phage T4 gene 32 protein UV-laser cross-linked to the oligonucleotide photoaffinity-labeled with 4-thiouridinediphosphate. In the first stage, the nucleic acid binding protein and its nucleic acid substrate or a photoactivatable analogue thereof are incubated under proper conditions to form the protein–nucleic acid complex. The sample is subsequently irradiated with UV light for a given period of time to create photochemically crosslinked protein–nucleic acid complexes.
Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a mult... more Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a multisubunit “cis” complex on isolated organelles. We now identify the v-SNAREs Vti1p and Ykt6p by mass spectrometry as additional components of the immunoisolated vacuolar SNARE complex. Immunodepletion of detergent extracts with anti-Vti1p removes all the Ykt6p that is in a complex with Vam3p, immunodepletion with anti-Ykt6p removes all the Vti1p that is complexed with Vam3p, and immunodepletion with anti-Nyv1p removes all the Ykt6p in complex with other SNAREs, demonstrating that they are all together in the same cis multi-SNARE complex. After priming, which disassembles the cis-SNARE complex, antibodies to any of the five SNARE proteins still inhibit the fusion assay until the docking stage is completed, suggesting that each SNARE plays a role in docking. Furthermore, vti1 temperature-sensitive alleles cause a synthetic fusion-defective phenotype in our reaction. Our data show that vacuo...
Proceedings of the National Academy of Sciences, 1996
The function of many of the uncharacterized open reading frames discovered by genomic sequencing ... more The function of many of the uncharacterized open reading frames discovered by genomic sequencing can be determined at the level of expressed gene products, the proteome. However, identifying the cognate gene from minute amounts of protein has been one of the major problems in molecular biology. Using yeast as an example, we demonstrate here that mass spectrometric protein identification is a general solution to this problem given a completely sequenced genome. As a first screen, our strategy uses automated laser desorption ionization mass spectrometry of the peptide mixtures produced by in-gel tryptic digestion of a protein. Up to 90% of proteins are identified by searching sequence data bases by lists of peptide masses obtained with high accuracy. The remaining proteins are identified by partially sequencing several peptides of the unseparated mixture by nanoelectrospray tandem mass spectrometry followed by data base searching with multiple peptide sequence tags. In blind trials, t...
Proceedings of the National Academy of Sciences, 2009
D-serine is a physiological coagonist of N-methyl D-aspartate receptors (NMDARs) that plays a maj... more D-serine is a physiological coagonist of N-methyl D-aspartate receptors (NMDARs) that plays a major role in several NMDAR-dependent events. In this study we investigate mechanisms regulating D-serine production by the enzyme serine racemase (SR). We now report that NMDAR activation promotes translocation of SR to the plasma membrane, which dramatically reduces the enzyme activity. Membrane-bound SR isolated from rat brain is not extracted from the membrane by high detergent and salt concentration, indicating a strong association. Colocalization studies indicate that most membrane-bound SR is located at the plasma membrane and dendrites, with much less SR observed in other types of membrane. NMDAR activation promotes translocation of the cytosolic SR to the membrane, resulting in reduced D-serine synthesis, and this effect is averted by blockade of NMDARs. In primary neuronal cultures, SR translocation to the membrane is blocked by a palmitoylation inhibitor, indicating that membrane...
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