A nonradioactive assay for protein tyrosine phosphatases (PTPs), employing a tyrosine-phosphorylated peptide as a substrate, has been developed and applied to analyze purified enzymes, cell extracts, and immunoprecipitates. The reaction was followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in a linear and positive ion mode with delayed extraction. MALDI-TOF MS detects a loss of peptide mass by 80 Da as a result of dephosphorylation and, more importantly, it yields phospho-peptide to dephosphorylated product peak intensity ratios proportional to their concentration ratios. A strong bias of the MALDI-TOF MS toward detection of the non-phospho-peptide allows accurate detection of small fractions of dephosphorylation. The method is highly sensitive and reproducible. It can be applied to general assays of protein phosphatases with various phospho-peptides as substrates.
"This suppression of ionization might be due to the higher hydrophilicity of oligonucleotide–peptide/protein heteroconjugates compared to the unmodified peptides/proteins. A similar problem is observed for phosphopeptides (Chen et al. 2001; Wind et al. 2001). Although MS has been used in a number of studies of cross-linked peptide–oligonucleotide species, there are only few reports on the use of tandem mass spectrometry for the structural analysis of such heteroconjugates. "
[Show abstract][Hide abstract] ABSTRACT: Protein–nucleic acid complexes are commonly studied by photochemical cross-linking. UV-induced cross-linking of protein to nucleic acid may be followed by structural analysis of the conjugated protein to localize the cross-linked amino acids and thereby idey the nucleic acid binding site. Mass spectrometry is becoming increasingly popular for characterization of purified peptide–nucleic acid heteroconjugates derived from UV cross-linked protein–nucleic acid complexes. The efficiency of mass spectrometry-based methods is, however, hampered by the contrasting physico-chemical properties of nucleic acid and peptide entities present in such heteroconjugates. Sample preparation of the peptide–nucleic acid heteroconjugates is, therefore, a crucial step in any mass spectrometry-based analytical procedure. This study demonstrates the performance of four different MS-based strategies to characterize E. coli single-stranded DNA binding protein (SSB) that was UV-cross-linked to a 5-iodouracil containing DNA oligomer. Two methods were optimized to circumvent the need for standard liquid chromatography and gel electrophoresis, thereby dramatically increasing the overall sensitivity of the analysis. Enzymatic degradation of protein and oligonucleotide was combined with miniaturized sample preparation methods for enrichment and desalting of cross-linked peptide–nucleic acid heteroconjugates from complex mixtures prior to mass spectrometric analysis. Detailed characterization of the peptidic component of two different peptide–DNA heteroconjugates was accomplished by matrix-assisted laser desorption/ionization mass spectrometry and allowed assignment of tryptophan-54 and tryptophan-88 as candidate cross-linked residues. Sequencing of those peptide–DNA heteroconjugates by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry ideied tryptophan-54 and tryptophan-88 as the sites of cross-linking. Although the UV-cross-linking yield of the protein–DNA complex did not exceed 15%, less than 100 pmole of SSB protein was required for detailed structural analysis by mass spectrometry.
[Show abstract][Hide abstract] ABSTRACT: Photochemical cross-linking is a commonly used method for studying the molecular details of protein-nucleic acid interactions. Photochemical cross-linking aids in defining nucleic acid binding sites of proteins via subsequent identification of cross-linked protein domains and amino acid residues. Mass spectrometry (MS) has emerged as a sensitive and efficient analytical technique for determination of such cross-linking sites in proteins. The present review of the field describes a number of MS-based approaches for the characterization of cross-linked protein-nucleic acid complexes and for sequencing of peptide-nucleic acid heteroconjugates. The combination of photochemical cross-linking and MS provides a fast screening method to gain insights into the overall structure and formation of protein-oligonucleotide complexes. Because the analytical methods are continuously refined and protein structural data are rapidly accumulating in databases, we envision that many protein-nucleic acid assemblies will be initially characterized by combinations of cross-linking methods, MS, and computational molecular modeling.
Mass Spectrometry Reviews 05/2002; 21(3):163-82. DOI:10.1002/mas.10024 · 7.71 Impact Factor
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