Journal ArticleDOI
Identification of phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis.
Xiaolong Zhang,Christopher J. Herring,Patrick R. Romano,Joanna Szczepanowska,Hanna Brzeska,and Alan G. Hinnebusch,Jun Qin +6 more
TLDR
A fast, sensitive, and robust procedure for the identification of precise phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis by a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF) and online capillary liquid chromatography electrospray tandem ion trap mass spectromaetry (LC/ESI/MS/MS).Abstract:
We report a fast, sensitive, and robust procedure for the identification of precise phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis by a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF) and on-line capillary liquid chromatography electrospray tandem ion trap mass spectrometry (LC/ESI/MS/MS). With this procedure, a single phosphorylation site was identified on as little as 20 ng (500 fmol) of the baculovirus-expressed catalytic domain of myosin I heavy-chain kinase separated by gel electrophoresis. The phosphoprotein is digested in the gel with trypsin, and the resulting peptides are extracted with >60% yield and analyzed by MALDI/TOF before and after digestion with a phosphatase to identify the phosphopeptides. The phosphopeptides are then separated and fragmented in an on-line LC/ESI ion trap mass spectrometer to identify the precise phosphorylation sites. This procedure eliminates any off-line HPLC separation and mi...read more
Citations
More filters
Journal ArticleDOI
Proteomics to study genes and genomes
TL;DR: Proteomics can be divided into three main areas: protein micro-characterization for large-scale identification of proteins and their post-translational modifications; ‘differential display’ proteomics for comparison of protein levels with potential application in a wide range of diseases; and studies of protein–protein interactions using techniques such as mass spectrometry or the yeast two-hybrid system.
Journal ArticleDOI
Mass Spectrometry in Proteomics
TL;DR: 4. Automated Interpretation of CID Spectra 282 5. Accurate Mass Tags 282 C. Protein Identification in Complex Mixtures 282 D. Analysis of Protein Expression 284 III.
Journal ArticleDOI
Analysis of proteins and proteomes by mass spectrometry
TL;DR: This work states that any human protein can now be identified directly from genome databases on the basis of minimal data derived by mass spectrometry, and that increased automation of sample handling, analysis, and the interpretation of results will generate an avalanche of qualitative and quantitative proteomic data.
Journal ArticleDOI
Requirement of ATM-Dependent Phosphorylation of Brca1 in the DNA Damage Response to Double-Strand Breaks
TL;DR: In this article, it was shown that the checkpoint protein kinase ATM (mutated in ataxia telangiectasia) was required for phosphorylation of Brca1 in response to ionizing radiation.
Journal ArticleDOI
Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome.
TL;DR: Several methods for enrichment of phosphorylated proteins and peptides are outlined and various options for their identification and quantitation are discussed with special emphasis on mass spectrometry-based techniques.
References
More filters
Journal ArticleDOI
Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels
TL;DR: Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining, and this work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.
Journal ArticleDOI
Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry
Matthias Wilm,Andrej Shevchenko,Tony Houthaeve,Stephen Breit,Lothar Schweigerer,Lothar Schweigerer,Theodore Fotsis,Matthias Mann +7 more
TL;DR: A simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electro-phoresis, using nano-electrospray3,4 tandem mass spectrometry5,6 and multiple-sequence stretches of up to 16 amino acids are obtained.
Journal ArticleDOI
Error-tolerant identification of peptides in sequence databases by peptide sequence tags.
Matthias Mann,Matthias Wilm +1 more
TL;DR: A new approach to the identification of mass spectrometrically fragmented peptides is demonstrated and an algorithm developed here that uses the sequence tag to find the peptide in a sequence database is up to 1 million-fold more discriminating than the partial sequence information alone.
Journal ArticleDOI
Reverse Transcriptase Motifs in the Catalytic Subunit of Telomerase
Joachim Lingner,Timothy P. Hughes,Andrej Shevchenko,Matthias Mann,Victoria Lundblad,Thomas R. Cech +5 more
TL;DR: The reverse transcriptase protein fold, previously known to be involved in retroviral replication and retrotransposition, is essential for normal chromosome telomere replication in diverse eukaryotes.
Journal ArticleDOI
The regulation of transcription by phosphorylation
Tony Hunter,Michael Karin +1 more
Related Papers (5)
Immobilized Gallium(III) Affinity Chromatography of Phosphopeptides
Matthew C. Posewitz,Paul Tempst +1 more