Search PubMed⌕ Search
Home/Archive/PubMed

PubMed · 12479833

Profiling substrate phosphorylation at the phosphopeptide level.

Andrea Gatti
View original source ↗Export metadata ↓

Abstract

The identification of substrates is a key aspect in the study of the biological function of protein kinases. The procedure here described is aimed at profiling substrate phosphorylation at the phosphopeptide level by sequentially involving (i). the assessment of the in vitro activity of individual protein kinases on a complex mix of immobilized proteins, (ii). the fractionation of the phosphopeptides being released upon proteolysis of substrates, and (iii). the final identification of the targeted sequences. In particular, the protein sample is spotted onto nitrocellulose membrane and then subjected to a solid-phase kinase assay in the presence of [32P]ATP, prior to solid-phase proteolytic digestion and two-dimensional phosphopeptide mapping. Radiolabeled phosphopeptides are subsequently isolated and sequenced to identify the substrates being targeted by the examined protein kinase. Using the gamma-isotype of p21-activated protein kinase (gamma-PAK) and its known in vitro substrates, I verified that both the specificity of substrate phosphorylation and its efficiency are similar upon solid- and liquid-phase conditions. To demonstrate the feasibility of the overall experimental system, I then employed a fairly crude cell extract as a source of candidate substrates and successfully identified the sequence of a putative substrate of gamma-PAK.

Explore related subjects

Peptide MappingPhosphopeptidesPhosphorylationSubstrate SpecificityJournal ArticleResearch Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Andrea Gatti. 2003-01-01. Profiling substrate phosphorylation at the phosphopeptide level.. https://doi.org/10.1016/s0003-2697(02)00426-8

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

Probabilistic enrichment of phosphopeptides by their mass defect.

The mass defect, that is, the difference between the nominal and actual monoisotopic masses, of a phosphorus in a phosphate group is greater than for most other atoms present in proteins. When the mass defects of tryptic peptides derived from the human proteome are plotted against their masses, phosphopeptides tend to fall off the regression line. By calculating the masses of all potential tryptic peptides from the human proteome, we show that regions of higher phosphorylation probability exist on such a plot. We developed a transformation function to estimate the mass defect of a peptide from its monoisotopic mass and empirically defined a simple formula for a user-selectable discriminant line that categorizes a peptide mass according to its probability of being phosphorylated. Our method performs similarly well on phosphopeptides derived from a database of experimentally validated phosphoproteins. The method is relatively insensitive to mass measurement error of up to 20 ppm. The approach can be used with a tandem mass spectrometer in real time to rapidly select and rank order the possible phosphopeptides from a mixture of unmodified peptides for subsequent phosphorylation site mapping and peptide sequence analysis.

Peptide Mapping↗
PubMed2006

A deflection system to reduce the interference from post-source decay product ions in photodissociation tandem time-of-flight mass spectrometry.

A deflection system consisting of four deflectors was designed and used to reduce the interference from post-source decay (PSD) product ions in photodissociation (PD) tandem time-of-flight (TOF) mass spectrometry. For simple protonated peptides generated by matrix-assisted laser desorption/ionization, the presence of PSD product ions at the laser irradiation spot was found to noticeably alter the minor peaks in the PD spectra even though the major ones were hardly affected. Other benefits from the use of the deflection system such as the improvement in the resolving power in PSD tandem mass spectra are discussed.

Peptide Mapping↗
PubMed2005

Optimization of a MALDI TOF-TOF mass spectrometer for intact protein analysis.

A MALDI TOF-TOF instrument was optimized and evaluated for intact protein analysis by tandem mass spectrometry. Ion source voltages and delay times were adjusted to affect an up to a 10-fold improvement in fragment ion yield compared to data obtained using default settings employed in peptide analysis. For large peptides (3-4.5 kDa), up to 90% of all possible b- and y-fragment ions were observed, which provides sufficient information for de novo sequencing and unambiguous protein identification. Product ion signals associated with preferential cleavages C-terminal to aspartic acid and glutamic acid residues and N-terminal to proline residues became dominant with increased protein molecular weight. Matrix effects were also evaluated and, among the eight matrices examined, alpha-cyano-4-hydroxycinnamic acid (CHCA) was found to produce the best intact protein tandem mass spectra for proteins up to 12 kDa. Optimized performance yielded detection limits of 50-125 fmol for proteins of 4 and 12 kDa, respectively. This improved performance has yielded an instrument with potential to be a useful tool in proteomic investigations via analysis of intact proteins.

Peptide Mapping↗