Analysis of protein phosphorylation by mass spectrometry.
The reversible phosphorylation of proteins is recognized as an essential post-translational modification regulating cell signaling and ultimately function of biological systems. Detection of phosphopeptides and localization of phosphorylation sites remains quite a challenge, even if the protein is purified to near homogeneity. Mass spectrometry has become a vital technique that is routinely utilized for the identification of proteins from whole cell lysates. Nonetheless, due to the minimal amount of phosphorylation found on proteins, enrichment steps for isolating phosphopeptides from complex mixtures have been the focus of many research groups world-wide. In this review, we describe some current methods for the enrichment of phosphopeptides that are compatible with mass spectrometry for assignment of phosphorylation sites. Phosphorylation modifications on proteins and peptides are either directly isolated by solid-phase approaches or chemically modified for selective isolation and/or improved characterization by mass spectrometry. These strategies hold the potential for rapid and sensitive profiling of phosphoproteins from a variety of sources and cellular conditions.
Garcia
Benjamin A
BA
Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA.
Shabanowitz
Jeffrey
J
Hunt
Donald F
DF
D002846:Chromatography, Affinity; D019295:Computational Biology / Q000379:methods; D030562:Databases, Protein; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D015398:Signal Transduction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013237:Stereoisomerism
D010455:Peptides; D010748:Phosphopeptides; D011506:Proteins