N-terminal isotope tagging strategy for quantitative proteomics: results-driven analysis of protein abundance changes.
Comparing the relative abundance of each protein present in two or more complex samples can be accomplished using isotope-coded tags incorporated at the peptide level. Here we describe a chemical labeling strategy for the incorporation of a single isotope label per peptide, which is completely sequence-independent so that it potentially labels every peptide from a protein including those containing posttranslational modifications. It is based on a gentle chemical labeling strategy that specifically labels the N-terminus of all peptides in a digested sample with either a d5- or d0-propionyl group. Lysine side chains are blocked by guanidination prior to N-terminal labeling to prevent the incorporation of multiple labels. In this paper, we describe the optimization of this N-terminal isotopic tagging strategy and validate its use for peptide-based protein abundance measurements with a 10-protein standard mixture. Using a results-driven strategy, which targets proteins for identification based on MALDI TOF-MS analysis of isotopically labeled peptide pairs, we also show that this labeling strategy can detect a small number of differentially expressed proteins in a mixture as complex as a yeast cell lysate. Only peptides that show a difference in relative abundance are targeted for identification by tandem MS. Despite the fact that many peptides are quantitated, only those few showing a difference in abundance are targeted for protein identification. Proteins are identified by either targeted LC-ES MS/MS or MALDI TOF/TOF. Identifications can be accomplished equally well by either technique on the basis of multiple peptides. This increases the confidence level for both identification and quantitation. The merits of ES MS/MS or MALDI MS/MS for protein identification in a results-driven strategy are discussed.
Zappacosta
Francesca
F
Proteomics and Biological Mass Spectrometry, Department of Computational, Analytical and Structural Sciences, GlaxoSmithKline, King of Prussia, Pennsylvania 19406, USA.
Annan
Roland S
RS
D002851:Chromatography, High Pressure Liquid; D007553:Isotope Labeling; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D011506:Proteins