Isotope-coded protein label.
A great variety of technologies using stable isotope labeling in combination with mass spectrometry have been described being tools to identify and relatively quantify proteins within complex mixtures. Here, we present a method, termed isotope-coded protein label (ICPL), which is capable of high-throughput quantitative proteome profiling on a global scale. Since ICPL is based on tagging stable isotope derivatives at the free amino groups of intact proteins, the method is applicable to any protein sample, including extracts from tissues or body fluids. All separation methods currently employed in proteome studies can be used to reduce complexity on the protein level. After enzymatic cleavage of the protein fractions, the ratios of peptides from different proteome states can be calculated by simple MS-based mass spectrometric analyses. Only peptides representing different expression levels in the different proteomic states are further analyzed by tandem-mass spectrometry to identify respective proteins. For quantification of proteins from multiplexed ICPL experiments, ICPLQuant was developed, a software package especially designed to cover the whole ICPL workflow. The ICPL method results in accurate and reproducible quantification of proteins and high sequence coverage, indispensable for a comprehensive detection of posttranslational modifications and discrimination of protein isoforms.
Max-Planck Institute of Biochemistry, Martinsried, Germany. kellerma@biochem.mpg.de
Josef
J
Kellermann
Friedrich
F
Lottspeich
D000818:Animals; D002247:Carbon Isotopes; D011232:Chemical Precipitation; D003545:Cysteine / Q000737:chemistry; D003627:Data Interpretation, Statistical; D003903:Deuterium; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000592:standards; D008745:Methylation; D010446:Peptide Fragments / Q000737:chemistry; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry* / Q000302:isolation & purification; D012015:Reference Standards; D012697:Serine Endopeptidases / Q000737:chemistry; D012984:Software; D013388:Succinimides
D002247:Carbon Isotopes; D010446:Peptide Fragments; D020543:Proteome; D013388:Succinimides; D003903:Deuterium; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D003545:Cysteine