Quantitative proteomics strategy involving the selection of peptides containing both cysteine and histidine from tryptic digests of cell lysates.
This paper describes a procedure for quantitative proteomics that selects peptides containing both cysteine and histidine residues from tryptic digests of cell lysates. Cysteine-containing peptides were selected first by covalent chromatography using thiol disulfide exchange. Following the release of cysteine-containing peptides from the covalent chromatography column with reductive cleavage, histidine-containing peptides were captured by passage through an immobilized metal affinity chromatography column loaded with copper. Quantification was achieved in a four-step process involving (i) differential labeling of control and experimental samples with isotopically differing forms of succinic anhydride, (ii) mixing the two globally labeled samples, (iii) fractionating the labeled peptides by reversed-phase liquid chromatography, and (iv) determining the isotope ratio in individual peptides by mass spectrometry. The results of these studies indicate that by selecting peptides containing both cysteine and histidine, the complexity of protein digests could be substantially reduced. Up-regulated proteins from plasmid bearing Escherichia coli that had been induced with isopropyl beta-thiogalacto-pyranoside were identified and quantified by the global internal standard technology (GIST) described above. Database searches were greatly simplified because the number of possible peptide candidates was reduced more than 95%.
Wang
Shihong
S
Department of Chemistry, Purdue University, West Lafayette, IN 47907-1393, USA.
Zhang
Xiang
X
Regnier
Fred E
FE
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism*
D010455:Peptides; D020543:Proteome; D006639:Histidine; D014357:Trypsin; D003545:Cysteine