Guanidination chemistry for qualitative and quantitative proteomics.
The application of guanidination chemistry, the conversion of lysine into homoarginine residues, is used to illustrate several important general considerations relating to the use of differential isotope labelling for relative quantification in proteomics. The derivatisation procedure has been optimised for automation using a liquid handling station designed for proteomics. Automated application of the procedure to the analysis of in-gel tryptic digests of multiple spots from the two-dimensional gel electrophoretic (2DE) analysis of proteins from the FDCP-mix cell line shows near-universal improvement in protein identification as a result of derivatisation. This chemistry has been extended for relative quantification, applicable to matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) and also tandem mass spectrometry (MS/MS). It provides a robust method for the quantitative comparison of two samples that have been separated by 2DE. A peptide pair may display poor detection during MS analysis, causing their reliable relative quantification to be difficult. In such circumstances, the additional selectivity of detection provided by MS/MS can substantiate identification and allow relative quantification of these species via product ion signal ratios.
Warwood
Stacey
S
Michael Barber Centre for Mass Spectrometry and Manchester Interdisciplinary Biocentre, University of Manchester, Manchester M1 7ND, UK.
Mohammed
Shabaz
S
Cristea
Ileana M
IM
Evans
Caroline
C
Whetton
Anthony D
AD
Gaskell
Simon J
SJ
D000818:Animals; D001120:Arginine / Q000737:chemistry; D002460:Cell Line; D022781:Hepatocytes / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D051379:Mice; D010449:Peptide Mapping / Q000379:methods*; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D020543:Proteome; D001120:Arginine; D008239:Lysine