Method for estimating the isotopic distributions of metabolically labeled proteins by MALDI-TOFMS: application to NMR samples.
We have developed an efficient method of estimating metabolic incorporation of heavy isotopes into proteins, including those where a single amino acid carries the label. The protein is digested with trypsin, and the resulting peptide mixture is examined directly by MALDI-TOF mass spectrometry. Peptides are chosen for analysis if they contain one or more labeled atoms and also exhibit clearly separated mass spectra. The known atomic composition of the peptide is then used to simulate ion distributions for various proportions of heavy isotope incorporation, to obtain the best match to the observed ion distribution. We demonstrate the method by comparing simulated and observed mass spectra of tryptic peptides of Escherichia coli citrate synthase labeled with 15N in several ways and show that the method is particularly applicable when only one amino acid is isotopically labeled.
Choudhary
Kajal
K
Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
Spicer
Victor L
VL
Donald
Lynda J
LJ
Duckworth
Harry W
HW
Ens
Werner
W
Loewen
Peter C
PC
Standing
Kenneth G
KG
D002950:Citrate (si)-Synthase / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes; D010446:Peptide Fragments / Q000032:analysis; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D002950:Citrate (si)-Synthase; D014357:Trypsin