Quantitative proteomics: measuring protein synthesis using 15N amino acid labeling in pancreatic cancer cells.
Pancreatic cancer MIA PaCa cells were cultured in the presence and absence of (15)N amino acids mixture for 72 h. During protein synthesis, the incorporation of (15)N amino acids results in a new mass isotopomer distribution in protein, which is approximated by the concatenation of two binomial distributions of (13)C and (15)N. The fraction of protein synthesis (FSR) can thus be determined from the relative intensities of the "labeled" (new) and the "unlabeled" (old) spectra. Six prominent spots were picked from 2-D gels of proteins from lysates of cells cultured in 0% (control), 50%, and 33% (15)N enriched media. These protein spots were digested and analyzed with matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. The isotopomer distribution of peptides after labeling can be fully accounted for by the labeled (new) and unlabeled (old) peptides. The ratio of the new and old peptide fractions was determined using multiple regression analysis of the observed spectrum as a linear combination of the expected new and the old spectra. The fractional protein synthesis rates calculated from such ratios of the same peptide from cells grown in 50% and 33% (15)N amino acid enrichments were comparable to each other. The FSR of these six identified proteins ranged between 44 and 76%.
Zhao
Yingchun
Y
Genomics & Functional Proteomics Laboratories, Osteoporosis Research Center, Creighton University Medical Center, 601 North 30th Street, Suite 6730, Omaha, Nebraska 68131, USA.
Lee
Wai-Nang Paul
WN
Lim
Shu
S
Go
Vay Liang
VL
Xiao
Jing
J
Cao
Rui
R
Zhang
Hengwei
H
Recker
Robert Roy
RR
Xiao
Gary Guishan
GG
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes / Q000032:analysis*; D010190:Pancreatic Neoplasms; D010455:Peptides / Q000032:analysis; D014176:Protein Biosynthesis; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured
D000596:Amino Acids; D009587:Nitrogen Isotopes; D010455:Peptides