Quantitative Proteomics: Measuring Protein Synthesis Using 15N Amino Acid Labeling in Pancreatic Cancer Cells
| Autor: | Wai-Nang Paul Lee, Gary Guishan Xiao, Hengwei Zhang, Vay Liang W. Go, Shu Lim, Jing Xiao, Robert Roy Recker, Rui Cao, Yingchun Zhao |
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| Rok vydání: | 2008 |
| Předmět: |
Proteomics
Molecular Sequence Data Quantitative proteomics Peptide Mass spectrometry Article Analytical Chemistry Pancreatic cancer Tumor Cells Cultured medicine Protein biosynthesis Humans Electrophoresis Gel Two-Dimensional Amino Acid Sequence Amino Acids Peptide sequence chemistry.chemical_classification Nitrogen Isotopes medicine.disease Amino acid Pancreatic Neoplasms chemistry Biochemistry Protein Biosynthesis Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Peptides |
| Zdroj: | Analytical Chemistry. 81:764-771 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/ac801905g |
| Popis: | 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%. |
| Databáze: | OpenAIRE |
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