Quantitative proteomics: measuring protein synthesis using 15N amino acid labeling in pancreatic cancer cells

Abstract

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%.

Figure 1

Gel image after 2-D electrophoresis. MIA PaCa cells were cultured in MEM medium enriched with natural amino acids (A), 50% ¹⁵N labeled algal amino acids (B), and 33% ¹⁵N labeled algal amino acids (C) for 3 days. The cell pellets were lysed in 2-D lysis buffer and applied to 2-D electrophoresis. The marked spots were selected, cut out, digested, and analyzed. See Material and Methods for details.

Figure 2

Mass spectra of 1699 m/z fragment from spot 6 of lysates of cells grown in the presence of natural amino acids (Panel A), 50% enriched (Panel B) and 33% enriched (Panel C) ¹⁵N algal amino acid mixtures. MS spectrum of control in Panel A shows the binomial distribution of isotopomic peaks largely due to natural existence of ¹³C. Incorporation of ¹⁵N resulted in obvious mass shift in isotopomic distribution in Panel B and C. The degree of mass shift is a function of ¹⁵N enrichment. Panel D shows MS/MS spectrum of this fragment in ‘lift” mode. The peptide sequence is easily identified as almost every y ion is observed.

Figure 3

Illustration of data analysis using example of 1699 m/z fragment from spot 6. Panel A: Theoretical and experimental distribution of the unlabeled fragment is compared. M₀ is the monoisotopic peak. Panel B: Mass spectra of unlabeled and labeled peptides are aligned showing mass shifts, which was determined by curve fitting. Mass shift is larger with higher ¹⁵N enrichment. Panel C: Spectrum of peptide from MIA cells grown in 50% ¹⁵N enriched medium is shown with spectra of existing and newly synthesized peptide obtained by concatenation process. Panel D: Spectrum of peptide from MIA cells grown in 33% ¹⁵N enriched medium is shown with spectra of existing and newly synthesized peptide obtained by concatenation process. Fractional synthesis rate can be calculated using multiple linear regression analysis.

Figure 4

Synthesis rate comparison among the six identified proteins after 72 hours. 1, Calreticulin precursor; 2, Alpha-enolase; 3, Triosephosphate isomerase; 4, Phosphoglycerate mutase I; 5, Prohibitin; 6, 40S ribosomal protein SA. Open columns represent 50% ¹⁵N labeling and filled columns represent 33% ¹⁵N labeling. The turnover rates of protein 1 and 5 are significantly lower than those of protein 2, 3, 4, and 6.