Accurate quantitation of protein expression and site-specific phosphorylation

Abstract

A mass spectrometry-based method is described for simultaneous identification and quantitation of individual proteins and for determining changes in the levels of modifications at specific sites on individual proteins. Accurate quantitation is achieved through the use of whole-cell stable isotope labeling. This approach was applied to the detection of abundance differences of proteins present in wild-type versus mutant cell populations and to the identification of in vivo phosphorylation sites in the PAK-related yeast Ste20 protein kinase that depend specifically on the G1 cyclin Cln2. The present method is general and affords a quantitative description of cellular differences at the level of protein expression and modification, thus providing information that is critical to the understanding of complex biological phenomena.

Figure 1

Method for quantitating differential protein expression. For illustration, proteins that remain unchanged in the two cell pools are assumed to be present in equal abundance. (In practice, proteins that remain unchanged in the two cell pools may not be present in equal abundance, and the ratios of the peptide peaks will be a constant value that is not equal to one.)

Figure 2

The measured ratio of unlabeled (¹⁴N) to labeled (¹⁵N) recombinantly expressed Abl-SH2 versus the calculated ratio showing the linearity of the method.

Figure 3

Examples of MALDI-MS spectra of tryptic peptides obtained from proteins derived from two pools of S. cerevisiae that differ only in their ability to express the G1 cyclin CLN2. (A) Elongation factor 1α; (B) Triosephosphate isomerase. The numbers “14” and “15” denote peaks originating from unlabeled (cln2⁻) and ¹⁵N-labeled (CLN2⁺) tryptic peptides. The ratios of the intensities of the pairs of unlabeled to labeled peaks were used to quantitate the relative levels of the proteins in the two cell pools. (Left Inset) Detail shows a pair of peaks from a single tryptic peptide. The lower mass cluster of peaks corresponds to isotopically resolved components of the unlabeled peptide whereas the upper mass cluster corresponds to the isotopic components of the ¹⁵N labeled peptide. Tests of the goodness of fit of the theoretical isotope distribution (Right Inset) to the experimental distribution (Left Inset) revealed that the level of incorporated ¹⁵N label was 93 ± 1%. The peak intensity for the unlabeled peptide was determined by integrating the intensities of each component in the lower isotopic cluster whereas that for the labeled peptide was determined by integrating the intensity of each component in the upper isotopic cluster.

Figure 4

Method for site-specific quantitation of changes in the level of phosphorylation on proteins. For illustration, peptides that remain unchanged in the two cell pools are assumed to be present in equal abundance, and the level of phosphorylation of peptide A is assumed to change from 30% (pool 1) to 70% (pool 2)—leading to a decrease in the measured intensity ratio of unphosphorylated peptide X and an increase for phosphorylated peptide Xp.

Figure 5

Percentage change of the normalized peak intensity ratio for four different Ste20_trunc peptides that undergo phosphorylation.

Figure 6

Analysis of function and interaction of Ste20 (wt and P-site mutant), Ste4, and Cln2. (A) Effect of CLN2 overexpression on Ste20p mobility with and without P-site mutations. A wt strain was transformed with a vector control (lane 1), with wt STE20 (lanes 2 and 3), or the 12-site P mutant (lanes 4 and 5), as well as with a plasmid encoding the CLN2 gene expressed from the GAL1 promoter (lanes 3 and 5). (B) The ste20 P-site mutant confers sensitivity to pheromone-induced growth arrest, which is repressed by GAL1∷CLN2. Serial dilutions of strain BOY491 (CLN⁺ ste20∷TRP1 GAL1∷CLN2) transformed with a vector control (row 1), wt STE20 (row 2), or the 12-site P-site mutant (row 3). Two separate transformants were tested. The plates used were SCDex (Top Left), SCDex with 0.3 μM α factor (Top Right), SCGal (Lower Left), or SCGal with 0.3 μM α factor (Lower Right). (C) Relative levels of α factor-induced FUS1 transcription (20) for wt STE20 and the 12-site P-site mutant. Transformants in strain BOY491 were grown in the presence (dark gray and white bars) or absence (black and light gray bars) of galactose. α factor (0.5 μM) was added (black and dark gray bars) before preparation of RNA and Northern blot analysis. The columns indicate the relative levels of FUS1 transcript detected, as compared with that observed for wt STE20 induced with pheromone. TCM1 was used as a loading control. (D) Cln2 coprecipitates with Ste20, and Ste4 expression reduces this association. The strain 2198–11C (CLN⁺) was transformed with either a vector control (lane 1) or with plasmids encoding wt GST-Ste20 (lanes 2 through 4) or 12-site mutant GST-ste20 (lanes 5 through 7). Transformants also contained either vector controls or plasmids overexpressing myc-tagged Cln2 and/or Ste4 from the GAL1 promoter, as indicated. The Ste20 protein was precipitated with glutathione agarose, and aliquots were analyzed by Western blotting to determine the level of Ste20 (A) or Cln2 (B) present. An aliquot of the whole cell extract also was analyzed to determine the relative level of Cln2. (E) Effect of CLN2 overexpression on Ste4/Ste20 association. The strain 2198–11C (CLN⁺, lanes 1, 2, and 4) or 2198–3A (CLN⁺ GAL1p∷CLN2, lanes 3 and 5) was transformed with either a vector control (lane 1) or with plasmids encoding wt GST-Ste20 (lanes 2 and 3) or the 12-site GST-ste20 mutant (lanes 4 and 5). Transformants also contained either a vector control or a plasmid overexpressing HA-tagged Ste4, as indicated. The Ste20 protein was precipitated with glutathione agarose, and aliquots were analyzed by Western blotting to determine the relative levels of Ste20 or Ste4 present. An aliquot of the whole cell extract (WCE) also was analyzed to determine the relative levels of Ste4.