Quantitation of changes in protein phosphorylation: a simple method based on stable isotope labeling and mass spectrometry

Abstract

Reversible protein phosphorylation plays an important role in many cellular processes. However, a simple and reliable method to measure changes in the extent of phosphorylation is lacking. Here, we present a method to quantitate the changes in phosphorylation occurring in a protein in response to a stimulus. The method consists of three steps: (i) enzymatic digestion in H(2)16O or isotopically enriched H(2)18O to label individual pools of differentially phosphorylated proteins; (ii) affinity selection of phosphopeptides from the combined digests by immobilized metal-affinity chromatography; and (iii) dephosphorylation with alkaline phosphatase to allow for quantitation of changes of phosphorylation by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We applied this strategy to the analysis of the yeast nitrogen permease reactivator protein kinase involved in the target of rapamycin signaling pathway. Alteration in the extent of phosphorylation at Ser-353 and Ser-357 could be easily assessed and quantitated both in wild-type yeast cells treated with rapamycin and in cells lacking the SIT4 phosphatase responsible for dephosphorylating nitrogen permease reactivator protein. The method described here is simple and allows quantitation of relative changes in the level of phosphorylation in signaling proteins, thus yielding information critical for understanding the regulation of complex protein phosphorylation cascades.

Figure 1

Regulation of NPR1 phosphorylation by the rapamycin-sensitive TOR signaling pathway. (A) Under good nutrient conditions, TOR promotes the association of TAP42 and SIT4, thereby inactivating the phosphatase activity of SIT4. NPR1 is, therefore, maintained in a highly phosphorylated state. (B) Rapamycin treatment leads to inactivation of TOR and to dissociation of the regulatory subunit TAP42 from SIT4. In turn, activated SIT4 dephosphorylates NPR1. Arrows indicate activation; bars indicate inhibition.

Figure 2

Outline of the strategy. Two protein pools differing in their extent of phosphorylation are digested with trypsin either in H₂¹⁶O or in H₂¹⁸O to obtain differential mass labeling. Equal amounts of the two pools are mixed, and phosphopeptides are selected with IMAC beads charged with Fe³⁺. To enable mass determinations of the bound peptides with high accuracy, the peptides are eluted from the IMAC beads with alkaline phosphatase. The two peptide pools can be distinguished by a shift of 4 Da in the isotope cluster, and the difference in the extent of phosphorylation is reflected by the peak areas of the two monoisotopic peaks.

Figure 3

Linearity assay. To simulate two protein pools with differing extent of phosphorylation, the relative amounts of ovalbumin digested in H₂¹⁸O or H₂¹⁶O was set to 16:1, and a MALDI spectrum was acquired for the T₃₁ phosphopeptide after IMAC selection and elution with alkaline phosphatase. (Inset) The plot of the ratios of the theoretical vs. the experimentally measured areas of the T₃₁ (2,008.8 Da) and the ¹⁸O-labeled T₃₁ (2,012.8 Da).

Figure 4

IMAC phosphopeptide selection from a tryptic digest of GST-NPR1. (A) MALDI-TOF spectrum of phosphopeptides in linear mode. *, Number of phosphates associated with the peptide. White circles mark unspecifically bound peptides. (B) MALDI-TOF spectrum of the phosphopeptides eluted from the IMAC beads in the presence of alkaline phosphatase. The spectrum was acquired in reflector mode. (Inset) Coomassie blue-stained SDS gel of purified GST-NPR1 without (−CIP) or with (+CIP) alkaline phosphatase treatment.

Figure 5

MALDI-TOF spectra of tryptic peptides T₆₈ (A), T_31–32 (B–D), and T₁₀ (E) of GST-NPR1. (A and B) The nonphosphorylated peptide T₆₈ (1,728.3 Da and 1,732.3 Da H₂¹⁶O/H₂¹⁸O digest) and the doubly phosphorylated T_31–32 peptide from untreated cells (1,227.4 Da and 1,231.4 Da H₂¹⁶O/H₂¹⁸O digest) indicate that equal amounts of protein were digested and equal amounts of phosphopeptides were selected. (C and D) T_31–32 phosphopeptide from untreated (1,227.4 Da) and from rapamycin-treated (1,231.4 Da) wild-type cells (C) or from sit4 mutant cells (D). (E) Phosphopeptide T₁₀ from untreated (3,269.4 Da) or rapamycin-treated (3,273.4 Da) wild-type cells. (F) Ratios between the isotope clusters of the T_31–32 (column h), the T₁₀ (column i) peptide from the wild-type strain treated with rapamycin, and the T_31–32 peptide from the sit4 mutant JC28-1B treated with rapamycin (column k) digested in H₂¹⁶O or H₂¹⁸O. Error bars represent the mean of three independent experiments. (G) Coomassie blue-stained SDS gel of GST-NPR1 (labeled with an asterisk) from wild-type cells (JC19-1A, wt, lanes 1 and 2) or sit4 mutant cells (JC28-1B, sit4, lanes 3 and 4), either untreated (−) or treated (+) with 200 nM rapamycin for 15 min.