An isotope labeling strategy for quantifying the degree of phosphorylation at multiple sites in proteins

Abstract

A procedure for determining the extent of phosphorylation at individual sites of multiply phosphorylated proteins was developed and applied to two polyphosphorylated proteins. The protocol, using simple chemical (Fischer methyl-esterification) and enzymatic (phosphatase) modification steps and an accessible isotopic labeling reagent (methyl alcohol-d 4 ), is described in detail. Site-specific phosphorylation stoichiometries are derived from the comparison of chemically identical but isotopically distinct peptide species analyzed by microspray liquid chomatography-mass spectrometry ( LC-MS) using a Micromass Q-TOF2 mass spectrometer. Ten phosphorylation sites were unambiguously identified in tryptic digests of both proteins, and phosphorylation stoichiometries were determined for eight of the ten sites using the isotope-coded strategy. The extent of phosphorylation was also estimated from the mass spectral peak areas for the phosphorylated and unmodified peptides, and these estimates, when compared with stoichiometries determined using the isotope-coded technique, differed only marginally (within ϳ20%). P rotein phosphorylation is an omnipresent and important dynamic phenomenon in living systems that affects protein structure, protein-protein interactions and catalytic activity during physiological processes [1]. Often, protein phosphorylation occurs simultaneously at many different sites in a single protein [2,3]. In these cases it is desirable to learn the extent to which each site is phosphorylated, to begin to allow correlation of phosphorylation status to function.While mass spectrometric (MS) techniques can be used to great effect in locating and sequencing phosphopeptides derived from proteolytic digests of polyphosphorylated proteins, differences in ionization and detection efficiencies, from peptide to peptide, make MS based quantification problematic [4]. Numerous isotopic-coding methodologies have been described for comparative proteomic applications in the past few years (reviewed in [5,6]). A technique utilizing differential protease-dependent incorporation of 18 O/ 16 O, followed by phosphopeptide enrichment, phosphatase treatment, and MALDI-MS analysis has been employed to examine changes in protein phosphorylation [7]. Zhang and coworkers described using differential phosphatase treatment and isotope coding to measure phosphorylation stoichiometry with MALDI-MS analysis [8]. Here we describe an alternative approach, which combines differential phosphatase treatment with a one step isotope coding strategy, to yield phosphorylation site stoichiometries. This method is coupled to microspray liquid chromatography/tandem mass spectrometry ( LC-MS/MS) to characterize phosphorylation sites in two poly-phosphorylated proteins from the Arabidopsis thaliana calcium dependent protein kinase super-family, CPK1 and CRK3 [9].We have applied a simple isotopic-coding strategy to determine the phosphorylation stoichiometry of phosphopeptides derived from purified polyphosphorylated proteins. The two-part procedure...

Phosphorylation Controls Autoinhibition of Cytoplasmic Linker Protein-170

“…The proteins were expressed in Escherichia coli BL21 and purified by sequential affinity columns on nickel-nitrilotriacetic acid agarose (QIAGEN, Valencia, CA) and glutathione-Sepharose 4B (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) as described by Hegeman et al , 2004. Purified protein (30 μg) was incubated in a manufacturer's reaction buffer and 1 mM ATP with PKA (2500 U; New England Biolabs, Ipswich, MA) for 3–18 h at 30°C.…”

“…Purified protein (30 μg) was incubated in a manufacturer's reaction buffer and 1 mM ATP with PKA (2500 U; New England Biolabs, Ipswich, MA) for 3–18 h at 30°C. The proteins were denatured by 8 M urea, diluted with 50 mM ammonium bicarbonate, pH 7.5, and digested with 0.6 μg of trypsin for 14 h as described by Hegeman et al (2004).…”

Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays

“…Fractions were assembled into 10 pools of approximately equal content as judged by 280 nm UV traces. Peptides from each of these pools were purified by solid phase extraction (Spec-PT-C18, Varian) before analysis by C18-reverse phase ESI tandem MS on a Q-TOF2 (Micromass, Manchester, U.K.) mass spectrometer with a previously described modified LC electrospray source (15). Briefly, chromatographic separation of peptides before MS was accomplished by using columns that were made with fused silica tubing (OD at 365 m and inner diameter at 100 m) with pulled tips (1 m of orifice), packed with Zorbax Eclipse XDB-C18, 5 m, 300 Å pore-size media (Agilent, Palo Alto, CA) to 12 cm (16).…”

Tobacco Calcium-dependent Protein Kinases Are Differentially Phosphorylated in Vivo as Part of a Kinase Cascade That Regulates Stress Response

“…The corresponding in vitro autophosphorylation sites (P-sites) have been mapped for several enzymes (13)(14)(15)(16) and were found with high frequency in the variable N termini (17). It has not yet been experimentally addressed whether CDPK autokinase reactions follow an intramolecular (unimolecular: kinase and P-site on the same polypeptide) or intermolecular (bimolecular: kinase and P-site on different polypeptide) mechanism or both.…”