Improved Sensitivity for Phosphopeptide Mapping Using Capillary Column HPLC and Microionspray Mass Spectrometry:  Comparative Phosphorylation Site Mapping from Gel-Derived Proteins

Abstract

Reversible protein phosphorylation regulates many cellular processes. Understanding how phosphorylation controls a given pathway usually involves specific knowledge of which amino acid residues are phosphorylated on a given protein. This is often a nontrivial task. In addition to the difficulties involved in purifying sufficient amounts of any given protein, most phosphoproteins contain multiple, substoichiometric sites of phosphorylation. In this paper, we describe substantial improvements made to our previously reported multidimensional electrospray MS-based phosphopeptide mapping technique that have resulted in a 20-fold increase in sensitivity for the overall process. Chief among these improvements are the incorporation of capillary chromatography and a microionspray source for the mass spectrometer into the first dimension of the analysis. In the first dimension of the process, phosphopeptides present in the proteolytic digest of a protein are selectively detected and collected into fractions during on-line LC/ESMS, which monitors for phosphopeptide specific marker ions. The phosphopeptide containing fractions are then analyzed in the second dimension by either MALDI-PSD or nano-ES with precursor ion scanning. The relative merits and limitations of these two techniques for phosphopeptide detection are demonstrated. The enhancement in sensitivity of the method under the new experimental conditions makes it suitable for phosphorylation mapping (from selective detection through sequencing) on gel-separated phosphoproteins where the level of phosphorylation at any given site is <200 fmol. Furthermore, this method detects serine, threonine, and tyrosine phosphorylation equally well. We have successfully employed this new configuration to map 11 in vivo sites of phosphorylation on the Saccharomyces cerevisiae protein kinase YAK1. YAK1 peptides containing all five YAK1 PKA consensus sites are phosphorylated, suggesting that YAK1 is an in vivo substrate for PKA. In addition, four peptides containing cdk sites and the autophosphorylation site at Tyr530 were found to be phosphorylated. Because the first dimension of this method generates a phosphorylation profile that can be used for a semiquantitative evaluation of site specific phosphoxylation, we evaluated its ability to detect site-specific changes in the phosphorylation profile of a protein in response to altered cellular conditions. This comparative phosphopeptide mapping strategy allowed us to detect a change in phosphorylation stoichiometry on the motor protein myosin-V in response to treatment with either mitotic or interphase Xenopus egg extracts and to identify the single functionally significant phosphorylation site that regulates myosin-V cargo binding.

IQGAP1 Promotes Neurite Outgrowth in a Phosphorylation-dependent Manner

“…Phosphorylation Site Analysis of IQGAP1-Phosphopeptide-specific LC-ESMS was performed as described (26). Tryptic peptides from IQGAP1 were separated by capillary reversed phase HPLC using acetonitrile/water gradients flowing at 4 l/min.…”

“…A small aliquot of the sample was analyzed by nanoelectrospray tandem mass spectrometry (25) to establish the protein as IQGAP1. The remaining sample was analyzed for phosphorylation content by using a phosphopeptidespecific liquid chromatograph-mass spectrometry technique in which the mass spectrometer detects phosphate released from phosphopeptides undergoing collision-induced dissociation in the ion source (26,32). These data demonstrated that endogenous IQGAP1 is phosphorylated on multiple sites in intact cells (Fig.…”

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

“…Moreover, PKA seems to additionally inhibit the function of Msn2 and Msn4 via the protein kinases Yak1 and Rim15. Both kinases are under negative control of PKA, supposedly by direct phosphorylation, and deletion of YAK1 or RIM15, like deletion of both MSN2 and MSN4, can suppress the lethality caused by loss of PKA activity (Garrett and Broach 1989;Garrett et al 1991;Reinders et al 1998;Griffioen et al 2001;Moriya et al 2001;Zappacosta et al 2002). A recent report suggests that Yak1 phosphorylates and thereby activates Msn2 through a still unknown mechanism, which apparently does not implicate the control of Msn2 subcellular localization (Lee et al 2008a).…”

Automated Identification and Quantification of Protein Phosphorylation Sites by LC/MS on a Hybrid Triple Quadrupole Linear Ion Trap Mass Spectrometer

“…The methodology described here is applicable to low nanogram amounts of phosphoproteins isolated by SDS-PAGE, although overall sensitivity is totally dependent upon the stoichiometry of phosphorylation at any given site. The selectivity of precursor ion scanning for phosphopeptides has been reported previously (20,21,23). However, there are a few cases where a precursor of 79 signal will be generated from non-phosphorylated peptides, most commonly from peptides containing cysteic acid and methionine sulfone.…”

“…However, sequencing information is difficult to obtain on a triple quadrupole mass spectrometer due to the limited sensitivity in full scan mode. This has led to the use of this precursor ion scan method in conjunction with fraction collection where phosphopeptide-containing fractions are then analyzed via a second round of mass spectrometry to sequence the phosphopeptides (19,20). The selective detection of tyrosine phosphorylated peptides has been reported by the use of precursor ion scanning in a quadrupole TOF mass spectrometer (21).…”