A general strategy for studying multisite protein phosphorylation using label-free selected reaction monitoring mass spectrometry

Abstract

The majority of eukaryotic proteins are phosphorylated in vivo and phosphorylation may be the most common regulatory post-translational modification. Many proteins are phosphorylated at numerous sites, often by multiple kinases, which may have different functional consequences. Understanding biological functions of phosphorylation events requires methods to detect and quantify individual sites within a substrate. Here we outline a general strategy that addresses this need and relies on the high sensitivity and specificity of selected reaction monitoring (SRM) mass spectrometry, making it potentially useful for studying in vivo phosphorylation without the need to isolate target proteins. Our approach uses label-free quantification for simplicity and general applicability, although it is equally compatible with stable isotope quantification methods. We demonstrate that label-free SRM-based quantification is comparable to conventional assays for measuring the kinetics of phosphatase and kinase reactions in vitro. We also demonstrate the capability of this method to simultaneously measure relative rates of phosphorylation and dephosphorylation of substrate mixtures, including individual sites on intact protein substrates in the context of a whole cell extract. This strategy should be particularly useful for characterizing the physiological substrate specificity of kinases and phosphatases, and can be applied to studies of other protein modifications as well.

FgCDC14 regulates cytokinesis, morphogenesis, and pathogenesis in Fusarium graminearum

“…ScCdc14 has been linked to the reversal of Cdk phosphorylation (minimal consensus Ser/Thr‐Pro) at the end of mitosis (Visintin et al ., ). Cdc14 enzymes exhibit a strict preference for the pSer‐Pro subset of Cdk sites, both in vitro and in vivo (Eissler et al ., 2011; 2014; Bremmer et al ., ). In vitro , ScCdc14 also exhibits a strong requirement for Lys/Arg at the +3 position relative to pSer (Bremmer et al ., ).…”

A Multiple Reaction Monitoring (MRM) Method to Detect Bcr-Abl Kinase Activity in CML Using a Peptide Biosensor

“…A key advantage of MRM is the ability to detect and reproducibly quantify targeted peptides and phosphopeptides [19], [20] with exquisite selectivity and sensitivity. A further advantage for the peptide biosensor assay was that the tryptic fragment arising from the “reporter” module sequence is unnatural, and thus not subject to confounding background from the native proteins in the cell lysate.…”

“…Lower detection limits for MRM can reach the fmol-amol range depending on the specific analytes and instrumentation involved. We and others[9], [19]–[21] have successfully detected and quantified peptides and their phosphorylated derivatives using this technique. Here we describe the development of the MRM method and demonstrate that the biosensor/MRM strategy can be employed to detect Bcr-Abl activity and inhibition in CML cells as a model for monitoring drug sensitivity in human leukemia.…”

Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway in plants

“…Approximately 0.3 cm sections were excised from SDS-PAGE gels from positions corresponding to the bands detected in immunoblots from identical gels with anti-PhADT3 antibodies. Gel sections were destained and digested with trypsin (Sequencing grade, Promega) overnight 75 and analyzed at the Proteomics Facilities (Bindley Bioscience Center, Purdue University) as follows. Samples were subjected to reverse-phase HPLC-ESI-MS/MS using the Dionex UltiMate 3000 RSLC nano System (Thermo Fisher Scientific) coupled to the Q-Exactive High Field (HF) Hybrid Quadrupole Orbitrap MS (Thermo Fisher Scientific) and a Nano-electrospray Flex ion source (Thermo Fisher Scientific).…”