Combining Alkaline Phosphatase Treatment and Hybrid Linear Ion Trap/Orbitrap High Mass Accuracy Liquid Chromatography−Mass Spectrometry Data for the Efficient and Confident Identification of Protein Phosphorylation

Abstract

Protein phosphorylation is a vital post-translational modification that is involved in a variety of biological processes. Several mass spectrometry-based methods have been developed for phosphoprotein characterization. In our previous work, we demonstrated the capability of a computational algorithm in mining phosphopeptide signals in large LC-MS data sets by measuring the mass shifts due to phosphatase treatment (Wu, H. Y.; Tseng, V. S.; Liao, P. C. J. Proteome Res. 2007, 6, 1812-1821). Mass accuracy seems to play an important role in efficiently selecting out phosphopeptide signals. In recent years, the hybrid linear ion trap (LTQ)/Orbitrap mass spectrometer, which provides a high mass accuracy, has emerged as a powerful instrument in proteomic analysis. Here, we developed a process to incorporate LC-MS data that was generated from an LTQ/Orbitrap mass spectrometer into our strategy for taking advantage of the accurate mass measurement. LTQ/Orbitrap raw files were converted to the open file format mzXML via the ReAdW.exe program. To find peaks that were contained in each mzXML file, an open-source computer program, msInspect, was utilized to locate isotopes and assemble those isotopes into peptides. An in-house program, LcmsFormatConverter, was utilized for signal filtering and format transformation. A proposed in-house program, DeltaFinder, was modified and used for defining signals with an exact mass shift due to the dephosphorylation reaction, which generated a table that listed potential phosphopeptide signals. The retention times and m/z values of these selected LC-MS signals were used to program subsequent LC-MS/MS experiments to get high-confidence phosphorylation site determination. Compared to our previous work finished by using a quadrupole/time-of-flight mass spectrometer, a larger number of phosphopeptides in the casein mixture were identified by using LTQ/Orbitrap data, demonstrating the merit of high mass accuracy in our strategy. In addition, the characterization of the lung cancer cell tyrosine phosphoproteome revealed that the use of alkaline phosphatase treatment combined with accurate mass measurement in this strategy increased data repeatability and confidence.

Attenuation of amyloid‐β generation by atypical protein kinase C‐mediated phosphorylation of engulfment adaptor PTB domain containing 1 threonine 35

“…1B-D). Our observations are in agreement with the finding of various mass spectrometric studies, namely, that GULP1 is a phosphoprotein (26)(27)(28). It has been reported that phosphorylation of AIPs alters APP processing, including residues within their PTB domains (14,19,29,30).…”

“…In vitro kinase assays were performed as previously described (22). GST-GULP1 [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43] and GST-GULP1 [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43] T35A were expressed in E. coli and purified with glutathione sepharose 4B according to the manufacturer's instructions (GE Healthcare). FLAG-tagged PKCz was isolated by immunoprecipitation using an anti-FLAG antibody (M2) (MilliporeSigma).…”

“…*P , 0.001. B) Bacterially expressed GST-GULP1[26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43] was incubated with PKCz immunoprecipitated from untreated(2) or 100 nM insulintreated (+) transfected cell lysate together with (g-[ 32 P])-ATP for 5 min at 30°C. RM is the reaction mix only without kinase.…”

Optimization of titanium dioxide and immunoaffinity-based enrichment procedures for tyrosine phosphopeptide using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

“…Because protein phosphorylation is regulated by an enormous range of protein kinases and phosphatases and maintains the dynamics of the living cell, characterization of the cellular phosphorylation status is important for understanding the complex network of signaling within a cell. Large-scale and comparative phosphoproteomics based on MS has emerged as an important tool [3][4][5]. Studies have produced and discriminated among cellular-protein phosphorylation that results in abnormalities leading to a variety of stimulations and diseases [6][7][8][9][10].…”

Development of an off-line capillary column IMAC phosphopeptide enrichment method for label-free phosphorylation relative quantification

“…Methyl esterification of acidic amino acids increases the phosphopeptide enrichment efficiency of IMAC by eliminating non-specific binding [1,6,13]. IMAC phosphopeptide enrichment followed by enzymatic de-phosphorylation strategies has been developed to increase the coverage of phosphoproteins in actual protein samples [14,15]. Sequential elution strategies aimed to separate monophosphorylated peptides from multi-phosphorylated peptides by employing different acid and base elution techniques have been also developed, expanding the coverage of phosphopeptides and phosphoproteins [16].…”