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.
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.
Wu
Hsin-Yi
HY
Department of Environmental and Occupational Health, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Tseng
Vincent Shin-Mu
VS
Chen
Lien-Chin
LC
Chang
Yu-Chen
YC
Ping
Peipei
P
Liao
Chen-Chung
CC
Tsay
Yeou-Guang
YG
Yu
Jau-Song
JS
Liao
Pao-Chi
PC
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000595:Amino Acid Sequence; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation* / Q000379:methods; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D012984:Software
D002364:Caseins; D010748:Phosphopeptides; D010750:Phosphoproteins; D000469:Alkaline Phosphatase