A novel titanium dioxide-polydimethylsiloxane plate for phosphopeptide enrichment and mass spectrometry analysis.
The phosphorylation of proteins is a major post-translational modification that is required for the regulation of many cellular processes and activities. Mass spectrometry signals of low-abundance phosphorylated peptides are commonly suppressed by the presence of abundant non-phosphorylated peptides. Therefore, one of the major challenges in the detection of low-abundance phosphopeptides is their enrichment from complex peptide mixtures. Titanium dioxide (TiO2) has been proven to be a highly efficient approach for phosphopeptide enrichment and is widely applied. In this study, a novel TiO2 plate was developed by coating TiO2 particles onto polydimethylsiloxane (PDMS)-coated MALDI plates, glass, or plastic substrates. The TiO2-PDMS plate (TP plate) could be used for on-target MALDI-TOF analysis, or as a purification plate on which phosphopeptides were eluted out and subjected to MALDI-TOF or nanoLC-MS/MS analysis. The detection limit of the TP plate was ∼10-folds lower than that of a TiO2-packed tip approach. The capacity of the ∼2.5 mm diameter TiO2 spots was estimated to be ∼10 μg of β-casein. Following TiO2 plate enrichment of SCC4 cell lysate digests and nanoLC-MS/MS analysis, ∼82% of the detected proteins were phosphorylated, illustrating the sensitivity and effectiveness of the TP plate for phosphoproteomic study.
Graduate Institute of Integrated Medicine, China Medical University, Taichung 40402, Taiwan; Proteomics Core Laboratory, Department of Medical Research, China Medical University Hospital, Taiwan. Electronic address: cjchen@mail.cmu.edu.tw.
Chao-Jung
CJ
Chen
Institute of Molecular Biology, National Chung Hsing University, Taichung 40402, Taiwan.
Chien-Chen
CC
Lai
Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan.
Mei-Chun
MC
Tseng
Proteomics Core Laboratory, Department of Medical Research, China Medical University Hospital, Taiwan.
Yu-Ching
YC
Liu
Graduate Institute of Integrated Medicine, China Medical University, Taichung 40402, Taiwan; Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung 40402, Taiwan.
Yu-Huei
YH
Liu
Proteomics Core Laboratory, Department of Medical Research, China Medical University Hospital, Taiwan.
Liang-Wei
LW
Chiou
College of Chinese Medicine, China Medical University, Taichung 40402, Taiwan; Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung 40402, Taiwan.
Fuu-Jen
FJ
Tsai
D002460:Cell Line; D004129:Dimethylpolysiloxanes / Q000737:chemistry*; D006801:Humans; D057230:Limit of Detection; D010748:Phosphopeptides / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014025:Titanium / Q000737:chemistry*
D004129:Dimethylpolysiloxanes; D010748:Phosphopeptides; C009495:titanium dioxide; C013830:baysilon; D014025:Titanium
Mass spectrometry; Phosphopeptide; Phosphoproteomics; Plate; Polydimethylsiloxane; Titanium dioxide