title: Systematic screening of protein modifications in four kinases using affinity enrichment and mass spectrometry analysis with unrestrictive sequence alignment.
abstract: Protein kinases transfer phosphate groups from ATP to substrate proteins, they are known to be involved in diverse cellular processes. They are also important therapeutic targets in pharmaceutical design. Previous studies indicated that multiple post-translational modifications (PTMs) exist in kinases in addition to phosphorylation, and these PTMs play an important role in regulating kinases activities. Nevertheless, a comprehensive analysis for PTMs of kinases is insufficient due to technical limitations, which prevent us from better understanding their functional regulation. Here, we have developed a novel strategy that combines glutathione S-transferase tag affinity enrichment with nano-liquid chromatography coupled with tandem mass spectrometry analysis and non-restrictive protein sequence alignment for identification of diverse PTMs in four yeast kinases. The method allows us to enrich and analyze the entire protein isomers and to minimize the loss of all isomers of protein sample during protein purification. In our study, nineteen phosphorylation sites and several other types of PTMs sites were localized in 4 protein kinases. In addition, we found that some interesting mass shifts can not match those of the known PTMs. It suggested the existence of some undescribed PTMs in the proteins. Accordingly, this study showed that the novel strategy holds a great potential for identification of full-spectrum PTMs in proteins. Our data serves as a stepping stone for future functional studies.
authors: Department of Chemistry, Nankai University, Tianjin, China. zhangkai730@yahoo.com.cn
authors: Kai
authors: K
authors: Zhang
authors: Yixin
authors: Y
authors: Zhu
authors: Xiwen
authors: X
authors: He
authors: Yukui
authors: Y
authors: Zhang
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D036103:Nanotechnology; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D005982:Glutathione Transferase; D011494:Protein Kinases