Mass Spectrometric Analysis of Phosphorylation Modification in Talin from Human Colorectal Carcinoma Tissues: Mass Spectrometric Analysis of Phosphorylation Modification in Talin from Human Colorectal Carcinoma Tissues

The phosphorylation modification of talin, especially the phosphorylation state of talin in pathological environment such as carcinoma, is closely relevant to carcinogenesis and metastasis process. In this study, talin protein was isolated from human colorectal carcinoma tissues by salt fractionation and ion exchange chromatography, followed by further purification by electrophoresis. The purified talin was subject to tryptic digestion. Either immobilized Fe3+ affinity chromatography or TiO2 affinity chromatography was used to absorb the phosphorylated peptides under acidic condition, which were then eluted with 1% ammonium hydroxide. The separation was performed on a Michrom Magic C-18 column with gradient elution using two mobile phase solutions (A: 99% water+1% ACN +0.1% formic acid; B: 99% ACN+1% water+0.1% formic acid). ESI mass spectrometer was used to detect the product ions of the eluted peaks under data-dependent acquisition mode. The results indicated that 8 phosphorylated peptides were captured by the immobilized Fe3+ affinity chromatography, whereas 9 phosphorylated peptides were captured by TiO2 affinity chromatography. The present study provides a rapid, accurate method for characterizing talin protein isolated from human colorectal carcinoma tissues.