利用磺基异硫氰酸苯酯化学辅助方法鉴定磷酸化肽修饰位点

利用MALDI-OF质谱结合磺基异硫氰酸苯酯(SPITC)化学辅助的从头测序（de novo sequencing）方法,将用固相金属离子亲和色谱(immobilized metal affinity chromatography,ＩＭＡＣ) 选择性地从混合物中亲和提取的磷酸肽进行磷酸化位点测定,该方法只有肽键断裂产生的带C端的碎片离子系列（y^＋ 离子系列）出现在质谱图中，图谱背景清晰,信噪比高,单纯的y^＋ 片段离子系列使得谱图解析变得非常容易,对于多磷酸化肽的磷酸化位点，不需借助于任何软件,只需简单地计算两峰之间的分子量之差即可确定.

Identification of Phosphopeptide Modification Sites Based on Chemically-Assisted Method Using 4-Sulfophenyl Isothiocyanate Derivatization by Mass Spectrometry

Phosphorylated peptides have been detected and phosphorylation modified sites have been identified very sensitively by combining immobilized metal affinity chromatography (IMAC) affinity capture with chemically assisted de novo sequencing followed 4-sulfophenyl isothiocyanate (SPITC) derivatization by MALDI-TOF mass spectrometry. The N-terminal sulfonation of phosphopeptide by SPITC enhanced MALDI-TOF-PSD fragmentation signals and produced a spectrum containing only y~ ions. This method facilitated de novo sequencing and spectrum interpretation. The characterization of phosphorylated sites can simply calculated from mass differences between the adjacent y-ion, and advanced software is not need.