Edman降解中异硫氰酸苯酯新修饰位点的发现和验证

Discovery and validation of a novel modification by phenyl isothiocyanate in Edman degradation

Edman降解是最早建立的一种用于多肽和蛋白质氨基端测序的方法,该方法现在仍被广泛用于生物化学领域。随着高通量蛋白质组学技术的发展和应用,该方法中的异硫氰酸苯酯反应被用于修饰蛋白质氨基端,并用于检测蛋白质水解位点。但还没有异硫氰酸苯酯是否可以修饰其他氨基酸侧链并影响多肽序列分析的研究。为了探究其修饰其他氨基酸的可能性,本文利用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)和液相色谱-串联质谱(LC-MS/MS)研究了异硫氰酸苯酯对一个模型肽的化学修饰。质谱数据解析后发现在高浓度异硫氰酸苯酯的反应条件下,组氨酸上可以引入一个新的异硫氰酸苯酯修饰位点。这一修饰位点的发现预示着通过改变实验条件或分析方法,可以更准确地利用Edman降解和蛋白质组学技术分析多肽和蛋白质。

Edman degradation is the earliest technique used for the peptide N-terminal sequen-cing. Moreover,this method is still widely applied in multiple disciplines of the life science research community. With the development and application of robust proteomic techniques,the reaction of phenyl isothiocyanate（ PITC）in this method developed by Edman has been used to modify the amines of peptides,and the identification of proteolytic cleavages of proteins occur-ring in cell signaling events,in a high throughput manner. However,there is no report about whether PITC can introduce modifications on the other amino acids,affecting the analysis and identification of proteins and peptide sequences. In order to explore the possibility of PITC modification on other amino acids,we used matrix-assisted laser desorption ionization-time of flight mass spectrometry （ MALDI-TOF-MS ） and liquid chromatography-mass spectrometry/mass spectrometry （ LC-MS/MS ） to investigate the reaction products between PITC and a model peptide under two different PITC conditions in this work. Through MS and MS/MS analy-ses,we found that at a high PITC concentration,PITC could additionally generate a new modi-fication on an amino acid,histidine. The discovery of this modification may help us to more accurately analyze protein and peptide sequences during the application of Edman degradation and proteomics analysis after the optimization of experimental conditions and improvement of analytical methods.