基质辅助激光解析电离质谱用于多肽和蛋白质亚硝基化修饰研究

Study on the Nitrosation of Peptide and Proteins with Nitrous Acid Using Matrix-Assisted Laser Desorption/Ionization-Time of Flight-Mass Spectrometry

亚硝基阴离子是生物体内重要的生物调控小分子,广泛分布于生物体的血液和组织中,能使蛋白质发生硝基化和亚硝基化修饰,并且调控生物体的多种生理过程。本研究应用高灵敏度的基质辅助激光解析电离-飞行时间串联质谱(MALDI-TOF-MS)技术,以合成的标准肽段和肌红蛋白为模型系统。研究了蛋白质和多肽主链N端氨基与赖氨酸侧链氨基对HNO2反应的敏感性,并考察了反应体系和不同的反应时间对HNO2与多肽的反应完全程度的影响。结果表明,HNO2能与肽段N端的伯氨基发生重氮化反应,形成的叠氮化中间产物不稳定,放出氮气形成碳正离子,碳正离子可进一步发生取代或消除反应。发生取代反应,水溶液中的羟基取代N-末端氨基形成M+1产物,既使得产物比原肽段分子量多1Da;发生消除反应,脱掉1分子的氨,又生成M-17烯烃衍生物,从而使产物比原肽段分子量少17Da。当采用1mol/L乙酸和1mol/L的亚硝酸钠作为反应体系时,反应5min,肽段的N端氨基即可完全与HNO2反应生成羟基取代产物和烯烃衍生物。而在上述条件下,未发现赖氨酸侧链氨基与HNO2反应,说明HNO2主要与肽段和蛋白的N端氨基发生反应。

Nitrite anions are key small molecules for regulating many physiological processes, and are abundant in blood and tissues. In the present work, the reactivity of ε-NH2 of N-terminus and ε-NH2 of lysine of peptides and proteins toward nitrous acids was studied using matrix assisted laser desorption/ionization-time of flight-mass spectrometry（MALDI-TOF-MS）. The synthesized standard peptides and standard protein （myoglobin） were used as model system to investigate the effect of different reaction system and different time on the reaction degree of peptides with nitrous acid. The results show that the diazo-reaction occurs between amino group of N-teminal of peptides and nitrous acid, and the intermediate formed in this reaction is a diazonium ion that is unstable to give nitrogen gas and a carbocation. The carbocation proceeds either to nucleophilic substitution or to elimination. The product of M ＋ 1 is formed by substitution. The other major product of M-17 observed in the present study is the alkene derivative formed by an elimination reaction. When the reaction system concludes acetic acid and sodium nitrite, the nitrous acid can completely react with amino group of N-termial peptides in 5 min. However,in the same situation, ε-NH2 of lysine of peptides could not react with nitrous acids . The results of this study can provide useful information for understanding the interaction of nitrous acid and proteins in depth.