Tracey B M, Shuker D E
Biomonitoring and Molecular Interactions Section, MRC Toxicology Unit, University of Leicester, U.K.
Chem Res Toxicol. 1997 Dec;10(12):1378-86. doi: 10.1021/tx970117+.
A synthetic peptide, VLSPADKTNWGHEYRMF(cmC)QIG, was reacted with 4-chlorobenzenediazonium hexafluorophosphate as a model for reactions of aromatic diazonium ions with proteins. At a ratio of diazonium ion to peptide of 0.8:1, three products could be seen by reversed-phase HPLC. Electrospray mass spectrometric analysis of the isolated products revealed that two of the products had the same mass of 2648 Da, being 138 Da higher than the parent peptide and corresponding to the addition of a 4-chlorobenzenediazo group. The third isolated product had a mass of 2787 Da which corresponded to the addition of two 4-chlorobenzenediazo groups (276 Da). Digestion of the monoadducted intact peptides with trypsin or endoproteinase Glu-C and HPLC separation of adduct oligopeptides followed by sequencing with electrospray ionization tandem mass spectrometry showed unambiguously that histidine and tyrosine residues were the major sites of modification. Incubation of human serum albumin with 4-chlorobenzenediazonium hexafluorophosphate at molar ratios of 1:1, 1:2, and 1:10 resulted in adduct formation as detected by shifts in the HPLC retention time of the protein and also by an increase in mass as determined by electrospray mass spectrometry.
一种合成肽VLSPADKTNWGHEYRMF(cmC)QIG，与4-氯苯重氮六氟磷酸盐反应，作为芳香族重氮离子与蛋白质反应的模型。在重氮离子与肽的比例为0.8:1时，通过反相高效液相色谱法可观察到三种产物。对分离出的产物进行电喷雾质谱分析表明，其中两种产物的质量相同，均为2648 Da，比母肽高138 Da，对应于添加了一个4-氯苯重氮基团。第三种分离出的产物质量为2787 Da，对应于添加了两个4-氯苯重氮基团（276 Da）。用胰蛋白酶或内肽酶Glu-C消化单加成的完整肽，对加合物寡肽进行高效液相色谱分离，然后用电喷雾电离串联质谱法测序，明确显示组氨酸和酪氨酸残基是主要的修饰位点。将人血清白蛋白与4-氯苯重氮六氟磷酸盐以1:1、1:2和1:10的摩尔比孵育，通过蛋白质高效液相色谱保留时间的变化以及电喷雾质谱法测定的质量增加检测到加合物的形成。
