Hogan Jason M, McLuckey Scott A
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-2084, USA.
J Mass Spectrom. 2003 Mar;38(3):245-56. doi: 10.1002/jms.458.
The M + 20H-M + 12H charge states of native and reduced porcine elastase, a 25.9 kDa serine protease, were subjected to collisional activation in a quadrupole ion trap. For most charge states, ion parking was used to increase the number of parent ions over that yielded directly by electrospray. Ion-ion proton transfer reactions were used to reduce product ion charge states largely to +1 to simplify spectral interpretation. Both forms of the protein show charge state dependent fragmentation behavior. The native protein, which contains four disulfide linkages, shows almost no evidence for fragmentation within the regions of the protein linked by disulfide bonds. However, at the lowest charge states studied, evidence for cleavage of a least one of the disulfide bonds was evident in the appearance of a c-type ion. The highest charge states of native elastase showed several prominent cleavages C-terminal to valine residues. As the charge state decreased, however, preferential cleavages at acidic amino acid residues became important. The reduced form of the protein did not show particularly prominent cleavages at valine residues. However, many of the same preferential cleavages at acidic amino acid residues noted for the native protein were also observed in the same charge states of the reduced protein. The reduced protein also showed additional cleavages from regions of the protein that are ordinarily protected by disulfide linkages in the native form.
25.9 kDa的丝氨酸蛋白酶——天然和还原型猪弹性蛋白酶的M + 20H - M + 12H电荷态,在四极离子阱中进行碰撞活化。对于大多数电荷态,采用离子驻留来增加母离子数量,超过电喷雾直接产生的数量。离子 - 离子质子转移反应用于将产物离子电荷态大幅降低至 +1,以简化光谱解析。该蛋白质的两种形式均表现出电荷态依赖性裂解行为。含有四个二硫键的天然蛋白质,在由二硫键连接的蛋白质区域内几乎没有裂解迹象。然而,在所研究的最低电荷态下,在c型离子的出现中明显有至少一个二硫键断裂的证据。天然弹性蛋白酶的最高电荷态在缬氨酸残基的C末端显示出几个显著的裂解。然而,随着电荷态降低,酸性氨基酸残基处的优先裂解变得重要。蛋白质的还原形式在缬氨酸残基处未显示出特别显著的裂解。然而,在还原型蛋白质的相同电荷态中也观察到了许多与天然蛋白质中酸性氨基酸残基处相同的优先裂解。还原型蛋白质还在通常由天然形式中的二硫键保护的蛋白质区域显示出额外裂解。