Méndez T J, Johnson J V, Nichols L S, Lang G H, Eyler J R, Powell D H, Richardson D E
Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, Florida 32611, USA.
Bioconjug Chem. 2000 Mar-Apr;11(2):182-94. doi: 10.1021/bc990117g.
The modification of ribonuclease A with the heterobifunctional cross-linker, 4-succinimdyloxycarbonyl-methyl-alpha-[2-pyridyldithio]-toluene (SMPT) is described. RNase A has 11 potential sites of modification by the SMPT reagent. Tracking the two-dimensional separation and proteolytic digestion of SMPT-modified RNase A with ESI/FTICR-MS and HPLC/ESI/QIT-MS demonstrates the detailed information about number of SMPT modifications and sites of modification that can be obtained by application of these techniques. Analysis of native and modified RNase A tryptic digests by ESI/FTICR-MS resulted in the identification of the sites of modification. Semiquantitative results of the reactivity of certain lysine residues toward the coupling reagent SMPT are presented. Two sites (lysines 1 and 37) are highly reactive, while three sites (lysines 41, 61, and 104) appear to be unreactive toward SMPT under the conditions used. Experimental results demonstrate that quantitative comparison of relative intensities of peptide sequences of different charge states is not possible. No correlation was found between number of basic residues and sensitivity to detection. Digestion of the modified and unmodified RNase A by subtilisin followed by examination by HPLC/ESI/QIT-MS and MS(n) enabled further investigation of modification on lysines 1 and 7, including modification at the epsilon- and alpha-amino positions on lysine 1.
本文描述了用异双功能交联剂4-琥珀酰氧基羰基甲基-α-[2-吡啶二硫基]-甲苯（SMPT）对核糖核酸酶A进行修饰的过程。核糖核酸酶A有11个可被SMPT试剂修饰的潜在位点。通过电喷雾电离/傅里叶变换离子回旋共振质谱（ESI/FTICR-MS）和高效液相色谱/电喷雾电离/四极杆离子阱质谱（HPLC/ESI/QIT-MS）追踪SMPT修饰的核糖核酸酶A的二维分离和蛋白酶解，可得到有关SMPT修饰数量和修饰位点的详细信息。通过ESI/FTICR-MS分析天然和修饰的核糖核酸酶A的胰蛋白酶消化产物，鉴定出了修饰位点。给出了某些赖氨酸残基与偶联试剂SMPT反应性的半定量结果。两个位点（赖氨酸1和37）反应性很高，而在所用条件下，三个位点（赖氨酸41、61和104）似乎对SMPT无反应。实验结果表明，无法对不同电荷态肽序列的相对强度进行定量比较。未发现碱性残基数量与检测灵敏度之间存在相关性。用枯草杆菌蛋白酶消化修饰和未修饰的核糖核酸酶A，然后通过HPLC/ESI/QIT-MS和串联质谱（MS(n)）进行检测，能够进一步研究赖氨酸1和7的修饰情况，包括赖氨酸1的ε-氨基和α-氨基位置的修饰。
