Covalent modification of stathmin by CCNU determined by FTMS analysis of modified proteins and tryptic peptides.
Chemical modification of proteins is often carried out to generate protein-small molecule conjugates for various applications. The high resolution and mass accuracy of a Fourier transform mass spectrometer is particularly useful for assessing the extent or sites of covalent modifications. As protein-small molecule reactions often produce products with variable numbers of the compound incorporated at different sites, a direct mass analysis of the reaction products at times yields mass spectra hard to interpret. Chromatographic separation at protein level could reduce the complexity of a sample, thus allowing more accurate mass spectrometric analysis. In this report, we demonstrate the utility of reversed-phase protein chromatography and FT-ICR mass spectrometry in analyzing CCNU (lomustine, 1-(2-chloroethyl)-3-cyclohexyl-1-nitroso-urea, MW: 233.7Da) modification of stathmin. With this combined approach, we determined the stoichiometry as well as sites of CCNU incorporation into the protein, demonstrating differential reactivity of several lysyl residues to CCNU alkylation.
Wu
Wells W
WW
Proteomics Core Facility, National Heart, Lung, and Blood Institute, NIH, Building 10, Room 8C103C, 10 Center Drive, Bethesda, MD 20892-1597, USA.
Wang
Guanghui
G
Liang
Xing-Jie
XJ
Park
John K
JK
Shen
Rong-Fong
RF
D000478:Alkylation; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D008130:Lomustine / Q000494:pharmacology*; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D050777:Stathmin / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
D018906:Antineoplastic Agents, Alkylating; D010455:Peptides; D050777:Stathmin; D008130:Lomustine; D014357:Trypsin; D008239:Lysine