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Accelerated on‐column lysine derivatization and cysteine methylation by imidazole reaction in a deuterated environment for enhanced product ion analysis
Author(s) -
Cindrić Mario,
Čepo Tina,
Škrlin Ana,
Vuletić Marko,
Bindila Laura
Publication year - 2006
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.2359
Subject(s) - chemistry , derivatization , chromatography , mass spectrometry , fragmentation (computing) , ion suppression in liquid chromatography–mass spectrometry , electrospray ionization , tandem mass spectrometry , computer science , operating system
The combination of separation techniques and mass spectrometry (MS) for peptide investigation allows superior sensitivity of detection and richer fragmentation data than available by direct MS analysis of a complex mixture. In this regard, liquid chromatography (LC) coupled to electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI) MS have evolved as versatile analytical tools in proteomics. Very often, however, the product ion mass spectrum is either incomplete or overfilled with ions, thus making sequence analysis difficult. Here we report overall ion intensity improvement of C‐terminal lysine‐containing peptides from Lys‐C digest by on‐column derivatization of lysines with 2‐methoxy‐4,5‐dihydro‐1 H ‐imidazole. The method is simple, fast and exhibits 100% efficiency of the reaction. Additionally, post‐source decay carried out on derivatized peptides gave rise almost exclusively to y‐series ion formation, at 100% sequence coverage and high intensity. The novelty of the method resides in the side reaction of this derivatization process, namely the methylation of cysteines. This facilitates the estimation of the disulfide bridge position in a protein and the fragmentation of cysteine‐containing peptide fragments. Additionally, by using this derivatization procedure, the loss of peptides, their degradation and/or oxidation, usually occurring in digest alkylation procedures, is greatly minimized. The new on‐column derivatization protocol is designed to be carried out on C 18 Spin Tubes or Cleanup C 18 Pipette Tips. We observed that use of buffered D 2 O solvent prevented unwanted oxidation and degradation reactions with respect to the stationary phase. This may be due to the fact that a deuteron is less polar than a proton, and thus the bonded silica stationary phase saturated with deuterons does not affect the reaction between ε‐amino or cysteine thiol groups and 2‐methoxy‐4,5‐dihydro‐1 H ‐imidazole. Complete tagging of the peptides by on‐column reaction could be obtained when using D 2 O, as compared to water‐based reaction. Methylation of cysteine residues was enhanced when β ‐mercaptoethanol was added in the reactant solution. Copyright © 2006 John Wiley & Sons, Ltd.