Design and synthesis of novel cleavable detergents for protein and peptide analysis by mass spectrometry.

摘要

This study introduces a novel technology addressing the fundamental problems encountered during the analysis of hydrophobic proteins using mass spectrometry. The limitations of MALDI mass spectrometry include the discrimination in favor of hydrophilic proteins due to the increased solubility of these proteins relative to their hydrophobic counterparts. The primary way in which biochemists handle the hydrophobic biomolecules is the use of detergents. In spite of the widespread use of detergents in biochemistry, the presence of these molecules often complicates further analysis by mass spectrometry. Specifically, MALDI mass spectrometry of samples containing detergents is inhibited because the strong interactions between proteins and the detergent prevent proper incorporation of the proteins into the matrix crystals.; This study presents a solution to this problem utilizing cleavable detergents. The added advantage of cleavable detergents over conventional detergents such as sodium dodecyl sulfate or n-octylglucoside is that the detergent properties that interfere with MALDI mass spectrometry can be eliminated prior to analysis. Detergents were designed which are cleaved using acid hydrolysis, nucleophilic attack of fluoride, and photolysis. Additionally, reagents were designed incorporating two of the known MALDI matrices into the cleavable detergent as the head of the molecule. Methods were developed that allow the molecules to be applied directly to the MALDI sample preparation process. Cleavage of the detergent has a dramatic effect on the crystallization of MALDI matrix and the resulting sensitivity of the MALDI-MS analysis. Application of these novel reagents to problems of crude protein mixture profiling using MALDI mass spectrometry was also tested. Western blot and 2D-gel analysis was used to characterize the difference in protein extraction when changing from conventional detergents to cleavable detergents. Cleavable detergents compared very well to the commercial detergents in both the number of proteins solubilized and the types of proteins analyzed. The MALDI-MS analysis of the RKO cell extracts using cleavable detergents show increases in the number of peaks observed in each spectrum and the signal intensity of the peaks common to both analyses.