MALDI QqTOF MS combined with off-line HPLC for characterization of protein primary structure and post-translational modifications.

The addition of off-line high-performance liquid chromatography to matrix-assisted laser desorption/ionization mass spectrometry greatly reduces congestion in the mass spectra, and also provides complete decoupling of the separation process from mass detection and measurement. This removes the time constraints inherent in on-line coupling, and so enables the detailed mass-spectrometric study of samples at later times. We describe here our use of this method to successfully characterize two "unknown" protein mixtures that were set as problems by the ABRF Proteomics Research Group (PRG) in the years 2003 and 2004.