Mass-biased partitioning to enhance middle down proteomics analysis.

A strategy is presented for enhancing the middle-down analysis of higher mass peptides recovered from complex protein mixtures. Following a 30-min digestion of multiple myeloma cell lysate by an acid cleavage reaction that is selective for aspartic acid, a 3000 Da membrane filter is used to bifurcate the peptide product mixture, and the heavier fraction is subjected to collisional activation with precursor selection that excludes charge states below +4. Filtration and charge state selection are shown to provide significant increases in the number of peptides identified in the mass range above 3000 Da and in information about protein sequences.