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The identification of proteins by tandem mass spectrometry relies on knowledge of the products produced by collision-induced dissociation of peptide ions. Most previous work has focused on fragmentation statistics for ion trap systems. We analyzed fragmentation in MALDI TOF/ TOF mass spectrometry, collecting statistics using acurated set of 2459 MS/MS spectra and applying bootstrap resampling to assess confidence intervals. We calculated the frequency of 18 product ion types, the correlation between both mass and intensity with ion type, the dependence of amide bond breakage on the residues surrounding the cleavage site, and the dependence of product ion detection on residues not adjacent to the cleavage site. The most frequently observed were internal ions, followed by y ions. A strong correlation between ion type and the mass and intensity of its peak was observed, with b and y ions producing the most intense and highest mass peaks. The amino acids P, W, D, and R had a strong effect on amide bond cleavage when situated next to the breakage site, whereas residues including I, K, and H had a strong effect on product ion observation when located in the peptide but not adjacent to the cleavage site, a novel observation.

Membrane proteomics, the large-scale analysis of membrane proteins, is often constrained by the difficulties of achieving fully resolvable separation and resistance to proteolysis, both of which could lead to low recovery and low identification rates of membrane proteins. Here, we introduce a novel integrated approach, GELFrEE Optimized FASP Technology (GOFAST) for large-scale and comprehensive membrane proteins analysis. Using an array of sample preparation techniques including gel-eluted liquid fraction entrapment electrophoresis (GELFrEE), filter-aided sample preparation (FASP), and microwave-assisted on-filter enzymatic digestion, we identified 2 090 proteins from the membrane fraction of a leukemia cell line (K562). Of these, 37% are annotated as membrane proteins according to gene ontology analysis, resulting in the largest membrane proteome of leukemia cells reported to date. Our approach combines the advantages of GELFrEE high-loading capacity, gel-free separation, efficient depletion of detergents, and microwave-assisted on-filter digestion, minimizing sample losses and maximizing MS-detectable sequence coverage of individual proteins. In addition, this approach also shows great potential for the identification of alternative splicing products.