ingel microwaveassisted acid hydrolysis of proteins combined with liquid chromatography tandem mass spectrometry for mapping protein sequences we report an enabling method for mapping the protein sequence with high sequence coverage this method combines the high separation power of gel electrophoresis for protein separation with the high sequence coverage capability of microwaveassisted acid hydrolysis maah mass spectrometry ms ingel maah using 25 trifluoroacetic acid was developed and optimized for degrading the gelseparated protein into small peptides suitable for tandem ms sequencing for bovine serum albumin bsa 67 kda with 4 g of protein loading onto a gel for separation followed by excising the protein gel band for ingel maah and then injecting 2 g of the resultant peptides into a liquid chromatography quadrupole timeofflight mass spectrometer for analysis 689 54 n 3 unique peptides were identified with a protein sequence coverage of 99 1 both the number of peptides detected and sequence coverage decreased as the sample amount decreased mainly due to background interference 316 59 peptides and 94 3 coverage for 2 g loading 136 19 and 76 5 for 1 g loading and 30 2 and 32 2 for 05 g loading to demonstrate the general applicability of the method 10 gel bands from gel electrophoresis of an albumindepleted human plasma sample were excised for ingel maah lcms analysis in total 19 relatively high abundance proteins with molecular weights ranging from 8 to 160 kd could be mapped with coverage of 100 for six proteins mw 8759 to 68425 da 9698 for five proteins mw 11458 to 36431 da 92 for three proteins mw 15971 to 36431 da 8087 for four proteins mw 42287 to 162134 da and 56 for one protein mw 51358 da finally to demonstrate the applicability of the method for more detailed analysis of complex protein mixtures twodimensional 2d gel electrophoresis was combined with ingel maah affinity purification and lcmsms to characterize six bovine alphas1casein phosphoprotein isoforms full sequence coverage was achieved for each protein and six new modification sites were found