Protein identification by in-gel digestion, high-performance liquid chromatography, and mass spectrometry: Peptide analysis by complementary ionization techniques

A biologically active protein fraction was isolated from rabbit intestine, purified by one-dimensional SDS-PAGE and stained with Coomassie Brilliant B lue. A predominant band of approximately 110-130 kDa was excised and digest ed in-gel with trypsin. The resulting peptides were extracted then separate d by microbore reversed-phase high-performance liquid chromatography (HPLC) . Mass spectrometric data from one HPLC fraction obtained by two different ionization techniques proved to be complementary. Matrix-assisted laser des orption/ ionization (MALDI) showed nine peptide masses, which by post sourc e decay analysis and database searching were attributed to two proteins. Na noflow electrospray analysis performed on a hybrid tandem mass spectrometer of quadrupole- quadrupole- orthogonal acceleration time-of-flight (QqTOF) geometry detected six additional peptide components. On the basis of the ad ditional peptides and superior quality collision-induced dissociation spect ra typical of this instrument type, two further proteins were identified. T he resolution afforded by the QqTOF instrument permitted charge state deter mination for the fragment ions while preserving the high detection sensitiv ity that was essential in obtaining the composition of this mixture of prot eins. (C) 2001 American Society for Mass Spectrometry.