Use of nonspecific cleavage products for protein sequence analysis as shown on calcyclin isolated from human granulocytes

In this paper, analysis strategies developed for a sequencing problem conce rning the identification of an S100 protein isolated from human granulocyte s are discussed. The analysis of a trypsinized lyophilized sample suggested the presence of a number of peptides which are non-tryptic in origin. Duri ng purification of proteins from cell lysates nonspecific cleavage can be o bserved which may reflect biological processes and can become an unavoidabl e analytical problem. Current mass spectrometric software is evaluated for the analysis of nonspecific digests in this context. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), high-performance liquid chromatography (HPLC)-MS/MS, and selected ion monitoring (SEA)-MS/MS have been used for peptide analysis and in addition HPLC-MS was carried out for protein analysis leading to the detection of an N-terminal modification of the protein. The success of the study is mainly due to the careful investig ation of nonspecific cleavage products. Data obtained from the routine mass spectrometric analysis of an in-gel-digest allowed the identification of t his protein as S100 calcium-binding protein A6-calcyclin whose expression i n granulocytes has not been described so far. (C) 2001 American Society for Mass Spectrometry.