A comprehensive on-line digestion-liquid chromatography/mass spectrometry/collision-induced dissociation mass spectrometry approach for the characterization of human fibrinogen

An automatic on-line digestion-liquid chromatography/mass spectrometry/coll ision-induced dissociation mass spectrometry (LC-MS/CID-MS) protocol has be en developed for detection of errors in the biosynthesis of human fibrinoge n, such as amino acid (AA) mismatch or incorrect posttranslational modifica tion (PTM). Using on-line digestion on an immobilized-enzyme column, the re action time is significantly reduced (less than 20 min) and the entire appr oach is suitable for automation. The two-loop MS experiments (full-scan acq uisition and sugar moieties monitoring by SIM) allow checking both the corr ect AA mapping via the peptides generated by the digestion of the PTM. Sinc e the protocol was designed for application on a routine basis, as a proof- of-concept detection of a rare case of 'abnormal' fibrinogen has been demon strated. The advantage of the proposed approach is exemplified by the fact that the DNA sequence information for the case investigated had not shown a ny evidence of the abnormality. Copyright (C) 2001 John Wiley & Sons, Ltd.