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/collision-induced dissociation mass spectrometry (LC-MS/CID-MS) protocol has been developed for detection of errors in the biosynthesis of human fibrinogen, such as amino acid (AA) mismatch or incorrect post-translational modification (PTM). Using on-line digestion on an immobilized-enzyme column, the reaction time is significantly reduced (less than 20 min) and the entire approach is suitable for automation. The two-loop MS experiments (full-scan acquisition and sugar moieties monitoring by SIM) allow checking both the correct AA mapping via the peptides generated by the digestion of the PTM. Since 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 demonstrated. The advantage of the proposed approach is exemplified by the fact that the DNA sequence information for the case investigated had not shown any evidence of the abnormality.
Vecchione
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G
Hospital "CCSS", Viale Cappuccini 1, 71013 S. Giovanni rotondo, Foggia, Italy.
Casetta
B
B
Santacroce
R
R
Margaglione
M
M
D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods; D004800:Enzymes, Immobilized; D005340:Fibrinogen / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006020:Glycopeptides / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D008970:Molecular Weight; D009862:Online Systems; D021122:Protein Subunits; D012016:Reference Values; D012794:Sialic Acids / Q000032:analysis; D014357:Trypsin
D004800:Enzymes, Immobilized; D006020:Glycopeptides; D007202:Indicators and Reagents; D021122:Protein Subunits; D012794:Sialic Acids; D005340:Fibrinogen; D014357:Trypsin