Mass spectrometry

We used mass spectrometry, that is often used for characterization of biomolecules [60–62], for detection of PTMs of fibrinogen. Samples of modified, control, native and patient fibrinogen were prepared by trypsin digestion. Briefly, after electrophoresis, the fibrinogen chains were cut out from gel in approximately 1 mm³ pieces. Destaining was performed by incubation with 0.1 M NH₄HCO₃/acetonitrile (1:1) and acetonitrile. Rehydratation was carried out by adding 0.1 M NH₄HCO₃. Tris(2-carboxyethyl)phosphine (50 mM) was used as the reduction reagent; whereas, 2-iodoacetamide (55 mM) was used as an alkylating agent. Trypsin digestion (12.5 ng/ml in 25 mM NH₄HCO₃) was performed with constant shaking for 16 h at 37 °C. Reaction was hindered by adding 50% acetonitrile/0.1% formic acid. Lyofilisated samples were stored at −80 °C.

For identifying the modified amino acid residue, HCT ultra ion-trap mass spectrometer with nanoelectrospray ionization, coupled to an UltiMate 3000 nanoLC system, was used. Data analyses were performed by The esquireControl v6.2 software for data acquisition, DataAnalysis v4.0 for data processing, and BioTools v3.2 (all Bruker Daltonics), collectively with MASCOT v2.2 (Matrix Science, London, UK) for database searching. For each environment, at least three independent samples were prepared. A PTM is considered to be a consequence of the reagent if it was not observed in the control or native fibrinogen. PTM was considered when occurred at least in one sample.