MALDI-TOF MS Analysis of rPIIIA Analogues

Qualitative analyses of rPIIIA and other analogues were performed using a previously described MALDI-TOF/MS-based method with modifications [47 (link)]. In summary, after centrifugation at 10,000× g for 10 min, the supernatant of the CFPS reaction mixture or other purified solutions treated with Enterokinase were desalted using ZIPTIP C18 (Millipore, Billerica, MA, USA) and subjected to MALDI-TOF MS. MALDI-TOF MS analysis was performed using an Ultraflex MALDI-TOF mass spectrometer (Bruker Daltonics, Billerica, MA, USA), equipped with a 50 Hz pulsed nitrogen laser (λ = 355 nm) and a 19 KV accelerating voltage operated in reflectron, positive ion mode. The samples were prepared by mixing 1 μL peptide solution with 1 μL of saturated matrix (α-cyano-4-hydroxycinnamic acid) in 0.1% TFA containing 30% ACN. Data collection and processing were respectively performed by FlexControl and FlexAnalysis software (V2.4, Bruker Daltonics, Billerica, MA, USA).