2.9. Protein extraction protocol and target plate preparation for MALDI-TOF MS analysis
The sample was prepared according to a short protein extraction protocol (Calderaro et al., 2014). The fractions obtained from 2D-AEC-SEC were treated as 30 µl of 70% formic acid from Merck co. (Darmstadt, Germany) in Mili-Q water / vigorous mixing, 30 µl pure acetonitrile from Merck co. (Darmstadt, Germany) /additional vigorous mixing, and finally centrifugation at 10,000 rpm for 5 minutes (Centrifuge 5415R, Eppendorf (Milano, Italy)). The purified FMDV was analyzed in a low (2–20 kD), medium (20–50 kD), and high (50-100 kD) molecular weight ranged by using MALDI TOF MS (Applied Biosystems 4800 MALDI-TOF/TOF) instrument. All of the samples were analyzed with MALDI TOF MS in a linear positive mode. Regarding the analysis in the low molecular weight range, equal volumes of supernatant were mixed with an equal volume of α-cyano-4-hydroxycinnamic acid matrix solution in 50% acetonitrile containing 0.1% trifluoroacetic acid (TFA) from Merck co.( Darmstadt, Germany), spotted (0.7 μL) onto one pre-spotted Mass Standards Calibration Opti-TOF™ insert MALDI plate, air dried, and analyzed with a MALDI-TOF MS. As for the high molecular weight range, a saturated solution of synapinic acid (SA) was used as the matrix (50 mg/mL SA in 30:70 ACN: TFA 0.1% Merck co. (Darmstadt, Germany) and equal volumes of supernatant and matrix solution were mixed. Then, 0.7 μL of the prepared solution was spotted onto the same target plate as air-dried at the room temperature.