Sinapinic acid 3 5 dimethoxy 4 hydroxycinnamic acid

Reserpine (metil-11,17α-dimetoxi-18β-((3,4,5-trimetobenzoilo)oxy)-3β,20α-yohimban-16β-carboxylate)
(R0875, Sigma Aldrich, USA) was dissolved in glacial acetic acid (A6283;
Sigma-Aldrich, USA) and diluted to a final concentration of 0.25%
acetic acid with distilled water.^{54 (link)} Sterile
saline solution was mixed with the acid (A6283; Sigma-Aldrich, USA)
until pH = 4 was reached.^{54 (link)} Sinapinic acid
(3,5-Dimethoxy-4-hydroxycinnamic acid) (D7927, Sigma-Aldrich, Germany)
was used as the matrix for MALDI-TOF MS analysis. Acetonitrile (ACN)
(271004, Merck, Germany) and trifluoroacetic acid (TFA) (T6508, Sigma-Aldrich,
Germany) were used for matrix preparation. Micro-90 concentrated cleaning
solution (Z281506, Sigma-Aldrich, Germany) was used for MALDI-TOF
MS target cleaning between different analyses.^{38 (link)} Red phosphorus (04004H, Riedel de Haën, Germany)
was used for MALDI-TOF MS calibration.^{68 (link)}

Before MALDI TOF analysis the gel filtration chromatography using the Sephadex G-50 (Fine, Sigma-Aldrich) column was made for separation of components of obtained AMP extract according to their molecular weights [7 (link), 12 (link)]. The fraction 1 (MW from 2500 to 17,500 Da) and the fraction 2 (MW from 2750 to 4750 Da) were collected, lyophilised, and stored at −20 °C for further analysis.
The mass analysis of the components of PRP was carried out by the MALDI method with sinapinic acid (3,5–dimethoxy-4-hydroxycinnamic acid, M = 224.2 Da, Sigma-Aldrich, Poland) used as a matrix, whereas antimicrobial peptides from the neutrophil crude extract were analyzed using the CCA matrix (α-cyano-4-hydroxycinnamic acid 189.2 Da, Sigma-Aldrich, Poland) [13 (link)].