Quantification of the PR-39 cathelicidin compound in porcine blood by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
BACKGROUND
The PR-39 porcine cathelicidin occurs naturally in animal neutrophils. Its main function is antimicrobial activity, which potentially can be used in antibiotic treatments in veterinary medicine. Investigations concerning such a use require the detection and quantification of PR-39 in a given sample. The aim of this work is to determine the concentration of PR-39 contained in porcine blood.


METHODS
Prior to matrix-assisted laser desorption/ionization (MALDI) analysis, the porcine blood sample was subjected to crude extraction in order to release the active form of PR-39 from the neutrophil granules. Next, gel filtration chromatography was performed to separate PR-39 from other cathelicidins present in porcine blood. Positive ion MALDI time-of-flight (TOF) mass spectra of the resulting portion of lyophilisate with unknown PR-39 content were acquired in linear mode. To quantify PR-39 in the lyophilisate sample, the standard addition method was applied. The PR-39 concentration obtained in the lyophilisate sample was then converted into the peptide concentration in porcine blood.


RESULTS
The linear fit function of the constructed calibration curve indicates an excellent correlation between the PR-39 peak intensity and the added quantity of synthetic PR-39 (R(2) = 0.994) and a low relative standard deviation of the slope = 1.98%. From the x-intercept of the straight line, we estimated the PR-39 concentration in porcine blood to be 20.5 ± 4.6 ng/mL.


CONCLUSIONS
The MALDI method was successfully applied for the quantitative analysis of PR-39 found in porcine blood. Compared with other available methods, it is relatively easy, inexpensive and not time-consuming. Despite the method having lower accuracy than the enzyme-linked immunosorbent assay (ELISA), the results obtained here, by a much simpler method, are in good agreement with the literature data.
Mass Spectrometry Laboratory, Institute of Physics, Maria Curie Sklodowska University, Pl. M. Curie-Skłodowskiej 1, 20-031, Lublin, Poland.
Anna
A
Smolira
Mass Spectrometry Laboratory, Institute of Physics, Maria Curie Sklodowska University, Pl. M. Curie-Skłodowskiej 1, 20-031, Lublin, Poland.
Stanisław
S
Hałas
Department of Pathophysiology, Chair of Preclinical Veterinary Sciences, Faculty of Veterinary Medicine, University of Life Sciences, Akademicka 12, 20-033, Lublin, Poland.
Joanna
J
Wessely-Szponder
D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000097:blood*; D002850:Chromatography, Gel; D016014:Linear Models; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013552:Swine; D054804:Cathelicidins
D023181:Antimicrobial Cationic Peptides; C072212:PR 39; D054804:Cathelicidins