Importance of the Matrix and the Matrix/Sample Ratio in MALDI-TOF-MS Analysis of Cathelicidins Obtained from Porcine Neutrophils

Abstract

Qualitative and quantitative mass spectrometric studies of biomolecules for example proteins, peptides, or lipids contained in biological samples like physiologic fluids are very important for many fields of science such as medicine, veterinary medicine, biology, biochemistry, molecular biology, or environmental sciences. In the last two decades, MALDI TOF MS — matrix-assisted laser desorption mass spectrometry, proved to be an especially convenient tool for these analyses. The main advantages of this method are its rapidity and high sensitivity which is particularly appreciated in the case of studies of complex biological specimen. A major challenge for many researchers is to maximize this sensitivity, among others, by appropriate procedures of sample preparation for the measurement. The objective of this work was to optimize these procedures, selecting the optimal matrix and optimum proportions of the sample and the matrix solution in a mixture of both solutions, aiming at the achievement of the maximum intensity of ion current. In this respect, five low molecular mass cathelicidins were studied: prophenin-2, protegrins 1–3, PR-39. All of them were obtained directly from the porcine blood. As a result of studies, the authors determined such experimental conditions when the intensity of investigated ionic current had the highest value.

Quantification of the PR‐39 cathelicidin compound in porcine blood by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

“…After removal of the signal peptide using signal peptidase, PR‐39 is stored in neutrophil granules as an inactive propeptide. To release the PR‐39 peptide in its active form the crude extraction process and proteolytic processing of the propeptide by elastase were performed as described previously . Briefly, fresh porcine blood was collected at an abattoir into containers with sodium citrate as an anticoagulant.…”

“…In the crude extract, besides PR‐39, a few other low molecular mass cathelicidins are present: proline‐phenylalanine‐rich prophenin‐2 (PF‐2, MW = 8807 Da) and cysteine‐rich protegrins 1 to 3 (PG‐1, MW = 2154.5 Da; PG‐2, MW = 1955.6 Da; PG‐3, MW = 2055.5 Da) . To separate them according to their molecular masses for accurate MALDI‐TOFMS detection of PR‐39, gel filtration chromatography was carried out.…”

“…To release the PR-39 peptide in its active form the crude extraction process and proteolytic processing of the propeptide by elastase were performed as described previously. [22] Briefly, fresh porcine blood was collected at an abattoir into containers with sodium citrate as an anticoagulant. The red blood cells were disrupted by the addition of 0.83% ammonium chloride in order to obtain leukocytes.…”

“…In the crude extract, besides PR-39, a few other low molecular mass cathelicidins are present: prolinephenylalanine-rich prophenin-2 (PF-2, MW = 8807 Da) and cysteine-rich protegrins 1 to 3 (PG-1, MW = 2154.5 Da; PG-2, MW = 1955.6 Da; PG-3, MW = 2055.5 Da). [22] To separate them according to their molecular masses for accurate MALDI-TOFMS detection of PR-39, gel filtration chromatography was carried out. For this purpose, a Sephadex G-50 column (Fine, Sigma-Aldrich, Poznań, Poland), designed to separate molecules with MW between 1.5 and 30 kDa, with 5% acetic acid as a running buffer was applied.…”

“…[19] In another paper, we described the optimization of the sample preparation procedure to maximize the sensitivity of detection of the investigated cathelicidin. [22] The purpose of the present investigations was to determine the PR-39 cathelicidin concentration in a porcine blood sample.…”