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.

Fig. 1

The scheme of the time of flight mass spectrometer used in the presented investigations. The apparatus was built by the author and coworkers in the Department of Molecular Physics, Institute of Physics, Maria Curie Sklodowska University

Fig. 2

The MALDI TOF MS sample holder with the matrix and the sample on its surface. The analyte ions created in the ion source near the sample holder surface are directed to the TOF mass analyzer

Fig. 3

Successive steps of obtaining the cathelicidin liophylisate sample from the porcine blood

Fig. 4

The schematic primary structure of cathelicidins

Fig. 5

MALDI TOF mass spectra of the synthetic PR-39 (4716 Da) obtained for different matrices: a) sinapinic acid (SA), b) succinic acid, c) nicotinic acid, d) 2,5-dyhydroxybenzoic acid (DHB), e) α-cyano-4-hydroxycinnamic acid (CCA), f) benzoic acid

Fig. 6

The intensity of the synthetic PR-39 ion mass peak as a function of the type of the matrix used in the sample preparation process

Fig. 7

Mass spectra of the lyophilisate sample with the PF-2 (8807 Da) ion mass peak obtained for different matrix/sample ratios (v/v): 1:1 (a),1:5 (b), 10:1 (c), 1:10 (d), 50:1 (e), 1:50 (f)

Fig. 8

The intensity of the PF-2 ion mass peak as a function of the matrix/sample ratio used in the sample preparation process

Fig. 9

The mass spectra of the lyophilisate sample containing PG-1 (1955, 6 Da), PG-2 (2055, 6 Da), PG-3 (2154, 5 Da), and PR-39 (4716 Da) ion mass peaks obtained for the matrix/sample ratios (v/v): 1:2 (a), 1:100 (b), 100:1 (c)

Fig. 10

The intensity of the: PG-1 (a), PG-2 (b), PG-3 (c), and PR-39 (d) ion mass peak as a function of the matrix/sample ratio used in the sample preparation process