On-Probe Sample Pretreatment for Direct Analysis of Lipids in Gram-Positive Bacterial Cells by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry

Abstract

On-probe sample pretreatment using trifluoroacetic acid as an additional reagent enabled the direct detection of phospholipids in whole bacteria by means of matrix-assisted laser desorption ionization mass spectrometry for not only gram-negative organisms but also gram-positive ones with a thicker peptidoglycan layer.Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has become a powerful method to characterize lipid components in bacterial cells (5). However, ordinary MALDI-MS requires a tedious sample pretreatment, such as solvent extraction of the lipids from the sample matrix or preparation of the sample suspension. Recently, a new analytical protocol for MALDI-MS called "on-probe sample pretreatment" was developed to achieve the direct detection of proteins in bacterial cells (1,3,7). In this technique, growing bacterial colonies are transferred directly from agar surfaces to the MALDI sample plate and then overlaid with a microvolume of matrix solution prior to irradiation with an N 2 laser. We applied a similar sampling protocol for the direct MALDI-MS measurement of phospholipids in bacterial cells (6) and successfully observed a series of ion peaks derived from phospholipids in gram-negative bacteria in the mass spectra. However, spectra of gram-positive bacteria did not show any lipid-related peaks, presumably due to their thicker peptidoglycan layer. In this work, direct detection of phospholipids in gram-positive bacteria was tried by means of MALDI-MS combined with the on-probe sample pretreatment using trifluoroacetic acid (TFA) as an additional reagent.Bacillus subtilis subsp. subtilis (NBRC 3007), a gram-positive bacterial sample purchased from National Institute of Technology and Evaluation, Japan, was used for stock cultures. Lyophilized cells of the bacterial sample were rehydrated with distilled water containing 1% NaCl, 0.5% yeast extract, and 1% polypeptone and then used to streak culture plates containing solid growth medium. The bacterial sample was cultured on LB agar and incubated at 37°C for 24 h.The procedure for on-probe sample pretreatment is basically similar to that described in our previous paper (6). Individual bacterial colonies weighing about 100 g were carefully removed from the agar surface with a tungsten wire loop and immediately spotted onto the MALDI plate. According to our previous paper (6), 2,5-dihydroxybenzoic acid (Aldrich) and sodium iodide (NaI; Wako) were used as a matrix and cationization reagent, respectively. A mixed solution of the matrix and cationization reagent was prepared by dissolving 2,5-dihydroxybenzoic acid and NaI in the solvent (2:1 mixture of chloroform:methanol) in concentrations of 500 mM and 100 mM, respectively. Here, TFA (Wako) was added to the solution in a concentration of 5% (vol/vol) to enhance the ion formation of phospholipids in the bacterial cell wall. Then, a ϳ1-l droplet of the solution was deposited onto each bacterial sample spot after the sample spot was additionally covered with 1 l of a TFA solution (5%). ...

Complementary analysis of lipids in whole bacteria cells by thermally assisted hydrolysis and methylation-GC and MALDI-MS combined with on-probe sample pretreatment

“…Individual bacteria colonies weighed about 100 mg were carefully removed from the agar surface with a tungsten wire loop and immediately spotted onto the MALDI plate. 2,5-Dihydroxybenzoic acid (DHB; Aldrich) and sodium iodide (NaI; Wako) were used as a matrix and cationization reagent, respectively [13,14]. A mixed solution of the matrix and cationization reagent was prepared by dissolving DHB and NaI in the solvent (2:1 mixture of chloroform:MeOH) in a concentration of 500 mM and 100 mM, respectively.…”

“…The procedure for the on-probe sample pretreatment is similar to that described elsewhere [13,14]. Individual bacteria colonies weighed about 100 mg were carefully removed from the agar surface with a tungsten wire loop and immediately spotted onto the MALDI plate.…”

“…In this sampling pretreatment, growing bacterial colonies are transferred directly from agar surfaces to the MALDI sample plate and then overlaid with a microvolume of matrix solution prior to irradiation with a N 2 laser. By using this sample pretreatment, the authors successfully observed a series of ion peaks of phospholipids in whole bacterial cells derived from phospholipids in Gram-negative [13] and Gram-positive [14] bacteria in the MALDI mass spectra. However, it is not an easy task to obtain accurately the quantitative data for acyl groups in phospholipids by MALDI-MS alone even using tandem MS technique.…”

Lipid fingerprinting of Bacillus spp. using online MALDI-TOF mass spectrometry

“…33 MALDI-TOF MS has also been applied to obtain abundant, accurate mass ion signals of the phospholipids in bacteria. [34][35][36][37][38][39] Therefore, the characterization of lipids in bacteria and other microorganisms plays a crucial role for taxonomic identification, differentiation of pathogenic species, and understanding of metabolic processes under normal or stressed growth conditions. However, there are few reports on the application of online MALDI-TOF MS coupling with lipid profiling for identification of bacterial species.…”

Direct Analysis of Minor Organic Components in Various Polymers and Biomaterials by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

“…Typical MALDI mass spectra of B. subtilis colony sample obtained by on-probe sample pretreatment using TFA 25) The peaks marked with closed circles and squares refer to PEs and PGs, respectively, and are assigned in Table 3. Novel methods for the direct analysis of small amounts of organic components in various polymeric and/or biomaterials were developed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using solid sampling techniques while avoiding troublesome solvent extraction.…”