Abstract :
[en] While the imaging of bacterial co-cultures, directly on agar media, has been reported in MALDI mass spectrometry imaging (MSI), such samples usually require laborious and time-consuming sample preparation prior to analysis. Furthermore, conventional preparation may cause sample deformation and/or degradation, and flaking leading to instrument clogging and potential damage. Other issues are directly associated with the use of an organic matrix in MALDI-MSI. As alternative methods are urgently required, we propose in this study a rapid and simple approach using a “desorption/ionization using through hole alumina membrane” (DIUTHAME) with a blotting sample preparation to image a Pseudomonas and Bacillus co-culture grown on agar, in both ionization modes, by surface-assisted laser desorption/ionization (SALDI) FT-ICR MSI.
For SALDI-MSI, the DIUTHAME membrane (Hamamatsu Photonics K.K.) was placed on the bacterial co-culture on the agar medium to allow direct transfer of the molecules from the sample to the membrane. The membrane was kept on the sample during 5 minutes, then removed from the sample, and finally stuck on an ITO-coated glass slide for analysis. For MALDI-MSI, microbial colonies on agar were cut directly from the Petri dish and deposited on an ITO-coated glass slide, previously covered with conductive copper tape. This assembly was dried and finally covered with CHCA matrix. MS images were acquired using a 9.4T SolariX XR mass spectrometer. The mass spectra were recorded in the positive and negative ionization modes, between m/z 100 and 2500.
In this study, a conventional MALDI-MSI sample preparation procedure used to study bacterial colonies on agar was compared to a new blotting method using a DIUTHAME membrane. The DIUTHAME procedure offers several advantages. First, this procedure is rapid and does not require advanced skills of the operator, compared to MALDI-MSI. Second, in the DIUTHAME blotting method, the analytes are directly transferred from the sample to the membrane prior to analysis, without sampling the agar medium. In this way, it circumvents (1) the degradation of labile compounds and the deformation of the sample caused by the drying step required in the MALDI procedure, and (2) the ion suppression caused by agar in MALDI-MSI. Third, the DIUTHAME also acts as the assisting material in SALDI-MSI. Hence, this technique does not require the addition of an organic matrix, which can cause ion suppression and interference with low-molecular weight compounds. DIUTHAME mass spectra are thus characterized by a clean background in the low m/z range and a reduced mass shift. Interestingly, lipopeptides were detected in both ionization modes, using both techniques, mainly as [M + Na]+ ions in the positive ion mode and [M – H]- ions in the negative ion mode. However, issues arise when visualizing the ion images acquired with the DIUTHAME membrane. Areas with very little to no signal can be seen on the ion images, around the colonies and at the interface. These “dark” areas correspond to regions where the sample has not been properly in contact with the membrane. The analysis with DIUTHAME also suffers from a lack of sensitivity, which affects the analysis of hardly ionizable and low-abundant compounds.
