The agar images and MS data presented in this note were provided by Erin Gemperline and Dr. Lingjun Li, Department of Chemistry and School of Pharmacy, University of Wisconsin- Madison, Madison, WI, USA.
Application & Background
MALDI imaging of microbial colonies on agar media is a growing application for mass spectrometry imaging; however, matrix deposition on agar is often complicated. Typically, matrix is applied to wet agar using a fine sieve to dust matrix powder over the top of the slide, which often yields low to medium signal intensity and poor reproducibility. Here we present a more reproducible method of applying 2,5-dihydroxybenzoic acid (DHB) to dried agar slices for MALDI imaging of metabolites from bacterial colonies.
Part of the bacterial colony and the surrounding agar were sliced out of the petri dish using a razor blade spatula and carefully laid on a glass slide using a metal spatula, ensuring there were no bubbles underneath the agar. The slides were dried down in a desiccator at room temperature overnight. Note that drying time will vary depending on the size of the agar slice and the effectiveness of the desiccator.
Tissue sections were then sprayed with DHB matrix (40 mg/mL, Methanol 50%, TFA 0.1%) using the HTX TM-Sprayer. The spray parameters are described in Table 1.
MALDI Mass Spectrometry Imaging
Spectra were collected across the entire agar/colony area using a MALDI- LTQ Orbitrap (Thermo Scienti!c, Waltham, MA, USA) analyzer equipped with a nitrogen laser in positive mode over a mass range of m/z 100 to 2000. The raster width was set to 100 μm for imaging.
MALDI MS images of compounds detected in the agar and the bacterial colony when matrix was applied using the method above were compared to images acquired when the matrix was applied via dry coating with a sieve.