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

Figure 1.  Bacterial colony grown on agar media.

Figure 1. Bacterial colony grown on agar media.

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.

Experimental

Sample Preparation

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.

Figure 2.  Agar before (left) and after (right) drying.

Figure 2. Agar before (left) and after (right) drying.

Figure 3.  Agar that has been drying too long. It has begun to crack and flake away from the glass slide.

Figure 3. Agar that has been drying too long. It has begun to crack and flake away from the glass slide.

Table 1.  Spray parameters for MALDI matrix deposition.

Table 1. Spray parameters for MALDI matrix deposition.

Matrix Deposition

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.

Figure 4.  High resolution image of DHB matrix crystal size and coverage on a glass slide when applied with the HTX TM-Sprayer compared to application via sieve.

Figure 4. High resolution image of DHB matrix crystal size and coverage on a glass slide when applied with the HTX TM-Sprayer compared to application via sieve.

Figure 5.  A bacterial colony on agar on a glass slide with matrix applied via dry sieve (left) and the HTX TM-Sprayer (right).

Figure 5. A bacterial colony on agar on a glass slide with matrix applied via dry sieve (left) and the HTX TM-Sprayer (right).

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.

Figure 6.  Mass spectra of bacterial colony samples when matrix is applied via the HTX TM-Sprayer or sieve. Metabolites with  m/z  100-2000 were acquired. The inlay zooms in on the higher mass region,  m/z  1000-2000, where dry sieving does not generate as many peaks as the HTX TM-Sprayer.

Figure 6. Mass spectra of bacterial colony samples when matrix is applied via the HTX TM-Sprayer or sieve. Metabolites with m/z 100-2000 were acquired. The inlay zooms in on the higher mass region, m/z 1000-2000, where dry sieving does not generate as many peaks as the HTX TM-Sprayer.

Results

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.

Figure 7. ( A) Optical image of the bacterial colony on agar with DHB applied via the HTX TM-Sprayer (top) or sieve (bottom). (B) TIC image comparing the DHB coverage of the two application methods. (C) Representative ion images of  m/z  230.095, 365.985, 664.113, and 1191.348 comparing metabolite distribution when matrix is applied with the HTX TM-Sprayer vs. sieve. Scale bar = 2 mm, Intensity scale = low abundance (blue) to high abundance (red).

Figure 7. (A) Optical image of the bacterial colony on agar with DHB applied via the HTX TM-Sprayer (top) or sieve (bottom). (B) TIC image comparing the DHB coverage of the two application methods. (C) Representative ion images of m/z 230.095, 365.985, 664.113, and 1191.348 comparing metabolite distribution when matrix is applied with the HTX TM-Sprayer vs. sieve. Scale bar = 2 mm, Intensity scale = low abundance (blue) to high abundance (red).