Supplemental information for:
Utilizing a Robotic Sprayer for High Lateral
and Mass Resolution MALDI FT-ICR MSI of
Microbial Cultures
Christopher R. Anderton,*† Rosalie K. Chu,† Nikola Tolić,† Alain Creissen,‡
Ljiljana Paša-Tolić†
†Environmental
Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA,
USA
‡HTX Technologies, LLC, Chapel Hill, NC, USA
Expanded Experimental Methods:
Reagents and Chemicals
Universal MALDI Matrix and α-cyano-4-hydroxycinnamic acid (CHCA) were purchased from Fluka
(St. Gallen, Germany), and 2,5-Dihydroxybenzoic acid (DHB) was purchased from Sigma (St. Louis,
MO). ISP2 agar (low salt media) was made as previously described [1]. Briefly, 2 g Dextrose
Monohydrate (Fisher Scientific, Fair Lawn, New Jersey) and 5 g Malt extract (Oxoid Ltd, Hants, UK)
were mixed with 2 g Yeast extract LD (Difco, BD and Co. Sparks, MD) and 7.5 g granulated Agar
(Difco) in 500 mL MilliQ water (EMD Millipore, Darmstadt, Germany) and autoclaved for 30 min at
120 oC to sterilize before pouring a thin layer of ISP2 agar into petri dishes (BD Falcon, Franklin
lakes, NJ).
Sample Culturing, Excision, and Mounting
Streaked B. subtilis WT 3610 plates were generously donated to us by the Dorrestein Lab. Isolates
were then grown at room temperature for 3 days in 10 mL LB Broth (Lennox, Mo BIO Laboratories,
Inc., Carlsbad, CA). The culture was then diluted 1:4 in 10x phosphate buffered saline (Sigma), 0.5 µl
pipetted on ISP2 agar plates in a 3x3 grid and grown at room temperature for 14 days. The cultures
used for high-lateral resolution where only grown for 6 days. Microbial colonies were cut and
removed from petri dishes and mounted onto grounded 96 well microToF plates (Bruker Daltonics,
Bremen, Germany) as previously described [1].
Laser Conditions for MALDI MSI
Lower lateral resolution images were collected with the small laser setting, where the laser beam was
randomly “walked” over a 250 µm area, and pixel size (as define by step size) was 300 µm. For sieve
and the CHCA sprayer methods an arbitrary laser power of 41% and 36%, respectively, were used
with 250 shots at 400 Hz per pixel. For DHB, an arbitrary laser power of 60% at 200 shots and 400 Hz
was used for each pixel. High-resolution images were collected with the minimal laser setting
(SmartBeam disabled). For CHCA, a 30 µm step size using 200 shots at 2000 Hz was employed with
52% arbitrary power. For DHB, a 20 µm step sizes was utilized with 250 shots at 2000 Hz and with
52% arbitrary power.
Supplemental figures:
Figure S1. Demonstrating adhesion of AY and LB agar after dehydration without the need to add
DHB to assist in adhesion.
Figure S2. Photographs of mounted and prepared microbial samples prior to MALDI MSI. (A) Was
prepared following the Sieve method and (B-C) were prepared using the HTX TM-Sprayer, where (B)
is CHCA, and (C) is DHB coated.
Figure S3. Additional ion images comparing the sieve method to the sprayer method (Figure 2).
While most species (aside from prophyrin) are detected outside of the B. subtilis colony using the
sieve, we see localization of all species within the colony with the sprayer method. Images are based
on a color scale where the higher the relative intensity, the brighter the respective color. All ion
images are normalized to the total ion current. Molecular identities are based on the Global Natural
Produces Social Molecular Networking Spectral Library (gnps.ucsd.edu).
Figure S4. Additional ion images using the sprayer method with DHB (From Figure 3). The overlay
image demonstrates the localization of porphyrin (m/z = 616.177; blue), surfactin minus leucine (m/z =
961.572; green), and surfactin (m/z = 1074.807, red). Agar at m/z = 867.2383 can be seen in purple,
PE (32:0) or PC (29:0) at m/z = 730.4784 in yellow, plipastatin at m/z = 1543.810 in aqua, and the
other ion images are unidentified. Images are based on a color scale where the higher the relative
intensity, the brighter the respective color. All ion images are normalized to the total ion current.
Molecular identities are based on the Global Natural Produces Social Molecular Networking Spectral
Library (gnps.ucsd.edu).
Figure S5. Additional high lateral resolution ion images using the sprayer method with CHCA (Figure
4A). All ion images are normalized to the total ion current.
Figure S6. Additional high lateral resolution ion images using the sprayer method with DHB (Figure
4A). All ion images are normalized to the total ion current.
References:
1.
Yang, J.Y., Phelan, V.V., Simkovsky, R., Watrous, J.D., Trial, R.M., Fleming, T.C., et al.:
Primer on Agar-Based Microbial Imaging Mass Spectrometry. J. Bacteriol. 194,
6023-6028 (2012)