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A primer on agar-based microbial imaging mass spectrometry Supplemental discussion How to minimize flaking – full contac
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A primer on agar-based microbial imaging mass spectrometry Supplemental discussion How to minimize flaking – full contact with non-polished target. From sample preparation through data acquisition, there are several ways to optimize adherence of the sample to the target plate and minimize flaking. A major portion of the sample preparation effort is geared toward ensuring that the dried agar is securely adhered to the MALDI target plate. We have provided a flowchart with checkpoints (Fig. S1) to maximize the ability to get consistent, reliable images and to minimize instrument down time. During microbial IMS sample preparation, a non-polished MALDI target plate must be used. The rough surface of a non-polished target plate increases the amount of surface area to which the growth media can adhere. Polished target plates can be made “non-polished” by sanding or applying steel wool to scratch the surface, but we have found that ground steel target plates work best. An example of flaking on a polished MALDI target plate is shown in Figure S1. The sample should remain in full contact with the target plate from pre-matrix application through data acquisition. If there are air bubbles prior to matrix application, we recommend using a spatula to gently lift up the corner of the agar sample until the bubble is reached and laying the agar back down again (Supplemental, Video 3). Culturing – use thin agar, adjust media contents. The goal of media preparations is to minimize flaking while maintaining the desired phenotype. In most cases, the adjustment to 1-1.5 mm agar thickness in the Petri dish has had no impact on the phenotype of the microbes or their interactions. When this alteration of growth
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conditions does have an undesired phenotypic impact, the amount of nutrients needs to be adjusted to achieve the equivalent phenotype. As a general rule, we use inverse linear scaling of nutrient concentration with respect to the media volume. For example, when decreasing media volume by half, the amount of nutrients is typically doubled. The optimization for the correct phenotypic response is necessary prior to IMS analysis. Also, determination of the IMS compatibility of media is necessary to ensure that background signals from media do not interfere with ion detection. Slightly increasing the water content of the agar media is effective in minimizing flaking. The result of increasing the water content of the agar is that it becomes more gel-like, which in turn increases surface area contact to the MALDI target plate. Water content is affected by the age of the agar, incubation conditions, and the percentage of agar in the media. Be aware, the phenotype and metabolic profile can change with changes in agar percentage. For instance, swarming motility generally occurs between 0.3% and 1% agar, and is inhibited over 1% agar concentrations (Fig. 1A; 17, 18). Other factors that may affect sample adherence in IMS affect the agar media properties including the development of vegetative hyphae and molecules produced by the microbe(s). Microbes that change agar properties or produce copious amounts of biosurfactants and/or lipopolysaccharides (LPS), such as Bacillus subtilis 3610 (2, 12, 22, 30, 33), Pseudomonas aeruginosa PAO1 and PA14 (11, 14, 16), Pantoea agglomerans (15), and Serratia marcescens (1, 23, 24), can also increase flaking. We hypothesize that the surfactants affect the interactions between the matrix, agar media, and target.
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Matrix saturation hypothesis. Saturating the microbial sample with Universal MALDI matrix (Sigma-Aldrich), a 1:1 mixture of 2,5-dihydroxybenzoic acid (DHB) and αcyano-4-hydroxycinnamic acid (CHCA) matrices, minimizes flaking and facilitates ionization of microbial molecules in microbial MALDI-TOF IMS. When each component of this mixture is applied individually, it is much more difficult to obtain quality images. DHB was absorbed into the agar resulting in samples adhering to the MALDI target with poor ionization during analysis, while CHCA remained on the sample surface resulting in ionization but with a much higher incidence of flaking, sample detachment from the target plate. Therefore, we hypothesize that DHB is acting as a weak glue to adhere the sample to the MALDI plate while the CHCA allows for better ionization of surface analyte (Fig. S2). Agar that is not saturated with matrix shrinks significantly during the drying process, supporting our hypothesis that a component of the matrix mixture anchors the sample to the target plate. The majority of the DHB in the mixture saturates the agar media, prevents shrinkage, and glues the sample to the target, whereas the majority of CHCA crystallizes on the top surface of the sample to assist in desorption and ionization of molecules. It should be possible to optimize the matrices as is done for other MALDI-TOF applications (9, 10, 13, 25) to ionize specific molecules of interest or maximize the number of microbial molecules detected while simultaneously maintaining sample adherence to the target plate. Achieving even matrix application on mucoid colonies (Fig. 3B, 4), samples with biosurfactants, or certain media that readily absorb matrix (Fig. S1; Fig. S3) such as YEPD (21) and Spider (15), can be challenging. This may be remedied by first coating
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the target plate with matrix prior to transfer of the sample to the target or by multiple applications during the dehydration process (Fig. S3). Other media, such as MSgg (6), appear to absorb and dissolve matrix upon dehydration (Fig. S1, 2a-b), which means that the agar is not saturated with matrix and shrinkage is apparent. Dehydration – check for flaking, sample height of about 1 mm. During the drying process, bubbles can form or regions can detach from the target. Visual inspection of the sample for air bubbles, cracks, and edges or regions that did not fully contact the target plate can be easily recognized by being a lighter color than the surrounding sample. The areas of opaqueness, peeling, and loose edges of dehydrated samples are the regions most prone to flaking and should detach when blowing away excess matrix (Fig. S1, 4-6, 9; Supplemental, Video 2). 2D MALDI-TOF IMS data acquisition – monitor live feed and vacuum pressure. Once the MALDI target plate containing the sample is inserted into the instrument, sample flaking will cause the live video feed to show a partially adhered or missing sample or the vacuum pressure will spike and data acquisition will pause. If flaking occurs inside the mass spectrometer, the user should abort the run, eject the target, and determine the number and size of the flakes. If the flakes are small in size (less than 1 mm x 1 mm) and few in number, the user may proceed with instrument usage at the risk of possibly damaging the machine. To minimize damage to the instrument after flaking, open up the mass spectrometer, recover the flake, and clean the instrument. As long as the sample adheres to the target plate, data acquisition is straightforward. When flaking occurs. When a flake inside the mass spectrometer contacts the
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voltage plate responsible for the acceleration of the ions into the flight tube in a time-offlight instrument, it dampens the charge differential responsible for ion acceleration resulting in fewer ions detected. Damage can occur to the instrument if the flake causes an electrical short circuit between the target plate and the voltage plate. In general, if the sample or a portion thereof detaches from the target and remains in the instrument after the target is ejected, the instrument will need to be cleaned resulting in undesired downtime. It is therefore important to minimize flaking. Data interpretation – black holes. A cause of black holes is uneven matrix coverage, as with mucoid, gram-negative bacteria such as P. agglomerans and S. marcescens. The center of these microbial colonies absorbs more matrix than the colony edges or surrounding agar, making it difficult to saturate the center with matrix. The uneven matrix coverage becomes more apparent post-dehydration and results in little to no signal in the center (Fig. 3B). Black holes are also observed when analyzing colonies that produce aerial hyphae or are excessively hard and dry and therefore cannot absorb matrix and physically mask the molecules underneath. One solution we have employed with filamentous fungi is to remove these structures with a swab doused in acetonitrile prior to matrix application. As previously stated, loss of signal is observed when a sample loses contact with the MALDI target and therefore a black hole could also be caused by an air bubble or lifted sample. Data interpretation – false gradients. The cause of false gradients may be due to charging or ion suppression. The matrix-covered colony or agar may insulate the charge difference between the target plate, which serves as the ion source, and the acceleration voltage. This results in sub-par ionization where samples are thicker, often
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where the colony lies. Charging of the sample also results in decreased ionization, but occurs over time as the IMS run progresses. The target plate retains charge in between spectrum acquisitions. Charging is more common with faster lasers (kHz) and indium tin oxide (ITO) coated glass slides used for tissue IMS compared to slower lasers (20 Hz) and stainless steel target plates used for microbial IMS. By decreasing the laser frequency from 1 kHz to 200 Hz or slower, enough time between raster spots is provided, allowing the electrical charge on the target plate to dissipate. These false gradients are usually irreproducible between independent IMS analyses (Fig. 3A, 6-7) and therefore are identified when multiple samples are analyzed in, preferably, different orientations and order of acquisition when more than one sample is placed on one MALDI plate. Molecular annotation. Microbial IMS not only detects peptides, but also small molecules, lipopeptides, metal-chelating siderophores, and other types of molecules. This approach to simultaneously detect multiple types of molecules leads to challenges in molecular annotation. In general, annotation of mass spectrometry data is an ongoing problem not just limited to IMS, and the issue of data annotation is apparent as annotation of 10-15% of an advanced “omics” dataset from a simple system, such as proteomics of a single organism with a small genome, requires a skilled informatician (20). In studies of vertebrates that contain microbiomes where host cells are outnumbered 10 to 1 by microbes (e.g. mouse or human samples), it is not atypical for a skilled mass spectrometrist and informatician to annotate only 5% of the data. Similar percentages of identifications are obtained in metabolomics, indicating a need to develop novel approaches to identify molecules in mass spectrometry datasets (5).
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Determining the origin of observed molecules. If an ion of interest from a dual-species interaction is spatially localized to or diffusing away from one of the colonies in the interaction, the colony nearest to the ion is assumed to be the producer and further experimentation either confirms or negates this. In this instance, IMS should be treated as a hypothesis-generating tool with observations verified by additional experimental approaches. To initially attempt the determination of the originating organism, interacting colonies and individual control colonies are prepared and analyzed on one target plate to observe cross correlations in mass signals. Based on the IMS data, pursuable candidate ions are selected and tandem MS is attempted directly from the IMS sample to obtain information about the nature of the compound from its fragment masses. Attempts are also made to extract, purify and determine the structure or structural motifs of the molecule via other tandem MS approaches and NMR (12, 19, 22, 32, 33). If the genomes are available, genome mining approaches (7, 26) may assist in the annotation of metabolic exchange factors observed in microbial IMS (4, 8, 31). Genome mining uses the knowledge about the biosynthetic enzymes involved to annotate and confirm the molecules produced. However, genome mining alone will not uncover which metabolites are actually produced. Peptidogenomics is another tool that can be utilized that correlates amino acid motifs annotated from tandem MS approaches with predicted metabolites from putative gene clusters (19). This tool involves searching a six-frame translation of the genome sequence for the sequence tag and is useful in identifying unannotated ribosomally encoded molecules. If the genus is unknown or the genome sequence is unavailable, identification via
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16S rRNA sequencing (29) or established MALDI-TOF protocols (27, 28) is attempted. Once the genus and/or species of the microbes are/is known, we search for known molecules associated with the masses of interest observed from IMS analysis. However, a majority of the masses from IMS do not match to characterized molecular entities when the organism has not been investigated thoroughly by previous investigators. The most crucial step in determining which microbe produces the molecule of interest is confirmation. Two main strategies are genetic knockout and complementation studies or assays with purified compound, in combination with more IMS. In a number of cases, the molecules of interest have roles in the development and defense of the colony; rendering the colony genetically incapable of producing such important molecules impacts development and fitness. Genetic complementation or incubation with pure compound may restore normal development and fitness, thereby confirming the biological importance of the molecules observed via IMS.
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Figure S1. How to minimize sample flaking inside instrument. At the first checkpoint
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we strongly recommend a non-polished MALDI target plate. If a polished target plate is used for microbial IMS, flaking will most likely occur (1, arrows in example pictures point to bubbles, cracks, and flakes). The second checkpoint assures that there is no air trapped between the sample and target plate. Once the sample on the target plate is bubble-free, matrix is applied until even and saturated (3a); then the sample is dehydrated (3b). There are media and microbes that may necessitate multiple rounds of matrix application during the dehydration process in order to achieve even coverage and saturation (refer to Fig. S3). If matrix coverage does not saturate the sample, the agar shrinks (2b) and sample preparation needs to be repeated. If there is sufficient matrix coverage after dehydration, use air to remove the loose matrix from the sample. This will also test whether or not the sample flaked during dehydration (4-6). If a portion of any sample flakes, repeat the sample preparation or carefully remove the flaking sample (11), then wipe the area with methanol and a Kimwipe, and proceed with sample inspection. Despite passing the previous checkpoints, there may be a bubble (7) or crack (8) in the dehydrated sample. The first attempts to test whether or not the sample may flake include tapping the bubble with a spatula (9) or blowing air over the cracked sample. If the sample remains intact, proceed to MALDI-TOF IMS and monitor the data acquisition for signs of flaking inside the instrument, such as sample fluttering or missing sample in the live feed, a spike in vacuum pressure, or a delay in the data acquisition run.
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Figure S2. Our hypothesis of action of dual-matrix. Samples saturated with a 1:1 mixture of 2,5-dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA) matrices are more likely to adhere to the target plate. We hypothesize that more DHB is absorbed into the agar and acts as an adhesive, whereas more CHCA stays on top of the sample and that saturation with matrix minimizes sample shrinkage during dehydration. The combination of these two matrices assists in adherence of the sample to the target and ionization of a variety of biomolecules from microbial samples on agar-based media.
Figure S3. Multiple matrix applications to saturate agar samples with matrix. A.
Candida albicans interacting with P. aeruginosa on Spider agar media on MALDI target plate. B. First matrix application shows even coverage and what appears to be saturation. C. After 1 h dehydration, the agar has absorbed much of the matrix and the sample is not saturated with matrix. D. Second matrix application shows even matrix coverage and saturation. E. The sample is completely dehydrated, matrix coverage is even, and the excess matrix has been removed.
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