MALDI TIMS IMS Reveals Ganglioside Molecular Diversity within Murine S. aureus Kidney Tissue Abscesses

Gangliosides play important roles in innate and adaptive immunity. The high degree of structural heterogeneity results in significant variability in ganglioside expression patterns and greatly complicates linking structure and function. Structural characterization at the site of infection is essential in elucidating host ganglioside function in response to invading pathogens, such as Staphylococcus aureus (S. aureus). Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) enables high-specificity spatial investigation of intact gangliosides. Here, ganglioside structural and spatial heterogeneity within an S. aureus-infected mouse kidney abscess was characterized. Differences in spatial distributions were observed for gangliosides of different classes and those that differ in ceramide chain composition and oligosaccharide-bound sialic acid. Furthermore, integrating trapped ion mobility spectrometry (TIMS) allowed for the gas-phase separation and visualization of monosialylated ganglioside isomers that differ in sialic acid type and position. The isomers differ in spatial distributions within the host–pathogen interface, where molecular patterns revealed new molecular zones in the abscess previously unidentified by traditional histology.


S-4
Table S1.ganglioside species that were also detected in the control mouse kidney.Ion images of these species can be seen in Figure S4.

Figure S2 .
Figure S2.Average MALDI TIMS mass spectrum of control (A) and 10 DPI S. aureus-infected (B) mouse kidney tissue section and an overlay of both spectra (C).Average FT-ICR mass spectrum of the abscessed region, collected from a serial section for high mass accuracy ganglioside identification (D).Major ganglioside classes detected in the infected kidney are identified in the overlay.

Figure S3 .
Figure S3.Ion mobility heat map of average mass spectrum of 10 DPI S. aureus-infected mouse kidney tissue section.

Figure S4 .
Figure S4.Example ion images of eight GM3 gangliosides in control and 10DPI S. aureus-infected mouse kidney tissue sections.GM3s were the only class of gangliosides detected above the limit of detection in the control samples.

Figure S5 .
Figure S5.Example ion images of seven GM2 gangliosides in control and 10DPI S. aureus-infected mouse kidney tissue sections.

Figure S6 .
Figure S6.On-tissue MALDI MS/MS of m/z 1370.78 reveals the structure of GM2 NeuGC (d34:1).The peak at m/z 1063.08 indicates the neutral loss of sialic acid (NeuGC -H2O), m/z 860.61 confirms the ceramide composition as (d34:1).No neutral loss of NeuAC sialic acid (-291.09)was observed in the fragmentation spectrum, indicating Neu5GCcontaining GM2 was the most likely the only isomer with m/z 1370.78.The absolute intensity of m/z 1370.78 is 1964.MALDI TIMS MS/MS IMS data were collected in negative ionization mode with the following parameters -isolation mass: m/z 1370.78 ± 1.5, CID: 55.0 eV, m/z range: m/z 200 -3000, area of data collection: 285 pixels.

Figure S7 .
Figure S7.Example ion images of seven GM1 gangliosides in control and 10 DPI S. aureus-infected mouse kidney tissue sections.

Figure S8 .
Figure S8.Example ion images of seven GD1 gangliosides in control and 10 DPI S. aureus-infected mouse kidney tissue sections.

Figure S9 .
Figure S9.Example ion images of seven GalNAc-GM1b gangliosides (A) and three extended series GM1b gangliosides (B) in control and 10 DPI S. aureus-infected mouse kidney tissue sections.

Figure S11 .
Figure S11.On-tissue MALDI TIMS MS/MS of m/z 1626.95(A) reveals the presence of both GM1a (B) and GM1b (C) isomers within a 10 DPI S.aureus-infected mouse kidney.The Extracted ion mobilograms of unique/preferential fragments confirm that GM1b (red) is more compact than GM1a (blue).

Table S2 .
Characteristic Fragment Ion and Neutral loss used for ganglioside species Identification This is not an exhaustive list of diagnostic fragments used in ganglioside identification; it only reflects fragment ions and neutral losses observed and used in this study.

Table S3 .
GM3 Gangliosides Identified in a 10 DPI Mouse Kidney Section; identified.

Table S4 .
GM2 Gangliosides Identified in a 10 DPI Mouse

Table S5 .
GM1 Gangliosides Identified in a 10 DPI Mouse Kidney Section

Table S6 .
GD1 Gangliosides Identified in a 10 DPI Mouse Kidney Section; *Denotes potential isomers that were not identified.

Table S7 .
GalNAc-GM1b and extended series GM1b Gangliosides Identified in a 10 DPI Mouse Kidney Section; *Denotes potential isomers that were not identified.