transmission-mode maldi-2 mass spectrometry imaging of ...10.1038/s41592-019-053… · overview...

Articleshttps://doi.org/10.1038/s41592-019-0536-2

Transmission-mode MALDI-2 mass spectrometry imaging of cells and tissues at subcellular resolutionM. Niehaus 1,4, J. Soltwisch 1,2,4, M. E. Belov3 and K. Dreisewerd 1,2*

1Institute for Hygiene, University of Münster, Münster, Germany. 2Interdisciplinary Center for Clinical Research, University of Münster, Münster, Germany. 3Spectroglyph LLC, Kennewick, WA, USA. 4These authors contributed equally: M. Niehaus, J. Soltwisch *e-mail: [email protected]

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Supplementary Figure 1

Overview bright field image of the mouse brain section evaluated in Fig. 2, 4 and S-3

Overview bright field image of the mouse cerebellum tissue section that was used to generate the ion images of

Fig. 2, Fig. 4, and Fig. S-2, respectively. The adjacent areas that were analyzed for producing the ion images are indicated by the black frames. For Fig. 2, the big rectangle to the right denotes the positive ion mode

measurements whereas the negative ion mode measurements (left) were conducted on three separate days. The HCCA matrix-coated tissue sample remained in the t-MALDI ion source during the full experimental circle, without that this caused any notable degradation in analytical performance.


Comparison of t-MALDI with t-MALDI-2

(a) t-MALDI (black trace, top panel) and t-MALDI-2 (red trace, bottom) mass spectra acquired from mouse

cerebellum in the positive ion mode at 1 µm pixel size. HCCA was used as a matrix. (b) Bright field image of the areas across which the mass spectra were acquired, the microscopic image was taken prior to spray-coating the HCCA MALDI matrix. (c) Ion images of selected lipids acquired in the t-MALDI (top) and t-MALDI-2

modes (bottom). Comparable experiments from four different mouse brain samples yielded similar results.


Comparison of [DAG(36:1)]+- and putative PC(36:1)-precursor-derived ion images

Ion images of four most prominent 36:1-phospholipid ion signals detected from mouse cerebellum by t-MALDI-2 in the positive ion mode and that of the [DAG(36:1)]+ dissociation product. The same data set as

used for producing Fig. 2 of the main text was evaluated. (a) m/z 605.552, [DAG(36:1)]+; (b) m/z 703.526, PA(36:1); (c) m/z 729.542, PC(36:1)-N(CH3)3 or isobaric PA(38:2); (d) 746.469; PE(36:1); (d) m/z 788.616, PC(36:1); (f) m/z 790.559, PS(36:1). All ions were detected as [M + H]+ species. All “Viridis” false color

pallets were adjusted to optimize the presentation of the individual ion images; hence, they are not to scale if the images were to be compared among each other. Comparable experiments from four different mouse brain

samples yielded similar results.


MSI and light microscopy data of mouse kidney tissue – part 1

(a) Microscopic image of a 6 µm-thick, H&E-stained section of a mouse kidney tissue. The stain was produced

following the t-MALDI measurements and removing the matrix by a washing step. The black rectangular area indicates the approximate regions with which t-MALDI-2-MSI experiments were conducted. (b) Autofluorescence image taken of the analyzed section after the MSI measurement. (c to f) Representative t-

MALDI-2-MS images acquired in the positive ion mode: (c) m/z 768.544, PE(38:4)+, (d) m/z 724.524, PE(O-36:5)/PE(P-36:4); (e) m/z 792.555, PE(40:6), (f) m/z 644.502, CerP(36:1-OH; presumably CerP(d18:1/C18:0-

)). All ions were detected as [M + H ]+ species. (g) Mass spectrum averaged over 580,000 pixels. Only a few selected major ion signals are annotated based on accurate mass. A more complete assignment of the detected lipids is provided in Table S-3. HCCA was used as the MALDI matrix and prepared using a spraying protocol.

The ablation laser was focused to an effective spot size of about 1.3 µm by diameter and the step size of the piezo stage was set to 1.2 µm. Comparable experiments from one different mouse kidney sample yielded

similar results.

.

Supplementary Figure 4 continued

MSI and light microscopy data of mouse kidney tissue – part 2

(a) Microscopic image of a 6 µm-thick, H&E-stained section of a mouse kidney tissue. The stain was produced following the t-MALDI measurements and removing the matrix by a washing step. The black rectangular area

indicates the approximate regions with which t-MALDI-2-MSI experiments were conducted. (b) Autofluorescence image taken of the analyzed section after the MSI measurement. (c to f) Representative t-

MALDI-2-MS images acquired in the positive ion mode: (c) m/z 768.544, PE(38:4)+, (d) m/z 724.524, PE(O-

36:5)/PE(P-36:4); (e) m/z 792.555, PE(40:6), (f) m/z 644.502, CerP(36:1-OH; presumably CerP(d18:1/C18:0-)). All ions were detected as [M + H ]+ species. (g) Mass spectrum averaged over 580,000 pixels. Only a few

selected major ion signals are annotated based on accurate mass. A more complete assignment of the detected lipids is provided in Table S-3. HCCA was used as the MALDI matrix and prepared using a spraying protocol.

The ablation laser was focused to an effective spot size of about 1.3 µm by diameter and the step size of the piezo stage was set to 1.2 µm. Comparable experiments from one different mouse kidney sample yielded similar results.


Single pixel MS/MS data recorded at 1 µm pixel size

(a) Bright-field microscopic image of a 6 µm-thick section of a mouse brain tissue before matrix coating. (b) t-

MALDI-2-MS/MS images of diagnostic fragments recorded from the same section following spray-coating with HCCA matrix. (c) Single pixel MS/MS spectrum of GalCer(d18:1/C22:1), m/z of the [M + H]+ precursor, 810.681; (d) MS/MS spectrum of PE(40:6), m/z of the [M + H]+ precursor, 792.554, recorded from 4 adjacent

pixels, and (e) MS/MS spectrum of PS(40:6), m/z of the [M + H]+ precursor, 836.543, also recorded from 4 pixel. Experiments were conducted on a single sample only.


SEM images of brain sections coated with HCCA matrix before and following t-MALDI-2-MSI

(a) SEM image of a 6 µm-thick mouse brain tissue section, spray-coated with HCCA matrix according to the

protocol outlined in the online methods section. The displayed region corresponds to the white matter of mouse cerebellum. (b) Ablation crater pattern formed during a t-MALDI-2-MSI experiment and use of 25 laser pulses. The t-MALDI-MSI experiment was conducted at a step size of 5.0 µm. (c) Enlarged view into the irradiated

sample. Experiments were conducted on a single sample only.


Tandem-MS data to demonstrate adduct formation with indium cations

An example tandem MS spectrum of an (unknown) analyte species with an indium adduct. Experiments were conducted on a single sample only.


Bright- field and t-MALDI-2-MSI-data of cultured Vero B4 cells in the negative ion mode

Bright field and exemplary ion images of a Vero B4 cell culture imaged by t-MALDI-2-MSI in the negative ion mode (cf. Fig. 5 of the main text for positive ion mode measurements). (a) Bright field image of sub-confluently grown cells with the DHB matrix applied by sublimation; the depicted area displays the region from which the

ion images in panels b to d were recorded. (b) M/z 742.538, PE(36:2); (c) m/z 788.545, PS(36:1); (d) m/z 861.550, PI(36:2); all ions were detected as [M – H]- species. A more complete list of tentatively annotated

lipids is provided in Table S-4. Comparable experiments from one different mouse kidney sample yielded similar results.


Snapshots of the t-MALDI setup

Snapshots of the custom-build t-MALDI-2 ion source. (a) The following components are depicted: 1- telescope lenses for beam manipulation. One of the lenses is attached to a piezo stage for fine adjustment of the laser

focus on the sample slide; 2- dielectric mirror in front of the quartz window guiding the laser beam into the ion source; 3- UV-grade mirrors; 4- focusing lens for the CCD camera. (b) The interior components of the ion

source. 1- 50x magnifying UV-grade microscope objective; 2- piezo sample stage; 3- ITO coated slide holder; 4- constant force spring to account for the weight of the attached sample holder and PEEK bracelet.

1

Table S-1: Putative lipids recorded by t-MALDI-2-MSI from mouse cerebellum tissue in the positive ion

mode – data refer to three separate MSI measurements conducted on two independently prepared brain

mouse brain samples from two different animals. They were conducted with mass resolving powers of

70,000 and 280,000 (@m/z 200), respectively, and some further variations in experimental settings for the

R=70,000 experiments.1)

m/zexp Proposed identity

Type of adduct ion and deviation with calculated m/z2) Intensity

R = 280.000

R = 70.000

R = 70.000

566.5509 N-acylsphingosine Cer(36:1) [+H], Δ -0.08 ppm; [+H], Δ -0.618 ppm; [+H], Δ -0.295 ppm 6.4E+02 3.0E+02 2.9E+02

604.5065 N-acylsphingosine Cer(36:1) [+K], Δ 0.945 ppm 1.4E+02

620.5977 N-acylsphingosine Cer(40:2) [+H], Δ -0.148 ppm; [+H], Δ -0.148 ppm 6.0E+03 7.6E+03

622.6130 N-acylsphingosine Cer(40:1) [+H], Δ -0.107 ppm; [+H], Δ 0.971 ppm 9.6E+02 1.3E+03

636.5324 N-acylsphingosine Cer(40:5) [+Na], Δ 1.09 ppm; [+Na], Δ 1.09 ppm; [+Na], Δ 1.377 ppm 2.5E+03 1.9E+02 4.1E+03

636.5933 N-acyl-4-hydroxysphinganine Cer(40:2)

[+H], Δ -1.421 ppm; [+H], Δ -0.942 ppm 7.4E+02 5.6E+02


[+H], Δ -0.135 ppm; [+H], Δ -0.04 ppm 4.8E+03 8.4E+03

644.5972 N-acylsphingosine Cer(42:4) [+H], Δ 0.615 ppm 1.9E+02



649.4802 Diacylglycerophosphate PA(32:0)

[+H], Δ 0.094 ppm 1.9E+02


654.5223 N-acylsphingosine Cer(40:4) [+K], Δ 0.724 ppm 1.7E+02


[+H], Δ -0.499 ppm 5.8E+02


[+H], Δ -0.092 ppm; [+H], Δ 0.367 ppm 2.0E+03 2.9E+03


[+H], Δ -0.054 ppm; [+H], Δ 0.22 ppm 6.2E+03 1.0E+04


[+K], Δ 1.133 ppm; [+K], Δ 0.587 ppm 3.7E+02 1.4E+02


[+Na], Δ 0.282 ppm 2.6E+02


[+K], Δ 0.985 ppm; [+K], Δ 1.167 ppm; [+K], Δ 1.076 ppm 1.0E+04 1.5E+04 7.5E+03

673.4802 Diacylglycerophosphate PA(34:2) /

Diacylglycerophosphocholine PC(32:1) – N(CH3)3

[+H], Δ 0 ppm; [+H], Δ 0.181 ppm; [+H], Δ 0.272 ppm 3.3E+03 2.1E+03 2.0E+03



[+H], Δ 0.037 ppm; [+H], Δ 0.76 ppm; [+H], Δ 0.67 ppm 4.5E+03 3.1E+03 4.5E+03

2



692.5224 Diacylglycerophosphoethanolamine PE(32:0)

[+H], Δ -0.053 ppm; [+H], Δ 0.211 ppm; [+H], Δ 0.299 ppm 2.8E+03 4.3E+03 1.7E+03



[+Na], Δ 0.483 ppm; [+Na], Δ 0.483 ppm; [+Na], Δ 0.133 ppm 3.6E+03 8.8E+03 2.9E+03




700.5284 Acylalkylglycerophosphoethanolamine PE(34:3)

[+H], Δ -1.158 ppm 1.5E+03


[+K], Δ 1.279 ppm 1.7E+02








[+H], Δ 0.107 ppm; [+H], Δ 4.271 ppm 6.5E+03 7.4E+03



[+K], Δ 0.952 ppm; [+K], Δ -0.335 ppm; [+K], Δ 0.094 ppm 9.4E+02 8.9E+02 8.0E+02





[+K], Δ 0.931 ppm 1.9E+03


[+H], Δ 0.119 ppm; [+H], Δ -1.926 ppm 9.1E+02 1.6E+03







[+Na], Δ 0.584 ppm; [+Na], Δ -0.934 ppm; [+Na], Δ -0.934 ppm 3.6E+03 8.3E+03 5.3E+03





[+H], Δ -0.83 ppm 1.1E+03






728.6023 Hexosyl-N-acylsphingosine HexCer(36:1)

[+H] , Δ 0.892 ppm; [+H] , Δ 3.908 ppm 1.4E+03 3.5E+03



3




732.5535 Diacylglycerophosphocholine PC(32:1)






[+K], Δ 0.373 ppm; [+K], Δ -3.434 ppm 6.9E+02 3.1E+03





[+K], Δ 0.265 ppm 6.3E+02





[+H], Δ 0.559 ppm; [+H], Δ 1.217 ppm; [+H], Δ -1.331 ppm 8.8E+02 5.4E+02 1.5E+03



744.5975 Hexosyl-N-acyl-4-hydroxysphinganine

HexCer(36:1)

[+H] , Δ 0.847 ppm; [+H] , Δ 3.142 ppm 2.0E+03 4.3E+03




[+H], Δ 0.084 ppm; [+H], Δ 2.123 ppm 2.4E+03 2.9E+03



[+Na], Δ 0.467 ppm 1.3E+03


[+H], Δ 0.124 ppm 1.6E+03


[+H], Δ -0.409 ppm 1.2E+03




[+Na], Δ 0.704 ppm; [+Na], Δ 0.785 ppm; [+Na], Δ -0.829 ppm 1.6E+03 9.6E+03 2.7E+03


[+H], Δ 0.19 ppm; [+H], Δ 0.029 ppm 2.6E+03 8.4E+03






[+H], Δ 0.142 ppm; [+H], Δ 0.866 ppm 3.1E+03 9.6E+03

760.5129 Diacylglycerophosphoserine PS(34:2)

[+H], Δ -0.822 ppm 1.7E+02





4


[+H], Δ -0.048 ppm 6.1E+02



[+K], Δ 1.222 ppm; [+K], Δ 1.063 ppm 2.9E+02 8.5E+02


[+K], Δ 0.13 ppm 1.0E+03





[+K], Δ 0.877 ppm; [+K], Δ 2.862 ppm 9.8E+02 7.5E+02



768.5898 Acylalkylglycerophosphocholine PC(36:4)

[+H], Δ 0.425 ppm 1.7E+02






HexCer(38:1)

[+H] , Δ 0.881 ppm; [+H] , Δ 2.224 ppm 2.5E+03 5.4E+03




[+H], Δ -1.293 ppm 1.5E+02



[+H], Δ 0.873 ppm 4.5E+02 6.1E+02


[+H] , Δ 0.819 ppm; [+H] , Δ 1.366 ppm 7.1E+02 7.2E+02






[+H] , Δ 0.849 ppm; [+H] , Δ 1.317 ppm 7.7E+03 1.3E+04




[+Na], Δ 1.754 ppm; [+Na], Δ -0.657 ppm 2.2E+03 8.3E+02


[+H] , Δ 0.956 ppm; [+H] , Δ 2.201 ppm 5.0E+03 1.1E+04


[+K], Δ 0.864 ppm 1.0E+03




[+H], Δ -0.033 ppm; [+H], Δ 0.199 ppm; [+H], Δ 0.122 ppm 1.3E+03 1.6E+03 2.5E+03





[+K], Δ 0.644 ppm 1.3E+02

5


[+H], Δ 0.153 ppm; [+H], Δ 0.462 ppm 8.9E+03 1.9E+04


[+H], Δ 0.094 ppm; [+H], Δ 0.248 ppm 1.3E+03 5.5E+03


[+K], Δ 0.143 ppm 2.2E+02


HexCer(40:3)

[+H] , Δ 2.463 ppm 5.7E+02




HexCer(40:2)

[+H] , Δ 0.807 ppm; [+H] , Δ 1.113 ppm 9.4E+03 1.6E+04


[+K], Δ 0.88 ppm; [+K], Δ 4.692 ppm 2.5E+03 2.8E+03


HexCer(40:1)

[+H] , Δ 0.913 ppm; [+H] , Δ 1.065 ppm; [+H] , Δ 1.141 ppm 4.7E+04 6.1E+02 8.3E+04


[+H], Δ 0.096 ppm 7.1E+02


[+K], Δ 0.932 ppm; [+K], Δ -0.279 ppm 7.4E+02 8.8E+02


[+Na], Δ -1.478 ppm; [+Na], Δ -1.1 ppm 7.3E+02 1.3E+03


[+H] , Δ 0.852 ppm; [+H] , Δ 1.23 ppm 9.0E+03 1.4E+04


[+K], Δ -0.065 ppm 4.3E+02


[+Na], Δ 0.449 ppm; [+Na], Δ 0.825 ppm 5.2E+02 2.3E+03









[+K], Δ 1.825 ppm; [+K], Δ 0.4 ppm 1.6E+02 1.5E+02


[+H], Δ 0.329 ppm 3.0E+02


[+H], Δ 0.135 ppm; [+H], Δ 0.657 ppm 8.6E+02 2.6E+03


[+K], Δ 0.776 ppm 7.2E+02


HexCer(40:1)

[+Na], Δ 0.234 ppm; [+Na], Δ -0.508 ppm 2.4E+03 4.5E+03


HexCer(42:3)

[+H] , Δ 0.96 ppm; [+H] , Δ 1.848 ppm 5.4E+03 1.0E+04




HexCer(42:2)


6


[+K], Δ 0.816 ppm; [+K], Δ 1.479 ppm 1.0E+03 1.3E+03


HexCer(42:1)



[+K], Δ 0.684 ppm; [+K], Δ 0.023 ppm 1.2E+03 2.0E+03


[+Na], Δ 0.871 ppm; [+Na], Δ 3.949 ppm 1.3E+03 2.2E+03


[+Na], Δ 1.849 ppm 1.4E+03


[+H], Δ 0.48 ppm; [+H], Δ 0.261 ppm 9.8E+02 2.2E+02


[+H], Δ -1.77 ppm 2.6E+03


[+H], Δ 0.118 ppm; [+H], Δ -0.675 ppm 1.4E+03 2.0E+03


[+H], Δ 0.075 ppm; [+H], Δ 1.513 ppm 9.2E+03 1.1E+04


[+K], Δ 0.809 ppm; [+K], Δ 3.105 ppm 4.4E+03 3.1E+03


HexCer(42:1)

[+Na], Δ 0.5 ppm; [+Na], Δ 0.07 ppm 3.0E+03 3.1E+03


HexCer(42:3)

[+K], Δ 0.222 ppm 1.2E+02


HexCer(42:2)

[+K], Δ 0.815 ppm; [+K], Δ 0.956 ppm 6.4E+03 7.3E+03


HexCer(42:1)


1) Lipids were annotated by the workflow described in the online methods. Briefly, the mean spectra of the 50 min-long imaging runs were loaded into the MZmine program and lipids were annotated via an automated database search (LipidSearch). As is discussed in the main text, some assigned signals, in particular (and if applicable) those of DAG and PA species are likely to (partially) represent the results of in-source decay fragmentation reactions.

2) The (up to three) assignments refer to the (up to three) individual MSI experiments indicated in the column to the right.

Table S-2: Putative lipids recorded by t-MALDI-2-MSI from mouse cerebellum tissue in the negative ion mode – data refer to two separate MSI measurements conducted on two independently prepared brain mouse brain samples from two different animals 1)

m/z Identity Type of adduct ion and deviation with calculated m/z2)) Intensity *

280.000 70.000


[-H], Δ -2.441 ppm; [-H], Δ -1.19 ppm 3.2E+03 1.4E+03

7


[-H], Δ 0.674 ppm 2.9E+02

647.4655 Diacylglycerophosphate PA(32:0) [-H], Δ -0.335 ppm; [-H], Δ 1.08 ppm 1.7E+03 7.3E+03


[-H], Δ 0.17 ppm; [-H], Δ 0.905 ppm 4.4E+02 6.4E+02

673.4810 Diacylglycerophosphate PA(34:1) / Diacylglycerophosphocholine

PC(32:0) – N(CH3)3

[-H], Δ -0.012 ppm; [-H], Δ 1.256 ppm 9.8E+03 4.3E+04


PC(34:1) – N(CH3)3

[-H], Δ -0.238 ppm; [-H], Δ 1.245 ppm 5.5E+03 3.1E+04


PC(34:0) – N(CH3)3

[-H], Δ 0.059 ppm; [-H], Δ 2.061 ppm 5.7E+03 2.1E+04


[-H], Δ -1.38 ppm 5.0E+02


[-H], Δ -0.064 ppm; [-H], Δ 0.107 ppm 5.9E+02 5.5E+03


[-H], Δ -0.368 ppm; [-H], Δ 1.246 ppm 3.4E+03 2.0E+04


PC(36:3) – N(CH3)3

[-H], Δ 1.626 ppm 2.6E+02


PC(36:1) – N(CH3)3

[-H], Δ 0.008 ppm; [-H], Δ 1.77 ppm 1.3E+03 3.5E+03


[-H], Δ -0.556 ppm 7.3E+02

741.4708 Diacylglycerophosphoglycerols PG(34:4)

[-H], Δ 0.512 ppm 1.1E+03


[-H], Δ -0.192 ppm; [-H], Δ 0.63 ppm 1.5E+03 1.1E+04


[-H], Δ -0.158 ppm; [-H], Δ 0.908 ppm 4.8E+03 3.7E+04


[-H], Δ -0.451 ppm 6.3E+02


[-H], Δ 1.589 ppm 1.3E+03


[-H], Δ 0.049 ppm 1.3E+03


[-H], Δ 2.389 ppm 1.4E+03


[-H], Δ 2.894 ppm 2.5E+02


[-H], Δ 1.435 ppm 3.4E+02


[-H], Δ 0.118 ppm; [-H], Δ -0.358 ppm 4.3E+02 1.5E+03


[-H], Δ -0.008 ppm; [-H], Δ 0.861 ppm 1.1E+03 7.2E+03


[-H], Δ -0.541 ppm; [-H], Δ 0.856 ppm 1.4E+03 1.7E+04


[-H], Δ -0.044 ppm; [-H], Δ 1.195 ppm 2.1E+03 1.1E+04


[-H], Δ -0.18 ppm; [-H], Δ 1.287 ppm 4.9E+02 1.5E+04

8

796.6293 Hexosyl-N-acyl-4-hydroxysphinganine HexCer(40:2)

[-H], Δ 1.238 ppm 3.2E+02


[-H], Δ -0.798 ppm; [-H], Δ -0.186 ppm 1.9E+03 6.0E+03

806.5474 3-O-sulfogalactosy-N-acylsphingosine SHexCer(36:1)

[-H], Δ -2.672 ppm 4.7E+02


[-H], Δ -0.733 ppm; [-H], Δ 0.249 ppm 7.8E+02 3.3E+03


[-H], Δ 1.182 ppm 1.9E+03


[-H], Δ 0.019 ppm; [-H], Δ 0.543 ppm 1.1E+03 7.8E+03


[-H], Δ 0.487 ppm 9.9E+02


[-H], Δ -0.594 ppm; [-H], Δ 1.256 ppm 1.1E+03 4.9E+03


[-H], Δ -0.931 ppm; [-H], Δ 0.767 ppm 2.2E+03 9.6E+03


[-H], Δ 1.245 ppm 4.2E+03


[-H], Δ 0.223 ppm 1.5E+03


[-H], Δ -1.108 ppm 7.4E+02


[-H], Δ -1.196 ppm 1.6E+03

878.6036 3-O-sulfogalactosy-N-acyl-4-hydroxysphinganine SHexCer(40:1)

[-H], Δ -0.971 ppm 1.1E+03

885.5480 Diacylglycerophosphoinositol PI(38:4)

[-H], Δ 2.049 ppm 4.6E+03


[-H], Δ -0.773 ppm; [-H], Δ -0.842 ppm 1.3E+03 8.4E+03


[-H], Δ -0.674 ppm; [-H], Δ 0.285 ppm 8.3E+02 5.7E+03


[-H], Δ -1.995 ppm 3.3E+02


[-H], Δ -0.617 ppm 2.3E+03

1) Lipids were annotated by the workflow described in the online methods. Briefly, the mean spectra of the 50 min-long imaging runs were loaded into the MZmine program and lipids were annotated via an automated database search (LipidSearch). As is discussed in the main text, some assigned signals, in particular (and if applicable) those of PA species are likely to (partially) represent the results of in-source decay fragmentation reactions.

2) The (up to two) assignments refer to the MSI runs indicated in the columns to the right.

9

Table S-3: Putative lipids recorded by t-MALDI-2-MSI from mouse kidney tissue in the positive ion mode – data refer to two separate MSI measurements conducted on two independently prepared brain mouse brain samples from the same animal 1)

m/z Identity Type of adduct ion and mass accuracy2) Intensity *

70.000 70.000 70.000

582.4855 N-acylsphingosine Cer(36:4) [+Na], Δ 1.376 ppm; [+Na], Δ 1.062 ppm 6,3E+02 4,0E+02

600.4754 N-acylsphingosine Cer(36:3) [+K], Δ 0.664 ppm 1,9E+02

608.5010 N-acylsphingosine Cer(38:5) [+Na], Δ 1.459 ppm 2,6E+02

626.4913 N-acylsphingosine Cer(38:4) [+K], Δ -0.103 ppm; [+K], Δ 0.482 ppm 1,1E+02 8,2E+02

648.6288 N-acylsphingosine Cer(42:2) [+H], Δ 0.218 ppm 1,4E+02


[+H], Δ 0.387 ppm; [+H], Δ 0.74 ppm 4,1E+02 2,1E+02


[+H], Δ 0.532 ppm 5,6E+02


[+H], Δ -1.113 ppm; [+H], Δ -0.772 ppm 1,9E+02 2,1E+02


[+H], Δ 0.204 ppm; [+H], Δ 0.459 ppm; [+H], Δ -0.052 ppm 4,3E+02 1,9E+02 5,6E+02


[+H], Δ 0.578 ppm; [+H], Δ 0.578 ppm; [+H], Δ 0.238 ppm 4,4E+02 3,8E+02 2,5E+03




[+H], Δ -2.888 ppm; [+H], Δ -0.108 ppm; [+H], Δ 0.06 ppm 3,6E+02 2,1E+03 1,4E+04


[+H], Δ -0.709 ppm 2,3E+02


[+H], Δ 0.018 ppm 1,7E+02




[+H], Δ -0.745 ppm; [+H], Δ -2.068 ppm; [+H], Δ -1.324 ppm 6,3E+02 1,6E+02 1,1E+03




[+Na], Δ 1.013 ppm; [+Na], Δ 0.766 ppm; [+Na], Δ 0.766 ppm 2,2E+03 3,2E+03 5,1E+03




[+H], Δ 0.188 ppm 2,8E+02




[+H], Δ 2.025 ppm 2,6E+02


[+Na], Δ -0.226 ppm 9,6E+02


[+H], Δ 1.144 ppm 1,1E+02


[+H], Δ 0.652 ppm 1,6E+02

10


[+H], Δ -2.999 ppm; [+H], Δ 0.498 ppm; [+H], Δ -0.722 ppm 2,8E+02 4,0E+02 4,7E+03


[+H], Δ -0.362 ppm; [+H], Δ 0.124 ppm; [+H], Δ 0.368 ppm 5,4E+02 1,8E+03 8,1E+03


[+Na], Δ -1.268 ppm; [+Na], Δ -1.026 ppm 2,2E+02 2,3E+02




[+K], Δ 1.393 ppm; [+K], Δ 1.232 ppm; [+K], Δ -1.745 ppm 8,9E+02 5,5E+02 2,7E+02






[+K], Δ -0.015 ppm; [+K], Δ 0.065 ppm 3,5E+02 4,1E+02


[+Na], Δ -1.001 ppm; [+Na], Δ 1.401 ppm 9,7E+02 2,7E+02


[+H], Δ 2.837 ppm 1,9E+02




[+Na], Δ -1.567 ppm; [+Na], Δ -0.691 ppm; [+Na], Δ -1.886 ppm 6,4E+03 9,8E+03 2,9E+04




[+Na], Δ 1.737 ppm 4,7E+02


[+K], Δ 1.005 ppm; [+K], Δ 0.531 ppm; [+K], Δ 0.847 ppm 5,3E+03 3,6E+03 3,3E+03

772.5252 AND/OR Acylalkylglycerophosphoetha

nolamine PE(38:6)



[+H], Δ 1.346 ppm; [+H], Δ 0.714 ppm 1,3E+02 1,6E+03


[+H], Δ 0.325 ppm 4,2E+03


nolamine PE(38:5)

[+Na], Δ -2.077 ppm 4,2E+03


[+H], Δ 1.375 ppm 5,8E+02




nolamine PE(38:4)





[+H], Δ 2.347 ppm 2,4E+02


nolamine PE(38:3)

[+Na], Δ -0.042 ppm 2,4E+02


[+Na], Δ -0.334 ppm; [+Na], Δ 0.057 ppm; [+Na], Δ -0.021 ppm 3,7E+02 1,6E+03 2,5E+03



11




[+H], Δ -2.042 ppm 2,5E+02




[+Na], Δ 0.815 ppm 1,6E+02




[+Na], Δ 0.38 ppm; [+Na], Δ 2.238 ppm 7,6E+02 1,1E+03


[+H], Δ 2.74 ppm 7,6E+02


[+H], Δ 1.537 ppm 1,7E+03


[+Na], Δ -0.053 ppm; [+Na], Δ -0.207 ppm; [+Na], Δ 1.569 ppm 2,1E+03 7,0E+03 4,5E+03


[+H], Δ 0.325 ppm; [+H], Δ -1.139 ppm; [+H], Δ 0.248 ppm 5,5E+03 2,3E+03 4,4E+03


[+H], Δ -2.052 ppm 5,1E+02


[+Na], Δ -1.141 ppm 2,1E+03

794.5695 AND/OR Diacylglycerophosphoethanol

amine PE(40:5)

[+H], Δ -1.411 ppm; [+H], Δ 1.2 ppm 1,1E+03 2,1E+03



796.5239 AND/OR Diacylglycerophosphocholine

PC(34:2)

[+K], Δ 2.047 ppm; [+K], Δ 2.89 ppm 3,6E+02 7,3E+02




[+K], Δ 2.991 ppm 1,1E+03


[+K], Δ 2.37 ppm 9,8E+01


[+K], Δ 0.493 ppm; [+K], Δ 0.57 ppm 2,5E+02 7,6E+02









806.5684 AND/OR Diacylglycerophosphocholine

PC(36:3)



[+H], Δ -1.83 ppm 6,5E+02




[+H], Δ 0.69 ppm 9,9E+01

12


[+Na], Δ -1.605 ppm 9,9E+01


[+Na], Δ -1.283 ppm; [+Na], Δ -0.605 ppm 8,5E+02 8,52E+03


[+Na], Δ -2.171 ppm; [+Na], Δ -2.397 ppm 2,4E+03 9,0E+03


[+H], Δ 0.118 ppm; [+H], Δ -1.159 ppm; [+H], Δ -0.107 ppm 2,4E+03 3,3E+03 9,0E+03




[+K], Δ 2.261 ppm; [+K], Δ 2.039 ppm 1,6E+02 5,7E+02


[+K], Δ -1.175 ppm 3,8E+02






[+K], Δ -0.565 ppm; [+K], Δ 0.611 ppm; [+K], Δ -2.402 ppm 3,3E+02 8,0E+02 4,7E+02


[+Na], Δ 2.362 ppm 4,4E+02


[+Na], Δ 1.067 ppm; [+Na], Δ 1.14 ppm 9,6E+02 3,9E+03


[+H], Δ -1.231 ppm 1,6E+02


[+Na], Δ 2.411 ppm; [+Na], Δ 1.533 ppm 1,4E+02 3,8E+02


[+H], Δ 0.699 ppm 2,9E+02


[+K], Δ 0.63 ppm; [+K], Δ 0.486 ppm; [+K], Δ -0.237 ppm 3,2E+02 9,2E+02 2,4E+02


[+K], Δ 2.008 ppm 2,0E+02







1) Lipids were annotated by the workflow described in the online methods. Briefly, the mean spectra of the 50 min-long imaging runs were loaded into the MZmine program and lipids were annotated via an automated database search (LipidSearch). As is discussed in the main text, some assigned signals, in particular (and if applicable) those of DAG and PA species are likely to (partially) represent the results of in-source decay fragmentation reactions.

2) The (up to three) assignments refer to the MSI runs indicated in the columns to the right.

13

Table S-4: Putative lipids recorded by t-MALDI-2-MSI from Vero B4 cell culture in the positive ion mode. Data refer to two separate MSI measurements conducted on different areas of the same cell culture 1)


70.000 70.000


PC(30:0) – N(CH3)3

[+K], Δ 1.041 ppm; [+K], Δ 0.506 ppm 9.7E+01 1.8E+02


PC(32:2) – N(CH3)3

[+K], Δ -2.694 ppm 1.4E+02


PC(32:1) – N(CH3)3

[+K], Δ 0.523 ppm; [+K], Δ 0.437 ppm 3.7E+02 1.0E+03


PC(32:0) – N(CH3)3

[+K], Δ 1.156 ppm; [+K], Δ 1.241 ppm 2.0E+02 4.5E+02


[+H], Δ -0.478 ppm; [+H], Δ -0.563 ppm 1.9E+02 4.2E+02


[+H], Δ 0.408 ppm; [+H], Δ 0.238 ppm 3.6E+02 5.8E+02


[+H], Δ 0.101 ppm 1.1E+02


PC(34:2) – N(CH3)3

[+K], Δ -0.786 ppm; [+K], Δ -0.786 ppm 2.9E+02 9.9E+02


PC(34:1) – N(CH3)3

[+K], Δ 0.901 ppm; [+K], Δ 0.736 ppm 1.0E+03 2.9E+03


PC(34:0) – N(CH3)3

[+K], Δ 2.99 ppm 1.7E+02


[+Na], Δ -1.289 ppm; [+Na], Δ -1.947 ppm 8.8E+01 2.6E+02


[+H], Δ 0.264 ppm; [+H], Δ 0.1 ppm 1.2E+03 3.2E+03


[+H], Δ 1.441 ppm; [+H], Δ 1.523 ppm 3.4E+02 7.7E+02


[+K], Δ 0.848 ppm; [+K], Δ 0.201 ppm 7.7E+01 2.0E+02


[+K], Δ 1.686 ppm; [+K], Δ 1.605 ppm 8.4E+01 1.7E+02


[+H], Δ -0.03 ppm; [+H], Δ -0.191 ppm 7.7E+01 2.2E+02


PC(30:1) – N(CH3)3

[+In], Δ -0.559 ppm 1.3E+02


[+H], Δ 0.324 ppm; [+H], Δ 0.083 ppm 1.7E+02 3.3E+02


PC(30:0) – N(CH3)3

[+In], Δ 0.517 ppm; [+In], Δ 0.437 ppm 4.0E+02 8.8E+02

763.3755 Diacylglycerophosphate PA(32:0) [+In], Δ 1.748 ppm 8.5E+01


PC(36:2) – N(CH3)3

[+K], Δ 0.903 ppm; [+K], Δ 1.143 ppm 1.6E+02 5.8E+02


[+K], Δ 0.975 ppm; [+K], Δ 0.658 ppm 9.2E+01 2.0E+02

14


[+H], Δ 0.477 ppm 2.0E+02


[+K], Δ 1.713 ppm; [+K], Δ 1.791 ppm 8.2E+01 2.0E+02


[+K], Δ 0.882 ppm; [+K], Δ 0.726 ppm 6.2E+02 1.7E+03


[+K], Δ 2.779 ppm 8.0E+01


PC(32:2) – N(CH3)3

[+In], Δ -0.509 ppm 1.8E+02


[+H], Δ 0.423 ppm 1.0E+02


PC(32:1) – N(CH3)3

[+In], Δ 0.455 ppm; [+In], Δ 0.532 ppm 1.6E+03 3.9E+03


PC(32:0) – N(CH3)3

[+In], Δ 1.336 ppm; [+In], Δ 1.258 ppm 1.2E+03 2.9E+03


[+K], Δ 1.281 ppm; [+K], Δ 1.051 ppm 9.7E+01 2.3E+02


[+K], Δ 1.615 ppm; [+K], Δ 1.157 ppm 2.3E+02 4.9E+02


[+K], Δ 1.613 ppm 2.1E+02


[+K], Δ 1.338 ppm; [+K], Δ 1.413 ppm 2.4E+02 7.1E+02


[+K], Δ 1.666 ppm 2.9E+02


PC(34:3) – N(CH3)3

[+In], Δ -0.161 ppm 1.3E+02


PC(34:2) – N(CH3)3

[+In], Δ 0.321 ppm; [+In], Δ 0.096 ppm 9.0E+02 2.6E+03


PC(34:1) – N(CH3)3

[+In], Δ 0.65 ppm; [+In], Δ 0.725 ppm 4.1E+03 1.0E+04


PC(34:0) – N(CH3)3

[+In], Δ 2.77 ppm; [+In], Δ 2.471 ppm 5.6E+02 1.7E+03


[+K], Δ 1.817 ppm 2.1E+02


[+In], Δ -0.202 ppm; [+In], Δ -0.569 ppm 8.4E+02 2.0E+03


[+In], Δ 0.562 ppm; [+In], Δ 0.562 ppm 1.7E+03 3.8E+03


[+K], Δ 2.057 ppm 1.2E+02


PC(36:4) – N(CH3)3

[+In], Δ 1.113 ppm 1.2E+02


PC(36:3) – N(CH3)3

[+In], Δ 2.449 ppm; [+In], Δ 2.304 ppm 9.4E+01 3.1E+02


PC(36:2) – N(CH3)3

[+In], Δ 1.023 ppm; [+In], Δ 0.95 ppm 8.1E+02 2.3E+03


[+In], Δ 1.015 ppm; [+In], Δ 0.871 ppm 3.2E+02 5.8E+02


[+In], Δ 2.841 ppm 1.4E+02

15


[+In], Δ -0.665 ppm; [+In], Δ -0.594 ppm 2.2E+02 5.4E+02


[+In], Δ 0.432 ppm; [+In], Δ 0.361 ppm 4.3E+03 1.1E+04


[+In], Δ 1.666 ppm; [+In], Δ 1.524 ppm 2.2E+03 5.4E+03


[+In], Δ -0.696 ppm 1.4E+02


[+In], Δ -0.036 ppm; [+In], Δ 0.244 ppm 3.0E+02 6.9E+02


[+In], Δ 1.04 ppm; [+In], Δ 1.04 ppm 4.7E+02 1.1E+03


PC(30:0) – N(CH3)3

[+2In-H], Δ 0.445 ppm 9.6E+01


[+In], Δ -0.587 ppm; [+In], Δ -0.173 ppm 1.1E+02 2.6E+02


[+In], Δ 0.544 ppm; [+In], Δ 0.406 ppm 6.7E+02 1.9E+03


[+In], Δ 0.428 ppm; [+In], Δ 0.021 ppm 1.6E+02 5.1E+02


PC(32:1) – N(CH3)3

[+2In-H], Δ 0.393 ppm; [+2In-H], Δ -0.082 ppm 1.6E+02 5.4E+02


PC(32:0) – N(CH3)3

[+2In-H], Δ 0.96 ppm; [+2In-H], Δ 1.028 ppm 5.1E+02 1.5E+03


[+In], Δ -0.145 ppm 1.0E+02


PC(34:2) – N(CH3)3

[+2In-H], Δ -1.105 ppm 1.1E+02


PC(34:1) – N(CH3)3



PC(34:0) – N(CH3)3



[+2In-H], Δ 0.684 ppm 1.1E+02


PC(36:1) – N(CH3)3

[+2In-H], Δ -0.1 ppm 1.0E+02




[+2In-H], Δ 2.469 ppm 1.5E+02

1) Lipids were annotated by the workflow described in the online methods. Briefly, the mean spectra of the 50 min-long imaging runs were loaded into the MZmine program and lipids were annotated via a database search (LipidSearch). As is discussed in the main text, some assigned signals, in particular (and if applicable) those of DAG and PA species are likely to (partially) represent the results of in-source decay fragmentation reactions.


16

Table S-5: Putative lipids recorded by t-MALDI-2-MSI from Vero B4 cell culture in the negative ion mode. Data refer to two separate MSI measurements conducted on different areas of the same cell culture 1)


70.000

671.4649 Diacylglycerophosphate PA(34:2) Diacylglycerophosphocholine

PC(32:1) – N(CH3)3

[-H], Δ 1.223 ppm 2.8E+02


PC(32:0) – N(CH3)3

[-H], Δ 2.434 ppm 1.5E+02


PC(34:1) – N(CH3)3

[-H], Δ 1.42 ppm 9.8E+02


[-H], Δ -0.013 ppm 3.9E+02


[-H], Δ 1.214 ppm 5.6E+02


[-H], Δ 0.301 ppm 2.8E+03


[-H], Δ 1.728 ppm 6.8E+02


[-H], Δ 0.21 ppm 1.8E+02


[-H], Δ 0.002 ppm 2.5E+02


[-H], Δ -0.044 ppm 8.9E+02


[-H], Δ -1.294 ppm 7.4E+02


[-H], Δ 0.505 ppm 4.1E+02

1)Lipids were annotated by the workflow described in the online methods. Briefly, the mean spectra of the 50 min-long imaging runs were loaded into the MZmine program and lipids were annotated via a database search (LipidSearch). As is discussed in the main text, some assigned signals, in particular (and if applicable) those of DAG and PA species are likely to (partially) represent the results of in-source decay fragmentation reactions.


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