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ARTICLES https://doi.org/10.1038/s41592-019-0536-2 Transmission-mode MALDI-2 mass spectrometry imaging of cells and tissues at subcellular resolution M. Niehaus  1,4 , J. Soltwisch  1,2,4 , M. E. Belov 3 and K. Dreisewerd  1,2 * 1 Institute for Hygiene, University of Münster, Münster, Germany. 2 Interdisciplinary Center for Clinical Research, University of Münster, Münster, Germany. 3 Spectroglyph LLC, Kennewick, WA, USA. 4 These authors contributed equally: M. Niehaus, J. Soltwisch *e-mail: [email protected] SUPPLEMENTARY INFORMATION In the format provided by the authors and unedited. NATURE METHODS | www.nature.com/naturemethods

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  • 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]

    SUPPLEMENTARY INFORMATION

    In the format provided by the authors and unedited.

    NATurE METhoDS | www.nature.com/naturemethods

    http://orcid.org/0000-0002-4876-6190http://orcid.org/0000-0002-0258-1561http://orcid.org/0000-0002-7619-808Xmailto:[email protected]://www.nature.com/naturemethods

  • 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.

  • Supplementary Figure 2

    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.

  • Supplementary Figure 3

    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.

  • Supplementary Figure 4

    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.

  • Supplementary Figure 5

    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.

  • Supplementary Figure 6

    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 on- line 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.

  • Supplementary Figure 7

    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.

  • Supplementary Figure 8

    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.

  • Supplementary Figure 9

    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

    638.6082 N-acyl-4-hydroxysphinganine Cer(40:1)

    [+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

    646.6133 N-acylsphingosine Cer(42:3) [+H], Δ -0.103 ppm; [+H], Δ 0.085 ppm 3.5E+03 4.2E+03

    648.6289 N-acylsphingosine Cer(42:2) [+H], Δ -0.064 ppm; [+H], Δ 0.124 ppm 1.2E+04 1.6E+04

    649.4802 Diacylglycerophosphate PA(32:0)

    [+H], Δ 0.094 ppm 1.9E+02

    650.6441 N-acylsphingosine Cer(42:1) [+H], Δ -0.026 ppm; [+H], Δ 1.381 ppm 2.9E+03 5.3E+03

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

    662.6085 N-acyl-4-hydroxysphinganine Cer(42:3)

    [+H], Δ -0.499 ppm 5.8E+02

    664.6237 N-acyl-4-hydroxysphinganine Cer(42:2)

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

    666.6394 N-acyl-4-hydroxysphinganine Cer(42:1)

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

    670.5171 N-acyl-4-hydroxysphinganine Cer(40:4)

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

    671.4626 Diacylglycerophosphate PA(32:0)

    [+Na], Δ 0.282 ppm 2.6E+02

    672.5326 N-acyl-4-hydroxysphinganine Cer(40:3)

    [+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

    675.4956 Diacylglycerophosphate PA(34:1) /

    Diacylglycerophosphocholine PC(32:0) – N(CH3)3

    [+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

    687.4361 Diacylglycerophosphate PA(32:0)

    [+K], Δ 0.808 ppm; [+K], Δ 0.985 ppm; [+K], Δ 1.074 ppm 6.8E+03 3.6E+03 3.5E+03

    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

    697.4782 Diacylglycerophosphate PA(34:1) /

    Diacylglycerophosphocholine PC(32:0) – N(CH3)3

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

    699.4953 Diacylglycerophosphate PA(36:3) /

    Diacylglycerophosphocholine PC(34:2) – N(CH3)3

    [+H], Δ 0.211 ppm; [+H], Δ 1.519 ppm; [+H], Δ 1.17 ppm 2.8E+03 3.1E+03 3.2E+03

    700.5284 Acylalkylglycerophosphoethanolamine PE(34:3)

    [+H], Δ -1.158 ppm 1.5E+03

    700.5637 N-acyl-4-hydroxysphinganine Cer(42:3)

    [+K], Δ 1.279 ppm 1.7E+02

    701.5113 Diacylglycerophosphate PA(36:2) /

    Diacylglycerophosphocholine PC(34:1) – N(CH3)3

    [+H], Δ 0.072 ppm; [+H], Δ 0.42 ppm; [+H], Δ 0.768 ppm 2.3E+04 2.0E+04 2.3E+04

    702.5425 Acylalkylglycerophosphoethanolamine PE(34:2)

    [+H], Δ 0.098 ppm; [+H], Δ 2.704 ppm; [+H], Δ 0.184 ppm 3.8E+03 2.5E+02 1.0E+04

    703.5257 Diacylglycerophosphate PA(36:1) /

    Diacylglycerophosphocholine PC(34:0) – N(CH3)3

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

    711.4365 Diacylglycerophosphate PA(34:2) /

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

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

    713.4516 Diacylglycerophosphate PA(34:1) /

    Diacylglycerophosphocholine PC(32:0) – N(CH3)3

    [+K], Δ 0.899 ppm; [+K], Δ 1.156 ppm; [+K], Δ 1.241 ppm 5.6E+04 4.2E+04 3.5E+04

    715.4673 Diacylglycerophosphate PA(34:0)

    [+K], Δ 0.931 ppm 1.9E+03

    716.5231 Diacylglycerophosphoethanolamine PE(34:2)

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

    718.5381 Diacylglycerophosphoethanolamine PE(34:1)

    [+H], Δ 0.068 ppm; [+H], Δ 0.153 ppm; [+H], Δ 0.068 ppm 1.1E+04 9.5E+03 1.3E+04

    720.5534 Diacylglycerophosphoethanolamine PE(34:0)

    [+H], Δ 0.018 ppm; [+H], Δ 0.781 ppm; [+H], Δ 0.95 ppm 1.1E+04 1.2E+04 8.7E+03

    723.4944 Diacylglycerophosphate PA(36:2) /

    Diacylglycerophosphocholine PC(34:1) – N(CH3)3

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

    725.5092 Diacylglycerophosphate PA(36:1) /

    Diacylglycerophosphocholine PC(34:0) – N(CH3)3

    [+Na], Δ 0.197 ppm; [+Na], Δ 1.29 ppm; [+Na], Δ 0.702 ppm 2.2E+03 2.9E+03 3.7E+03

    726.5438 Acylalkylglycerophosphoethanolamine PE(36:4)

    [+H], Δ -0.83 ppm 1.1E+03

    727.5261 Diacylglycerophosphate PA(38:3) /

    Diacylglycerophosphocholine PC(36:2) – N(CH3)3

    [+H], Δ 0.271 ppm; [+H], Δ 2.453 ppm; [+H], Δ 2.117 ppm 1.8E+03 2.5E+02 2.1E+03

    728.5588 Acylalkylglycerophosphoethanolamine PE(36:3)

    [+H], Δ 0.128 ppm; [+H], Δ 0.212 ppm; [+H], Δ 0.045 ppm 5.2E+03 1.2E+03 1.5E+04

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

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

    729.5417 Diacylglycerophosphate PA(38:2) /

    [+H], Δ 0.054 ppm; [+H], Δ 1.393 ppm; [+H], Δ 3.484 ppm 5.3E+03 9.3E+02 4.7E+03

  • 3

    Diacylglycerophosphocholine PC(36:1) – N(CH3)3

    730.5742 Acylalkylglycerophosphoethanolamine PE(36:2)

    [+H], Δ 0.162 ppm; [+H], Δ 0.914 ppm; [+H], Δ 0.413 ppm 8.0E+03 7.9E+02 2.0E+04

    732.5535 Diacylglycerophosphocholine PC(32:1)

    [+H], Δ 0.018 ppm; [+H], Δ 0.851 ppm; [+H], Δ 0.351 ppm 1.0E+03 8.1E+02 7.8E+02

    734.5692 Diacylglycerophosphocholine PC(32:0)

    [+H], Δ 0.052 ppm; [+H], Δ 0.551 ppm; [+H], Δ 0.551 ppm 1.3E+04 2.3E+04 1.2E+04

    737.4535 Diacylglycerophosphate PA(36:3) /

    Diacylglycerophosphocholine PC(34:2) – N(CH3)3

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

    739.4672 Diacylglycerophosphate PA(36:2) /

    Diacylglycerophosphocholine PC(34:1) – N(CH3)3

    [+K], Δ 0.901 ppm; [+K], Δ 1.231 ppm; [+K], Δ 1.231 ppm 4.7E+04 3.7E+04 3.6E+04

    740.4995 Acylalkylglycerophosphoethanolamine PE(34:2)

    [+K], Δ 0.265 ppm 6.3E+02

    741.4824 Diacylglycerophosphate PA(36:1) /

    Diacylglycerophosphocholine PC(34:0) – N(CH3)3

    [+K], Δ 0.933 ppm; [+K], Δ 2.167 ppm; [+K], Δ 2.085 ppm 2.7E+04 1.3E+04 1.7E+04

    742.5380 Diacylglycerophosphoethanolamine PE(36:3)

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

    744.5536 Diacylglycerophosphoethanolamine PE(36:2)

    [+H], Δ 0.1 ppm; [+H], Δ 0.346 ppm; [+H], Δ 0.182 ppm 9.7E+03 4.5E+03 1.6E+04

    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

    746.5691 Diacylglycerophosphoethanolamine PE(36:1)

    [+H], Δ 0.133 ppm; [+H], Δ 0.705 ppm; [+H], Δ 0.46 ppm 2.3E+04 1.2E+04 3.2E+04

    748.5843 Diacylglycerophosphoethanolamine PE(36:0)

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

    751.5250 Diacylglycerophosphate PA(38:2) /

    Diacylglycerophosphocholine PC(36:1) – N(CH3)3

    [+Na], Δ 0.467 ppm 1.3E+03

    752.5588 Acylalkylglycerophosphoethanolamine PE(38:5)

    [+H], Δ 0.124 ppm 1.6E+03

    754.5748 Acylalkylglycerophosphoethanolamine PE(38:4)

    [+H], Δ -0.409 ppm 1.2E+03

    756.4942 Diacylglycerophosphoethanolamine PE(34:1)

    [+K], Δ 0.96 ppm; [+K], Δ 0.153 ppm; [+K], Δ 0.476 ppm 1.3E+03 6.5E+02 1.3E+03

    756.5518 Diacylglycerophosphocholine PC(32:0)

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

    756.5901 Acylalkylglycerophosphoethanolamine PE(38:3)

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

    758.5086 Diacylglycerophosphoethanolamine PE(34:0)

    [+K], Δ 0.83 ppm; [+K], Δ 1.795 ppm; [+K], Δ 3.646 ppm 4.7E+03 2.3E+03 2.7E+03

    758.5693 Diacylglycerophosphocholine PC(34:2)

    [+H], Δ 0.131 ppm; [+H], Δ 1.82 ppm; [+H], Δ -1.478 ppm 1.3E+03 5.5E+02 2.1E+03

    758.6054 Acylalkylglycerophosphoethanolamine PE(38:2)

    [+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

    760.5848 Diacylglycerophosphocholine PC(34:1)

    [+H], Δ 0.163 ppm; [+H], Δ 0.645 ppm; [+H], Δ 0.484 ppm 1.7E+04 2.2E+04 2.4E+04

    762.5991 Diacylglycerophosphocholine PC(34:0)

    [+H], Δ 0.196 ppm; [+H], Δ 2.437 ppm; [+H], Δ 3.717 ppm 3.3E+03 7.1E+03 4.0E+03

  • 4

    764.5225 Diacylglycerophosphoethanolamine PE(38:6)

    [+H], Δ -0.048 ppm 6.1E+02

    765.4828 Diacylglycerophosphate PA(38:3) /

    Diacylglycerophosphocholine PC(36:2) – N(CH3)3

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

    766.5152 Acylalkylglycerophosphoethanolamine PE(36:3)

    [+K], Δ 0.13 ppm 1.0E+03

    767.4982 Diacylglycerophosphate PA(38:2) /

    Diacylglycerophosphocholine PC(36:1) – N(CH3)3

    [+K], Δ 0.854 ppm; [+K], Δ 1.57 ppm; [+K], Δ 1.888 ppm 1.3E+04 4.8E+03 1.1E+04

    768.5295 Acylalkylglycerophosphoethanolamine PE(36:2)

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

    768.5535 Diacylglycerophosphoethanolamine PE(38:4)

    [+H], Δ 1.049 ppm; [+H], Δ 0.732 ppm; [+H], Δ -0.857 ppm 1.2E+03 4.2E+03 4.2E+03

    768.5898 Acylalkylglycerophosphocholine PC(36:4)

    [+H], Δ 0.425 ppm 1.7E+02

    772.5250 Diacylglycerophosphocholine PC(32:0)

    [+K], Δ 0.847 ppm; [+K], Δ 1.163 ppm; [+K], Δ 1.242 ppm 1.1E+04 1.8E+04 1.1E+04

    772.5848 Diacylglycerophosphoethanolamine PE(38:2)

    [+H], Δ 0.161 ppm; [+H], Δ 1.267 ppm; [+H], Δ -0.155 ppm 3.3E+03 2.0E+02 6.3E+03

    772.6291 Hexosyl-N-acyl-4-hydroxysphinganine

    HexCer(38:1)

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

    774.6003 Diacylglycerophosphoethanolamine PE(38:1)

    [+H], Δ 0.193 ppm; [+H], Δ 0.666 ppm; [+H], Δ 0.666 ppm 5.6E+03 1.3E+03 1.0E+04

    774.6381 Acylalkylglycerophosphocholine PC(36:1)

    [+H], Δ -1.293 ppm 1.5E+02

    775.5272 Diacylglycerophosphate PA(42:7) /

    Diacylglycerophosphocholine PC(40:6) – N(CH3)3

    [+H], Δ 0.873 ppm 4.5E+02 6.1E+02

    780.6345 Hexosyl-N-acylsphingosine HexCer(40:3)

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

    782.5095 Diacylglycerophosphoethanolamine PE(36:2)

    [+K], Δ 0.804 ppm; [+K], Δ 1.116 ppm; [+K], Δ 0.882 ppm 1.3E+03 5.6E+02 1.7E+03

    782.5671 Diacylglycerophosphocholine PC(34:1)

    [+Na], Δ 0.401 ppm; [+Na], Δ 0.869 ppm; [+Na], Δ 0.635 ppm 2.2E+03 8.2E+03 4.7E+03

    782.6501 Hexosyl-N-acylsphingosine HexCer(40:2)

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

    784.5249 Diacylglycerophosphoethanolamine PE(36:1)

    [+K], Δ 0.834 ppm; [+K], Δ 1.301 ppm; [+K], Δ 1.379 ppm 7.5E+03 2.8E+03 7.2E+03

    784.5828 Diacylglycerophosphocholine PC(34:0)

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

    784.6654 Hexosyl-N-acylsphingosine HexCer(40:1)

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

    786.5408 Diacylglycerophosphoethanolamine PE(36:0)

    [+K], Δ 0.864 ppm 1.0E+03

    786.6003 Diacylglycerophosphocholine PC(36:2)

    [+H], Δ 0.267 ppm; [+H], Δ 1.742 ppm; [+H], Δ -0.276 ppm 9.3E+02 1.3E+02 2.2E+03

    788.5435 Diacylglycerophosphoserine PS(36:2)

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

    788.6159 Diacylglycerophosphocholine PC(36:1)

    [+H], Δ 0.144 ppm; [+H], Δ 0.608 ppm; [+H], Δ 1.073 ppm 5.6E+03 2.0E+03 1.2E+04

    789.4832 Diacylglycerophosphate PA(40:5) /

    Diacylglycerophosphocholine PC(38:4) – N(CH3)3

    [+K], Δ 0.644 ppm 1.3E+02

  • 5

    790.5590 Diacylglycerophosphoserine PS(36:1)

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

    792.5536 Diacylglycerophosphoethanolamine PE(40:6)

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

    796.5621 Acylalkylglycerophosphoethanolamine PE(38:2)

    [+K], Δ 0.143 ppm 2.2E+02

    796.6283 Hexosyl-N-acyl-4-hydroxysphinganine

    HexCer(40:3)

    [+H] , Δ 2.463 ppm 5.7E+02

    798.5405 Diacylglycerophosphocholine PC(34:1)

    [+K], Δ 0.851 ppm; [+K], Δ 1.386 ppm; [+K], Δ 1.386 ppm 1.2E+04 1.6E+04 1.5E+04

    798.6451 Hexosyl-N-acyl-4-hydroxysphinganine

    HexCer(40:2)

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

    800.5549 Diacylglycerophosphocholine PC(34:0)

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

    800.6607 Hexosyl-N-acyl-4-hydroxysphinganine

    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

    802.6320 Diacylglycerophosphoethanolamine PE(40:1)

    [+H], Δ 0.096 ppm 7.1E+02

    806.5099 Diacylglycerophosphoethanolamine PE(38:4)

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

    806.6496 Hexosyl-N-acylsphingosine HexCer(40:1)

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

    808.6658 Hexosyl-N-acylsphingosine HexCer(42:3)

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

    810.5416 Diacylglycerophosphoethanolamine PE(38:2)

    [+K], Δ -0.065 ppm 4.3E+02

    810.5984 Diacylglycerophosphocholine PC(36:1)

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

    810.6814 Hexosyl-N-acylsphingosine HexCer(42:2)

    [+H] , Δ 0.881 ppm; [+H] , Δ 1.182 ppm; [+H] , Δ 1.258 ppm 3.7E+04 4.2E+02 6.5E+04

    812.5551 Diacylglycerophosphoethanolamine PE(38:1)

    [+K], Δ 0.867 ppm; [+K], Δ 1.693 ppm; [+K], Δ 4.998 ppm 9.5E+02 1.2E+02 1.1E+03

    812.6963 Hexosyl-N-acylsphingosine HexCer(42:1)

    [+H] , Δ 0.835 ppm; [+H] , Δ 2.337 ppm; [+H] , Δ 2.938 ppm 1.5E+04 2.5E+02 3.6E+04

    813.4828 Diacylglycerophosphate PA(42:7) /

    Diacylglycerophosphocholine PC(40:6) – N(CH3)3

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

    816.5746 Diacylglycerophosphoserine PS(38:2)

    [+H], Δ 0.329 ppm 3.0E+02

    818.5902 Diacylglycerophosphoserine PS(38:1)

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

    822.6219 Hexosyl-N-acylsphingosine HexCer(40:1)

    [+K], Δ 0.776 ppm 7.2E+02

    822.6436 Hexosyl-N-acyl-4-hydroxysphinganine

    HexCer(40:1)

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

    824.6604 Hexosyl-N-acyl-4-hydroxysphinganine

    HexCer(42:3)

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

    826.5719 Diacylglycerophosphocholine PC(36:1)

    [+K], Δ 0.809 ppm; [+K], Δ 1.326 ppm; [+K], Δ 1.326 ppm 2.4E+03 1.1E+03 3.8E+03

    826.6763 Hexosyl-N-acyl-4-hydroxysphinganine

    HexCer(42:2)

    [+H] , Δ 0.841 ppm; [+H] , Δ 1.357 ppm; [+H] , Δ 1.136 ppm 2.9E+04 2.9E+02 5.0E+04

  • 6

    828.5147 Diacylglycerophosphoserine PS(36:1)

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

    828.6919 Hexosyl-N-acyl-4-hydroxysphinganine

    HexCer(42:1)

    [+H] , Δ 0.869 ppm; [+H] , Δ 1.237 ppm; [+H] , Δ 1.311 ppm 5.5E+04 8.8E+02 1.0E+05

    830.5099 Diacylglycerophosphoethanolamine PE(40:6)

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

    832.6622 Hexosyl-N-acylsphingosine HexCer(42:2)

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

    834.6783 Hexosyl-N-acylsphingosine HexCer(42:1)

    [+Na], Δ 1.849 ppm 1.4E+03

    836.5433 Diacylglycerophosphoserine PS(40:6)

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

    844.6805 Diacylglycerophosphocholine PC(40:1)

    [+H], Δ -1.77 ppm 2.6E+03

    846.6221 Diacylglycerophosphoserine PS(40:1)

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

    848.6368 Diacylglycerophosphoserine PS(40:0)

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

    850.6522 Hexosyl-N-acylsphingosine HexCer(42:1)

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

    850.6746 Hexosyl-N-acyl-4-hydroxysphinganine

    HexCer(42:1)

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

    862.6172 Hexosyl-N-acyl-4-hydroxysphinganine

    HexCer(42:3)

    [+K], Δ 0.222 ppm 1.2E+02

    864.6323 Hexosyl-N-acyl-4-hydroxysphinganine

    HexCer(42:2)

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

    866.6476 Hexosyl-N-acyl-4-hydroxysphinganine

    HexCer(42:1)

    [+K], Δ 0.913 ppm; [+K], Δ 1.547 ppm; [+K], Δ 1.547 ppm 1.9E+04 2.6E+02 2.2E+04

    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

    634.5791 N-acyl-4-hydroxysphinganine Cer(40:2)

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

  • 7

    636.5932 N-acyl-4-hydroxysphinganine Cer(40:1)

    [-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

    664.6246 N-acyl-4-hydroxysphinganine Cer(42:1)

    [-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

    699.4967 Diacylglycerophosphate PA(36:2) / Diacylglycerophosphocholine

    PC(34:1) – N(CH3)3

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

    701.5119 Diacylglycerophosphate PA(36:1) / Diacylglycerophosphocholine

    PC(34:0) – N(CH3)3

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

    714.5089 Diacylglycerophosphoethanolamine PE(34:2)

    [-H], Δ -1.38 ppm 5.0E+02

    716.5236 Diacylglycerophosphoethanolamine PE(34:1)

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

    718.5389 Diacylglycerophosphoethanolamine PE(34:0)

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

    723.4958 Diacylglycerophosphate PA(38:4) / Diacylglycerophosphocholine

    PC(36:3) – N(CH3)3

    [-H], Δ 1.626 ppm 2.6E+02

    727.5277 Diacylglycerophosphate PA(38:2) / Diacylglycerophosphocholine

    PC(36:1) – N(CH3)3

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

    740.5240 Diacylglycerophosphoethanolamine PE(36:3)

    [-H], Δ -0.556 ppm 7.3E+02

    741.4708 Diacylglycerophosphoglycerols PG(34:4)

    [-H], Δ 0.512 ppm 1.1E+03

    742.5391 Diacylglycerophosphoethanolamine PE(36:2)

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

    744.5546 Diacylglycerophosphoethanolamine PE(36:1)

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

    746.5709 Diacylglycerophosphoethanolamine PE(36:0)

    [-H], Δ -0.451 ppm 6.3E+02

    747.5170 Diacylglycerophosphoglycerols PG(34:1)

    [-H], Δ 1.589 ppm 1.3E+03

    760.5134 Diacylglycerophosphoserine PS(34:1)

    [-H], Δ 0.049 ppm 1.3E+03

    762.5061 Diacylglycerophosphoethanolamine PE(38:6)

    [-H], Δ 2.389 ppm 1.4E+03

    764.5214 Diacylglycerophosphoethanolamine PE(38:5)

    [-H], Δ 2.894 ppm 2.5E+02

    768.5538 Diacylglycerophosphoethanolamine PE(38:3)

    [-H], Δ 1.435 ppm 3.4E+02

    770.5706 Diacylglycerophosphoethanolamine PE(38:2)

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

    772.5858 Diacylglycerophosphoethanolamine PE(38:1)

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

    786.5289 Diacylglycerophosphoserine PS(36:2)

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

    788.5443 Diacylglycerophosphoserine PS(36:1)

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

    790.5388 Diacylglycerophosphoethanolamine PE(40:6)

    [-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

    798.6463 Hexosyl-N-acyl-4-hydroxysphinganine HexCer(40:1)

    [-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

    808.6668 Hexosyl-N-acylsphingosine HexCer(42:2)

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

    810.6813 Hexosyl-N-acylsphingosine HexCer(42:1)

    [-H], Δ 1.182 ppm 1.9E+03

    816.5758 Diacylglycerophosphoserine PS(38:1)

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

    822.6455 Hexosyl-N-acyl-4-hydroxysphinganine HexCer(42:3)

    [-H], Δ 0.487 ppm 9.9E+02

    824.6613 Hexosyl-N-acyl-4-hydroxysphinganine HexCer(42:2)

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

    826.6773 Hexosyl-N-acyl-4-hydroxysphinganine HexCer(42:1)

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

    834.5280 Diacylglycerophosphoserine PS(40:6)

    [-H], Δ 1.245 ppm 4.2E+03

    844.6071 Diacylglycerophosphoserine PS(40:1)

    [-H], Δ 0.223 ppm 1.5E+03

    862.6088 3-O-sulfogalactosy-N-acylsphingosine SHexCer(40:1)

    [-H], Δ -1.108 ppm 7.4E+02

    872.6396 Diacylglycerophosphoserine PS(42:1)

    [-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

    888.6242 3-O-sulfogalactosy-N-acylsphingosine SHexCer(42:2)

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

    890.6393 3-O-sulfogalactosy-N-acylsphingosine SHexCer(42:1)

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

    904.6202 3-O-sulfogalactosy-N-acyl-4-hydroxysphinganine SHexCer(42:2)

    [-H], Δ -1.995 ppm 3.3E+02

    906.6346 3-O-sulfogalactosy-N-acyl-4-hydroxysphinganine SHexCer(42:1)

    [-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

    692.5221 Diacylglycerophosphoethanolamine PE(32:0)

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

    702.5429 Acylalkylglycerophosphoethanolamine PE(34:2)

    [+H], Δ 0.532 ppm 5,6E+02

    715.4551 Diacylglycerophosphoglycerols PG(32:4)

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

    716.5223 Diacylglycerophosphoethanolamine PE(34:2)

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

    718.5378 Diacylglycerophosphoethanolamine PE(34:1)

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

    720.5535 Diacylglycerophosphoethanolamine PE(34:0)

    [+H], Δ 0.272 ppm; [+H], Δ 0.442 ppm; [+H], Δ 0.526 ppm 2,7E+03 1,8E+03 2,1E+03

    724.5283 Acylalkylglycerophosphoethanolamine PE(36:5)

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

    728.5594 Acylalkylglycerophosphoethanolamine PE(36:3)

    [+H], Δ -0.709 ppm 2,3E+02

    732.5538 Diacylglycerophosphocholine PC(32:1)

    [+H], Δ 0.018 ppm 1,7E+02

    734.5692 Diacylglycerophosphocholine PC(32:0)

    [+H], Δ 0.384 ppm; [+H], Δ 0.384 ppm; [+H], Δ 0.301 ppm 7,4E+03 8,1E+03 8,9E+03

    738.5079 Diacylglycerophosphoethanolamine PE(36:5)

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

    740.5223 Diacylglycerophosphoethanolamine PE(36:4)

    [+H], Δ 0.445 ppm; [+H], Δ 0.28 ppm; [+H], Δ 0.197 ppm 8,1E+03 4,9E+03 1,4E+04

    742.5356 Diacylglycerophosphoethanolamine PE(34:0)

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

    744.5532 Diacylglycerophosphoethanolamine PE(36:2)

    [+H], Δ 0.919 ppm; [+H], Δ 0.919 ppm; [+H], Δ 0.509 ppm 3,4E+03 2,4E+03 9,3E+03

    746.5118 Acylalkylglycerophosphoethanolamine PE(38:8)

    [+H], Δ 0.188 ppm 2,8E+02

    746.5681 Diacylglycerophosphoethanolamine PE(36:1)

    [+H], Δ 2.831 ppm; [+H], Δ 1.604 ppm; [+H], Δ 1.114 ppm 5,5E+02 7,6E+02 5,4E+03

    746.6043 Acylalkylglycerophosphocholine PC(34:1)

    [+H], Δ 2.025 ppm 2,6E+02

    748.5259 Acylalkylglycerophosphoethanolamine PE(36:4)

    [+Na], Δ -0.226 ppm 9,6E+02

    748.5842 Diacylglycerophosphoethanolamine PE(36:0)

    [+H], Δ 1.144 ppm 1,1E+02

    749.5322 Diacylglycerophosphoglycerols PG(34:1)

    [+H], Δ 0.652 ppm 1,6E+02

  • 10

    750.5440 Acylalkylglycerophosphoethanolamine PE(38:6)

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

    752.5588 Acylalkylglycerophosphoethanolamine PE(38:5)

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

    754.5371 Diacylglycerophosphocholine PC(32:1)

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

    756.5513 Diacylglycerophosphocholine PC(32:0)

    [+Na], Δ 0.866 ppm; [+Na], Δ 0.946 ppm; [+Na], Δ 0.785 ppm 2,8E+03 8,0E+03 5,4E+03

    758.5100 Diacylglycerophosphoethanolamine PE(34:0)

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

    758.5682 Diacylglycerophosphocholine PC(34:2)

    [+H], Δ 1.418 ppm; [+H], Δ 1.016 ppm; [+H], Δ 2.384 ppm 9,1E+02 3,5E+03 1,4E+03

    760.5844 Diacylglycerophosphocholine PC(34:1)

    [+H], Δ 0.966 ppm; [+H], Δ 0.645 ppm; [+H], Δ 0.966 ppm 1,9E+03 1,1E+04 1,5E+03

    762.4840 Acylalkylglycerophosphoethanolamine PE(36:5)

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

    762.5048 Diacylglycerophosphoethanolamine PE(36:4)

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

    762.5986 Diacylglycerophosphocholine PC(34:0)

    [+H], Δ 2.837 ppm 1,9E+02

    764.5221 Diacylglycerophosphoethanolamine PE(38:6)

    [+H], Δ 0.83 ppm; [+H], Δ 0.351 ppm; [+H], Δ 0.51 ppm 7,1E+03 3,1E+03 4,2E+03

    766.5373 Diacylglycerophosphoethanolamine PE(36:2)

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

    768.5535 Diacylglycerophosphoethanolamine PE(38:4)

    [+H], Δ 0.414 ppm; [+H], Δ 0.494 ppm; [+H], Δ 0.414 ppm 3,3E+04 2,3E+04 5,6E+04

    768.5870 Acylalkylglycerophosphocholine PC(34:1)

    [+Na], Δ 1.737 ppm 4,7E+02

    772.5252 Diacylglycerophosphocholine PC(32:0)

    [+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)

    [+Na], Δ 0.808 ppm; [+Na], Δ 0.334 ppm; [+Na], Δ 0.65 ppm 5,3E+03 3,6E+03 3,3E+03

    772.5843 Diacylglycerophosphoethanolamine PE(38:2)

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

    774.5430 Acylalkylglycerophosphoethanolamine PE(40:8)

    [+H], Δ 0.325 ppm 4,2E+03

    774.5430 AND/OR Acylalkylglycerophosphoetha

    nolamine PE(38:5)

    [+Na], Δ -2.077 ppm 4,2E+03

    774.5997 Diacylglycerophosphoethanolamine PE(38:1)

    [+H], Δ 1.375 ppm 5,8E+02

    776.5582 Acylalkylglycerophosphoethanolamine PE(40:7)

    [+H], Δ 0.356 ppm; [+H], Δ 0.671 ppm; [+H], Δ 1.614 ppm 6,2E+02 2,3E+02 2,0E+03

    776.5582 AND/OR Acylalkylglycerophosphoetha

    nolamine PE(38:4)

    [+Na], Δ -1.725 ppm; [+Na], Δ -2.04 ppm; [+Na], Δ -0.782 ppm 2,3E+02 6,2E+02 2,0E+03

    778.4781 Diacylglycerophosphoethanolamine PE(36:4)

    [+K], Δ 0.979 ppm; [+K], Δ 1.058 ppm; [+K], Δ 0.979 ppm 5,8E+02 1,1E+03 8,2E+02

    778.5727 Acylalkylglycerophosphoethanolamine PE(40:6)

    [+H], Δ 2.347 ppm 2,4E+02

    778.5727 AND/OR Acylalkylglycerophosphoetha

    nolamine PE(38:3)

    [+Na], Δ -0.042 ppm 2,4E+02

    780.5520 Diacylglycerophosphocholine PC(34:2)

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

    782.5093 Diacylglycerophosphoethanolamine PE(36:2)

    [+K], Δ 1.896 ppm; [+K], Δ 1.74 ppm; [+K], Δ 0.024 ppm 2,7E+02 2,4E+02 3,8E+02

  • 11

    782.5679 Diacylglycerophosphocholine PC(34:1)

    [+Na], Δ -0.691 ppm; [+Na], Δ -0.613 ppm; [+Na], Δ -0.067 ppm 2,8E+03 5,2E+03 1,4E+04

    784.5139 Diacylglycerophosphoserine PS(36:4)

    [+H], Δ -2.042 ppm 2,5E+02

    784.5815 Diacylglycerophosphocholine PC(34:0)

    [+Na], Δ 1.754 ppm; [+Na], Δ 2.61 ppm; [+Na], Δ 2.221 ppm 2,0E+02 6,5E+02 1,3E+03

    786.5043 Diacylglycerophosphoethanolamine PE(38:6)

    [+Na], Δ 0.815 ppm 1,6E+02

    786.5998 Diacylglycerophosphocholine PC(36:2)

    [+H], Δ 1.121 ppm; [+H], Δ 0.811 ppm; [+H], Δ 1.587 ppm 3,7E+02 4,2E+03 3,5E+02

    788.5196 Diacylglycerophosphoethanolamine PE(38:5)

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

    788.5203 Diacylglycerophosphoethanolamine PE(40:8)

    [+H], Δ 2.74 ppm 7,6E+02

    788.6152 Diacylglycerophosphocholine PC(36:1)

    [+H], Δ 1.537 ppm 1,7E+03

    790.5359 Diacylglycerophosphoethanolamine PE(38:4)

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

    792.5539 Diacylglycerophosphoethanolamine PE(40:6)

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

    792.5918 Acylalkylglycerophosphocholine PC(38:6)

    [+H], Δ -2.052 ppm 5,1E+02

    794.5685 Diacylglycerophosphoethanolamine PE(38:2)

    [+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

    796.5237 Acylalkylglycerophosphoethanolamine PE(40:8)

    [+Na], Δ 1.856 ppm; [+Na], Δ 2.929 ppm; [+Na], Δ 2.699 ppm 3,6E+02 2,6E+02 7,3E+02

    796.5239 AND/OR Diacylglycerophosphocholine

    PC(34:2)

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

    796.5845 Diacylglycerophosphoethanolamine PE(40:4)

    [+H], Δ 0.003 ppm; [+H], Δ 0.999 ppm; [+H], Δ 1.152 ppm 6,1E+02 4,5E+02 3,1E+03

    798.5391 Diacylglycerophosphocholine PC(34:1)

    [+K], Δ 2.991 ppm 1,1E+03

    800.4614 Diacylglycerophosphoethanolamine PE(38:7)

    [+K], Δ 2.37 ppm 9,8E+01

    802.4785 Diacylglycerophosphoethanolamine PE(38:6)

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

    804.4939 Diacylglycerophosphoethanolamine PE(38:5)

    [+K], Δ 0.751 ppm; [+K], Δ 1.206 ppm; [+K], Δ 0.448 ppm 8,2E+02 9,5E+02 2,5E+03

    804.5515 Diacylglycerophosphocholine PC(36:4)

    [+Na], Δ 0.359 ppm; [+Na], Δ 0.662 ppm; [+Na], Δ 0.511 ppm 4,5E+02 2,8E+03 3,1E+03

    806.5093 Diacylglycerophosphoethanolamine PE(38:4)

    [+K], Δ 1.159 ppm; [+K], Δ 0.78 ppm; [+K], Δ 1.31 ppm 5,2E+03 5,5E+03 6,3E+03

    806.5684 Diacylglycerophosphocholine PC(38:6)

    [+H], Δ 0.955 ppm; [+H], Δ 1.561 ppm; [+H], Δ 1.258 ppm 1,5E+03 3,9E+03 3,6E+03

    806.5684 AND/OR Diacylglycerophosphocholine

    PC(36:3)

    [+Na], Δ -1.351 ppm; [+Na], Δ -1.049 ppm; [+Na], Δ -0.746 ppm 1,5E+03 3,6E+03 3,9E+03

    808.5138 Diacylglycerophosphoserine PS(38:6)

    [+H], Δ -1.83 ppm 6,5E+02

    808.5829 Diacylglycerophosphocholine PC(36:2)

    [+Na], Δ 0.193 ppm; [+Na], Δ 0.796 ppm; [+Na], Δ 0.268 ppm 6,3E+02 1,1E+03 5,6E+03

    810.5274 Diacylglycerophosphoserine PS(38:5)

    [+H], Δ 0.69 ppm 9,9E+01

  • 12

    810.5274 Diacylglycerophosphoserine PS(36:2)

    [+Na], Δ -1.605 ppm 9,9E+01

    810.5996 Diacylglycerophosphocholine PC(36:1)

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

    812.5436 Diacylglycerophosphoserine PS(36:1)

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

    812.5439 Diacylglycerophosphoserine PS(38:4)

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

    820.5249 Diacylglycerophosphocholine PC(36:4)

    [+K], Δ 1.095 ppm; [+K], Δ 1.393 ppm; [+K], Δ 1.021 ppm 4,6E+02 7,4E+02 5,7E+02

    824.5554 Diacylglycerophosphocholine PC(36:2)

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

    826.6102 Acylalkylglycerophosphoethanolamine PE(40:1)

    [+K], Δ -1.175 ppm 3,8E+02

    828.4928 Diacylglycerophosphoethanolamine PE(40:7)

    [+K], Δ 1.54 ppm; [+K], Δ 2.203 ppm; [+K], Δ 2.497 ppm 3,0E+02 1,6E+03 4,2E+02

    828.5509 Diacylglycerophosphocholine PC(38:6)

    [+Na], Δ 1.38 ppm; [+Na], Δ 1.011 ppm; [+Na], Δ 1.453 ppm 2,0E+02 2,3E+03 6,6E+02

    830.5109 Diacylglycerophosphoethanolamine PE(40:6)

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

    830.5656 Diacylglycerophosphocholine PC(38:5)

    [+Na], Δ 2.362 ppm 4,4E+02

    832.5823 Diacylglycerophosphocholine PC(38:4)

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

    834.5290 Diacylglycerophosphoserine PS(40:7)

    [+H], Δ -1.231 ppm 1,6E+02

    834.5972 Diacylglycerophosphocholine PC(38:3)

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

    836.5430 Diacylglycerophosphoserine PS(40:6)

    [+H], Δ 0.699 ppm 2,9E+02

    844.5256 Diacylglycerophosphocholine PC(38:6)

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

    846.4670 Diacylglycerophosphoserine PS(38:6)

    [+K], Δ 2.008 ppm 2,0E+02

    848.5562 Diacylglycerophosphocholine PC(38:4)

    [+K], Δ 1.406 ppm; [+K], Δ 1.118 ppm; [+K], Δ 0.974 ppm 2,7E+02 1,9E+02 9,6E+02

    850.4993 Diacylglycerophosphoserine PS(38:4)

    [+K], Δ 0.981 ppm; [+K], Δ 0.407 ppm; [+K], Δ 1.412 ppm 1,7E+02 2,2E+02 2,9E+02

    887.5623 Diacylglycerophosphoinositol PI(38:4)

    [+H], Δ 2.014 ppm; [+H], Δ 2.495 ppm; [+H], Δ 2.564 ppm 6,9E+02 5,1E+02 1,7E+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 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)

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

    70.000 70.000

    685.4205 Diacylglycerophosphate PA(32:1) / Diacylglycerophosphocholine

    PC(30:0) – N(CH3)3

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

    709.4230 Diacylglycerophosphate PA(34:3) / Diacylglycerophosphocholine

    PC(32:2) – N(CH3)3

    [+K], Δ -2.694 ppm 1.4E+02

    711.4364 Diacylglycerophosphate PA(34:2) / Diacylglycerophosphocholine

    PC(32:1) – N(CH3)3

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

    713.4515 Diacylglycerophosphate PA(34:1) / Diacylglycerophosphocholine

    PC(32:0) – N(CH3)3

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

    716.5229 Diacylglycerophosphoethanolamine PE(34:2)

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

    718.5379 Diacylglycerophosphoethanolamine PE(34:1)

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

    732.5537 Diacylglycerophosphocholine PC(32:1)

    [+H], Δ 0.101 ppm 1.1E+02

    737.4529 Diacylglycerophosphate PA(36:3) / Diacylglycerophosphocholine

    PC(34:2) – N(CH3)3

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

    739.4674 Diacylglycerophosphate PA(36:2) / Diacylglycerophosphocholine

    PC(34:1) – N(CH3)3

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

    741.4814 Diacylglycerophosphate PA(36:1) / Diacylglycerophosphocholine

    PC(34:0) – N(CH3)3

    [+K], Δ 2.99 ppm 1.7E+02

    742.5375 Diacylglycerophosphoethanolamine PE(34:0)

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

    744.5537 Diacylglycerophosphoethanolamine PE(36:2)

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

    746.5683 Diacylglycerophosphoethanolamine PE(36:1)

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

    754.4785 Diacylglycerophosphoethanolamine PE(34:2)

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

    756.4933 Diacylglycerophosphoethanolamine PE(34:1)

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

    758.5695 Diacylglycerophosphocholine PC(34:2)

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

    759.3460 Diacylglycerophosphate PA(32:2) / Diacylglycerophosphocholine

    PC(30:1) – N(CH3)3

    [+In], Δ -0.559 ppm 1.3E+02

    760.5849 Diacylglycerophosphocholine PC(34:1)

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

    761.3609 Diacylglycerophosphate PA(32:1) / Diacylglycerophosphocholine

    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

    765.4829 Diacylglycerophosphate PA(38:3) / Diacylglycerophosphocholine

    PC(36:2) – N(CH3)3

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

    770.5096 Diacylglycerophosphocholine PC(32:1)

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

  • 14

    772.5847 Diacylglycerophosphoethanolamine PE(38:2)

    [+H], Δ 0.477 ppm 2.0E+02

    780.4932 Diacylglycerophosphoethanolamine PE(36:3)

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

    782.5096 Diacylglycerophosphoethanolamine PE(36:2)

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

    784.5237 Diacylglycerophosphoethanolamine PE(36:1)

    [+K], Δ 2.779 ppm 8.0E+01

    785.3616 Diacylglycerophosphate PA(34:3) / Diacylglycerophosphocholine

    PC(32:2) – N(CH3)3

    [+In], Δ -0.509 ppm 1.8E+02

    786.6004 Diacylglycerophosphocholine PC(36:2)

    [+H], Δ 0.423 ppm 1.0E+02

    787.3765 Diacylglycerophosphate PA(34:2) / Diacylglycerophosphocholine

    PC(32:1) – N(CH3)3

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

    789.3915 Diacylglycerophosphate PA(34:1) / Diacylglycerophosphocholine

    PC(32:0) – N(CH3)3

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

    796.5249 Diacylglycerophosphocholine PC(34:2)

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

    798.5404 Diacylglycerophosphocholine PC(34:1)

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

    806.5089 Diacylglycerophosphoethanolamine PE(38:4)

    [+K], Δ 1.613 ppm 2.1E+02

    808.5248 Diacylglycerophosphoethanolamine PE(38:3)

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

    810.5402 Diacylglycerophosphoethanolamine PE(38:2)

    [+K], Δ 1.666 ppm 2.9E+02

    811.3770 Diacylglycerophosphate PA(36:4) / Diacylglycerophosphocholine

    PC(34:3) – N(CH3)3

    [+In], Δ -0.161 ppm 1.3E+02

    813.3924 Diacylglycerophosphate PA(36:3) / Diacylglycerophosphocholine

    PC(34:2) – N(CH3)3

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

    815.4076 Diacylglycerophosphate PA(36:2) / Diacylglycerophosphocholine

    PC(34:1) – N(CH3)3

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

    817.4217 Diacylglycerophosphate PA(36:1) / Diacylglycerophosphocholine

    PC(34:0) – N(CH3)3

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

    824.5557 Diacylglycerophosphocholine PC(36:2)

    [+K], Δ 1.817 ppm 2.1E+02

    830.4194 Diacylglycerophosphoethanolamine PE(34:2)

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

    832.4343 Diacylglycerophosphoethanolamine PE(34:1)

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

    834.5398 Diacylglycerophosphoethanolamine PE(40:4)

    [+K], Δ 2.057 ppm 1.2E+02

    837.3916 Diacylglycerophosphate PA(38:5) / Diacylglycerophosphocholine

    PC(36:4) – N(CH3)3

    [+In], Δ 1.113 ppm 1.2E+02

    839.4062 Diacylglycerophosphate PA(38:4) / Diacylglycerophosphocholine

    PC(36:3) – N(CH3)3

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

    841.4230 Diacylglycerophosphate PA(38:3) / Diacylglycerophosphocholine

    PC(36:2) – N(CH3)3

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

    846.4496 Diacylglycerophosphocholine PC(32:1)

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

    848.4636 Diacylglycerophosphocholine PC(32:0)

    [+In], Δ 2.841 ppm 1.4E+02

  • 15

    856.4353 Diacylglycerophosphoethanolamine PE(36:3)

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

    858.4500 Diacylglycerophosphoethanolamine PE(36:2)

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

    860.4647 Diacylglycerophosphoethanolamine PE(36:1)

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

    870.4510 Diacylglycerophosphocholine PC(34:3)

    [+In], Δ -0.696 ppm 1.4E+02

    872.4659 Diacylglycerophosphocholine PC(34:2)

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

    874.4808 Diacylglycerophosphocholine PC(34:1)

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

    875.2569 Diacylglycerophosphate PA(32:1) / Diacylglycerophosphocholine

    PC(30:0) – N(CH3)3

    [+2In-H], Δ 0.445 ppm 9.6E+01

    884.4664 Diacylglycerophosphoethanolamine PE(38:3)

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

    886.4813 Diacylglycerophosphoethanolamine PE(38:2)

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

    900.4971 Diacylglycerophosphocholine PC(36:2)

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

    901.2728 Diacylglycerophosphate PA(34:2) / Diacylglycerophosphocholine

    PC(32:1) – N(CH3)3

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

    903.2877 Diacylglycerophosphate PA(34:1) / Diacylglycerophosphocholine

    PC(32:0) – N(CH3)3

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

    904.4560 Diacylglycerophosphoserine PS(36:1)

    [+In], Δ -0.145 ppm 1.0E+02

    927.2896 Diacylglycerophosphate PA(36:3) / Diacylglycerophosphocholine

    PC(34:2) – N(CH3)3

    [+2In-H], Δ -1.105 ppm 1.1E+02

    929.3039 Diacylglycerophosphate PA(36:2) / Diacylglycerophosphocholine

    PC(34:1) – N(CH3)3

    [+2In-H], Δ 0.5 ppm; [+2In-H], Δ 0.303 ppm 1.0E+03 3.3E+03

    931.3188 Diacylglycerophosphate PA(36:1) / Diacylglycerophosphocholine

    PC(34:0) – N(CH3)3

    [+2In-H], Δ 1.05 ppm; [+2In-H], Δ 1.378 ppm 5.7E+02 2.2E+03

    946.3301 Diacylglycerophosphoethanolamine PE(34:1)

    [+2In-H], Δ 0.684 ppm 1.1E+02

    957.3356 Diacylglycerophosphate PA(38:2) / Diacylglycerophosphocholine

    PC(36:1) – N(CH3)3

    [+2In-H], Δ -0.1 ppm 1.0E+02

    972.3462 Diacylglycerophosphoethanolamine PE(36:2)

    [+2In-H], Δ 0.314 ppm; [+2In-H], Δ 0.189 ppm 3.4E+02 1.0E+03

    974.3597 Diacylglycerophosphoethanolamine PE(36:1)

    [+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.

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

  • 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)

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

    70.000

    671.4649 Diacylglycerophosphate PA(34:2) Diacylglycerophosphocholine

    PC(32:1) – N(CH3)3

    [-H], Δ 1.223 ppm 2.8E+02

    673.4797 Diacylglycerophosphate PA(34:1) Diacylglycerophosphocholine

    PC(32:0) – N(CH3)3

    [-H], Δ 2.434 ppm 1.5E+02

    699.4960 Diacylglycerophosphate PA(36:2) Diacylglycerophosphocholine

    PC(34:1) – N(CH3)3

    [-H], Δ 1.42 ppm 9.8E+02

    714.5079 Diacylglycerophosphoethanolamine PE(34:2)

    [-H], Δ -0.013 ppm 3.9E+02

    716.5227 Diacylglycerophosphoethanolamine PE(34:1)

    [-H], Δ 1.214 ppm 5.6E+02

    742.5390 Diacylglycerophosphoethanolamine PE(36:2)

    [-H], Δ 0.301 ppm 2.8E+03

    744.5536 Diacylglycerophosphoethanolamine PE(36:1)

    [-H], Δ 1.728 ppm 6.8E+02

    760.5132 Diacylglycerophosphoserine PS(34:1)

    [-H], Δ 0.21 ppm 1.8E+02

    786.5291 Diacylglycerophosphoserine PS(36:2)

    [-H], Δ 0.002 ppm 2.5E+02

    788.5447 Diacylglycerophosphoserine PS(36:1)

    [-H], Δ -0.044 ppm 8.9E+02

    861.5510 Diacylglycerophosphoinositol PI(36:2)

    [-H], Δ -1.294 ppm 7.4E+02

    863.5651 Diacylglycerophosphoinositol PI(36:1)

    [-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.

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

    SpringerNature_NatMeth_536_ESM.pdfESM1_ISIESM1_to_merge