Timstof flex

Tentative identification of the m/z features was carried out by MS/MS measurement on whole-mount breast cancer tissue sections. The measurements were performed in situ using a Rapiflex in LIFT mode (m/z 772.4 and 1198.7) and a timsTOFflex (m/z 1428.7 and 1495.7) mass spectrometer (Bruker Daltonics). Laser power for fragmentation was set at 70–80% in positive ionization mode, 2000 shots at a laser frequency of 5 kHz, and a beam scan of 25 µm². For the identification, the MS/MS spectra (Figures S1 and S2) were submitted to MASCOT MS/MS Ion Search, where the SwissProt database was searched to match tryptic peptide sequences to the respective intact proteins, defining homo sapiens as the taxonomic class. The MS/MS spectrum search parameters included a mass tolerance of 1 Da, MS/MS tolerance of ±1 Da, up to two missed cleavages, methionine oxidation, protein N-terminal acetylation, and proline oxidation as variable modifications.

MALDI-IMS-MS negative ion mode data were acquired on a timsTOF fleX (Bruker Daltonics GmbH & Co. KG) using timsControl 4.1 and flexImaging 7.2. The imaging run was exported from flexImaging 7.2 via “Save run file as…” to make the geometry files available for MS² measurements.
R2c_1a-d: Imaging data were acquired with a laser field size of 30 µm. The imaging raster size was 50 µm to keep material for subsequent MS² acquisition (see Supplementary Fig. S13a). Resulting in a final image resolution of 50 µm. The laser frequency was 10,000 Hz with 100 shots and 1 burst per pixel. The laser parameters were set to ‘Custom’ (Application), a 0% (Power Boost), single (Smart Beam), enabled (Beam Scan), and 26 µm (Scan Range). The mass range was set to 100–1500 m/z, the 1/K₀ range was set to 0.65–1.75 Vs/cm² and a 150 ms ramp time. Tune parameters were set to 50 V (MALDI plate offset), −70 V (Deflection delta), 500 Vpp (Funnel 1 RF), 0.0 eV (isCID), 350 Vpp (Funnel 2 RF), 350 Vpp (Multipole Vpp), 10 eV (Collision energy), 1100 Vpp (Collision cell RF), 5 eV (Ion energy), 100 m/z (Low mass), 80 µs (Transfer time), 5 µs (Pre pulse storage). TIMS parameters were set to 20 V (Δt1), 120 V (Δt2), −70 V (Δt3), −100 V (Δt4), 0 V (Δt5), −100 V (Δt6), −220 V (Collision cell in). After MS¹ acquisition, data analysis was performed in MZmine 3.

A trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) instrument (timsTOFflex, Bruker Daltonics, Bremen, Germany) was used. The instrument was equipped with an ESI-to-MALDI switchable dual source. According to the manufacturer, the AP interface of all current Bruker instruments is identical to the above, and thus, the APFD source is fully compatible among these. The mass spectrometer was controlled by the Bruker timsControl software (V 2.0) and data analysis was performed using the Bruker DataAnalysis software (V 6.0).
External mass calibrations were established in ESI mode by using Agilent Tune Mix (G1969-85,000) for the m/z 100–2500 range [43 (link), 44 ]. Mass accuracy was in the order of 3 ppm.

Samples were prepared as previously described for targeted collagen and extracellular matrix peptide imaging^{55 (link),101 (link)–104 (link)}. Briefly, deglycosylated samples^{105 ,106 (link)} were antigen retrieved with 10 mM Tris, pH 9, autosprayed (M5, HTX-Technologies) with 0.1 µg/µL collagenase type III (Worthington) dissolved in ammonium bicarbonate pH 7.4, 1 mM CaCl₂, and incubated at 38.5 °C with ≥85% humidity for 5 h. The matrix α-Cyano-4-hydroxycinnamic acid (CHCA, Sigma-Aldrich) was dissolved in 1.0% trifluoracetic acid (Sigma), 50% acetonitrile (LC-MS grade, Fisher Chemical) and autosprayed (M5, HTX Technologies) onto tissue. Tissues were imaged on a MALDI-QTOF (timsTOF-flex, Bruker) in positive ion mode over m/z range 700–2500. Laser was adjusted to 20 µm² and each pixel consisted of 300 laser shots. Images were collected with a laser step size of 60 µm. Data was visualized in SCiLS Lab Software (v2022b, Bruker) and processed for image segmentation and principal component analysis. Peak data were exported by mean spectrum processed as peak maximum and further statistical comparisons were done using Metaboanalyst 5.0 and GraphPad Prism 9.0. Exported peak intensities were visualized as heatmaps using the TM4 MultiExperiment Viewer suite^{107 (link)} with clustering by Manhattan metric and single linkage.

Mass spectra were acquired on a timsTOF fleX (Bruker Daltonics, Billerica, MA) in negative ion mode. Using FlexImaging 5.1 software, hippocampal sections were outlined as individual ROIs with a pixel size of 20 μm. Most metabolites were acquired in a single imaging run (m/z range 50–1000); however, a separate method was developed to target small metabolites not identified well by the first method, such as hypoxanthine (m/z range 50–400). In both methods, each MSI pixel consisted of 1000 laser shots. The laser Smartbeam parameter was set to single with a frequency of 10 kHz. Based on the mass range and selectivity of biomolecules for the two MALDI methods, the instrument parameters were optimized, e.g., quadrupole, ion transfer funnels, collision cell, and focus pre-TOF.
Before each acquisition, the instrument was calibrated by MALDI using the three ¹⁵N standards spiked into the matrix, as described above. For the small molecule method, a small spot of lactate, pyruvate, and ¹⁵N₁-glutamate was dried onto the ITO slide before matrix application, and these three ions were used for calibration of the lower m/z range.

Data acquisition was performed on a timsToF fleX (Bruker Daltonics, Bremen, Germany) in positive ion mode in the range of m/z 900–3200.
Spectra were recorded using 600 shots per pixel with a laser repetition rate of 10 kHz using a 50 × 50 µm step size. Transfer parameters were as follows: Funnel 1 RF 500 Vpp; Funnel 2 RF 500 Vpp; Multipole RF 500 Vpp; Deflection Delta 70 V; MALDI Plate Offset: 50 V. Quadrupole parameters: Ion energy 5.0 eV; Low Mass m/z 900. Focus PreToF Parameters: Transfer Time 145 µs; PrePulse Storage 28 µs.
External calibration was done in the electrospray mode using ESI-Low Concentration Tuning Mix (Agilent Technologies, Santa Clara, CA, USA)