Flexcontrol

MSI was performed using a RapifleX MALDI Tissuetyper time-of-flight (TOF) mass spectrometer (Bruker Daltonics). A peptide calibration standard mix (bradykinin, angiotensin II, angiotensin I, substance P, bombesin, ACTH clip 1–17, ACTH clip 18–39, and somatostatin 28 (Bruker Daltonics)) was used for external calibration. Each spectrum was automatically generated at a spatial resolution of 50 µm using flexControl (Bruker Daltonics) in the mass range of m/z = 600–3200. 500 laser shots were acquired for each spectrum at 1 kHz, with a laser power of 65–80%. The measurement regions were defined using flexImaging (Bruker Daltonics). Following the MSI measurements, matrix was removed by two washes in 99.99% methanol (Carl Roth GmbH, Karlsruhe, Germany) for 2 min each, followed by two washings in 99.99% ethanol (Carl Roth GmbH) for 10 s.

A saturated solution of sinapinic acid (SA) as matrix in 30:70 (v/v) acetonitrile:water, 0.1% trifluoroacetic acid (TFA) and a protein solution in 30:70 (v/v) acetonitrile:water, 0.1% TFA were prepared and mixed in a 1:1 ratio. A total of 1 µL of the previous mixture was deposited into a polished stainless-steel target (Bruker, Bremen, Germany) and allowed to dry. Then, 1 µL of SA matrix solution was deposited into the sample and allowed to dry. The same procedure was followed for the Protein Standard Calibration I solution (Bruker) used for Calibration. The target was introduced in a Autoflex MALDI-TOF (Bruker), spectra were acquired in lineal mode (Flex control, Bruker) and processed by Flex Analysis (Bruker).

30 μg protein were runned in the same conditions as for silver staining. Gels were fixed with 50% methanol and 2% o-phosphoric acid over night. After that, gels were washed three times with distilled water during 20 min and incubated with 34% methanol, 2% o-phosphoric acid and 17% ammonium sulfate during 1 hour. Gels were stained with 34% methanol, 2% o-phosphoric acid, 17% ammonium sulfate and 0,066% Coomasie G-250 during at least 5 days. Finally, gels were distained with 25% methanol during 1 min and kept in distilled water until excising. Identified spots were excised from the gel and spotted on a MALDI AnchorChip target plate according to the manufacturer's instructions. Analysis was performed on an OrbitrapXL MALDI TOF instrument (Thermofisher) using Bruker Flex-control, Flex-analysis, and Biotools software. Calibration was performed externally using SIGMA Proteo- Mass™ Peptide MALDI–MS Calibration Kit. MALDI mass spectra were recorded within an m/z range of 700–3500 in the positive ion reflector mode. Mass spectra were obtained by averaging up to 300 individual laser shots. Each analysis was performed in triplicate.

MALDI-TOF/TOF-MSI was performed using a RapifleX MALDI-TOF/TOF system (Bruker Daltonics). Negative-ion-mode mass spectra were acquired at a pixel size of 5 × 5 µm² over a mass range of m/z 80–1000. Prior to analysis, the instrument was externally calibrated using red phosphorus. Spectra were acquired with 15 laser shots per pixel (for 5 × 5 µm² measurements) at a laser repetition rate of 10 kHz. Data acquisition was performed using flexControl (Version 4.0, Bruker Daltonics) and flexImaging 5.0 (Bruker Daltonics). Sections present on the same slide were measured in a randomized order. The m/z features present in MALDI-TOF-MSI dataset were further used for identity assignment of metabolites and lipid species. The m/z values were imported into the Human Metabolome Database^{47 (link)} (https://hmdb.ca/) after re-calibration in mMass and annotated for metabolites and lipids species with an error ≤ ±20 ppm. The ¹³C-labeled peaks were selected by comparing the spectrum of control and ¹³C-labeling experiments, and annotated based on the presence of un-labeled metabolites and their theoretical m/z values. Peak intensities of the selected features were exported for all the measured pixels from SCiLS Lab 2016b (version 2016b, Bruker Daltonics), which were used for the following analysis. Single ion visualizations were also obtained from SCiLS Lab.

Data collection

Water contact angle: Krüss Advance 1.6.2.0; Krüss GmbH

AFM: MultiMode 8-HR; Bruker Corporation

ToF-SIMS: Surfacelab 7.0.106074; IONTOF GmbH

MS: flexControl 4.0.35; Bruker Corporation

IR: Opus 7.8; Bruker Corporation

UV–Vis: UV WinLab 6.0.4; PerkinElmer, Inc./Gen5; BioTek Instruments, Inc.

Microscopy: BZ-II Viewer 1.5.0.0; Keyence Corporation

Data analysis:

Water contact angle: Krüss Advance 1.6.2.0; Krüss GmbH

AFM: NanoScope Analysis 1.50 (build R3.119069); Bruker Corporation

ToF-SIMS: Surfacelab 7.0.106074; IONTOF GmbH

MS: flexAnalysis 4.0.14; Bruker Corporation

IR: Opus 7.8; Bruker Corporation

UV–Vis: Office 264 ProPlus Version 1908 Build 11929.20648; Microsoft/OriginPro 2019b Build 9.6.5.169; OriginLab Corporation

Microscopy: BZ-II Analyzer 2.1; Keyence Corporation/Office 264 ProPlus Version 1908 Build 11929.20648; Microsoft/OriginPro 2019b Build 9.6.5.169; OriginLab Corporation

The LDI-TOF-MS analysis was performed using ultrafleXtreme mass spectrometer (Bruker Daltonics, Hamburg, Germany) and ZnO NCs suspended in water were spotted on the ground steel target (Bruker Daltonik, Bremen, Germany) according to the previously described protocol [10 (link)] with the required modification (without the α-Cyano-4-hydroxycinnamic acid (HCCA) matrix). Protein Calibration Standards I (Bruker Daltoniks, Bremen, Germany), Peptide Calibration Standard and two signals characteristic for the matrix [M-H]⁺ and [2M-H]⁺ were used for the external calibration, according to the standardized Bruker sample preparation procedure. Molecular fingerprint (MF) spectra of ZnO NCs were recorded in reflectron positive mode, within a m/z range of 100–3500, and we applied an acceleration voltage of 25 kV. Fragment spectra were recorded using the LIFT default method (Bruker Daltonics, Hamburg, Germany) at 100% of laser power, global attenuator 50% with calibration on the immonium ions [29 (link),30 (link)]. The voltage on the LIFT electrodes was 19.0 and 2.7 kV, respectively. MS spectra were registered in FlexControl (Bruker Daltonics, Hamburg, Germany), while the FlexAnalysis (Bruker Daltonics, Hamburg, Germany) was used for data analysis.