# Chemical Images on Fingerprints Revealed with Mass Spectrometry
## 2 Materials and Methods  
A MALDI 4800 TOF-TOF (AB SCIEX, Old Connecticut Path, Framingham, MA 01701, USA) was used. The instrument worked in reflectron mode, achieving a resolving power (RP) of 25,000 and a mass accuracy (MA) of 50 ppm. The ion source was equipped with a 355 nm Nd:YAG laser (200 Hz repetition rate) and operated under a vacuum (0.7 \(\upmu\)Torr). The optics were oriented to focus the laser beam (50 \(\upmu\)m) orthogonally on the target, obtaining an energy density of 500-1000 eV/mm\({}^{2}\). Ions were extracted and accelerated by applying a high-voltage pulse (20 kV) after a predetermined time delay of 0.35 ms. The spectrometer digitalized data every 2 ns, averaging one spectrum every four laser shots. The electron multiplier was set to 0.85 V. The instrument was controlled by software with a 4000-Series SCIEX Explorer.  
Before any experiment, the instrument was tuned with a CAL-MIX5 and MALDI methodology was optimized using caffeine and stearic acid as reference standards for positive and negative ion modes. A matrix suspension of a photoactive matrix (Evonik P25 TiO\({}_{2}\), 0.5 \(\upmu\)g/\(\upmu\)L) was prepared using H\({}_{2}\)O and CH\({}_{3}\)OH (75:25 \(v/v\)). Approximately 1 \(\upmu\)L of TiO\({}_{2}\) suspension was drop-cast on the laser target, which was then dried upside-down for 2 h to ensure even TiO\({}_{2}\) coverage. In this way, the MALDI target was functionalized with the nano-material. Finally, 1 \(\upmu\)L of each reference standard was deposited on the TiO\({}_{2}\) spot.  
The limits of detection (LODs) were assessed in positive and negative ion modes, reducing the amounts of calibrants spotted on the MALDI target. Assuming an S/N ratio equal to 3, the LODs were estimated as 400 \(\text{fmol}/\text{mm}^{2}\) for caffeine (positive ion mode) and 250 \(\text{fmol}/\text{mm}^{2}\) for stearic acid (negative ion mode). The relative spectra are shown in Figure S1.  
The imaging experiment on endogenous compounds in LSM started with a donor who touched the MALDI target with the index finger. Approximately 2 mg of TiO\({}_{2}\) nanoparticles were spread on the target. Some nanoparticles were trapped in the LFM, but the excess nanomaterial was carefully removed under a gentle nitrogen stream. The mass spectrometer was set up in order to collect each pixel using 10 individual laser shoots. The molecular distribution of the analytes was determined by recording a pixel every 30 \(\upmu\)m. Acquisition occurred in negative ion mode, revealing skin fatty acids. The shooting rate was approximately 104 laser shoots per mm\({}^{2}\). Time to digital converter (TDC) was set to 4 ns/bin for data storage. Dedicated software (BioMap, Novartis) was used to merge all the spectra, leading to the final chemical image. Attribution of the MS signals was based on accurate mass measurements and confirmed by analyzing the same sample with an LTQ/Orbitrap (Thermo Fisher Scientific, San Jose, CA, USA) mass analyzer, equipped with a DESI ion source (Prosolia, Indianapolis, IN, USA). The LTQ/Orbitrap operated at a resolution of 100,000, achieving a 5 ppm mass accuracy.  
The imaging experiment of exogenous compounds used most of the instrumental conditions reported above. An over-the-counter drug (formulation: acetaminophen, aspirin, and caffeine) was used. The experiment started by asking a subject to touch the drug tablet with their finger and then deposit the fingerprint on the MALDI target. Approximately 2 mg of TiO\({}_{2}\) was spread on the target, revealing the LMFs. Images were acquired in positive reflectron ion mode, revealing traces of target compounds embedded in human skin.