This dataset contains MALDI-TOF MS Main Spectra Profile (MSP) generated for Stenotrophomonas sp. strain PUT21, a taxonomically distinct isolate representing a potentially novel lineage within the genus Stenotrophomonas. MALDI-TOF MS analyses and MSP generation were performed using a Bruker MALDI-TOF MS platform following standard Bruker Biotyper protocols recommended by the manufacturer for bacterial identification and taxonomic studies. Spectral acquisition, preprocessing, quality control, and MSP generation were conducted using default parameters, without non-standard or experimental modifications. The dataset provides reference protein mass spectral profiles intended to support comparative analyses, taxonomic evaluation, and future species delineation. At the time of deposition, no reference MSP corresponding to strain PUT21 was available in the Bruker MALDI Biotyper reference library. The spectrum provided here constitutes an original, publicly accessible reference profile. Sample Preparation and Spectra Acquisition Stenotrophomonas sp. strain PUT21 was cultured on TSA plates at 30 °C for 18 h. Protein extraction was performed using the standard ethanol/formic acid (EtOH/FA) protocol recommended by the MALDI Biotyper manufacturer (Bruker Daltonics). Spectra were acquired in linear positive ion mode within a mass range of 2,000–20,000 m/z using a Microflex LT/SH instrument controlled by flexControl 3.4 software (Bruker Daltonics). External calibration was performed using the Bruker Bacterial Test Standard according to the manufacturer’s instructions. At least eight technical replicates were included for MSP generation. Raw spectra were processed using default preprocessing parameters in flexAnalysis 3.4. The Main Spectrum Profile (MSP) was generated using the automated function implemented in MALDI Biotyper Compass Explorer Module 4.1.80 (Bruker Daltonics). All spectra were visually inspected for quality prior to MSP creation. Strain deposition Stenotrophomonas sp. strain PUT21 is deposited in the Collection of Plasmids and Microorganisms | KPD under accession number KPD 2002. Strain-related datasets The corresponding genome sequence is available in GenBank under accession number CP197645. --------------------------Author contributions: Data acquisition, sample preparation, and mass spectrometry analysis (including MSP generation) were performed by Anna-Karina Kaczorowska.Bacterial strain was provided by Mateusz Łężyk and Sivasankar Palaniappan, who performed genome sequencing and genomic characterization, and contributed to biological material selection.

 Funding note The MALDI-TOF MS data presented in this dataset were generated using infrastructure acquired through a Capital Equipment Grant (No. 6830/IA/SP/2018) awarded by the Polish Ministry of Science and Higher Education. The acquisition, implementation, and scientific use of the Microflex LT/SH and MALDI Biotyper system were initiated and coordinated by Anna-Karina Kaczorowska.

The MSP file is provided in the proprietary *.btmsp format generated by the MALDI Biotyper software (Bruker Daltonics). The *.btmsp file can be directly imported into the MALDI Biotyper system for identification and comparative analyses. For details regarding file import and compatibility, users are referred to the manufacturer's documentation.