2.9. Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS) and relative proteome quantitation

Peptide separation and mass spectrometric analyses were carried out as previously described [26, 35]. Briefly, protein samples were re-suspended in 20 µL of 97.5 % H₂O/2.4% acetonitrile/0.1% formic acid and subjected to liquid chromatography and electrospray ionization tandem MS (LC-ESI-MS/MS). A linear 65-min gradient ranging from 5–55% of 80% acetonitrile at a flow rate of 110 nL/min was used. The electrospray voltage and ion transfer capillary temperature was 1.8 kV and 230 °C, respectively. All samples were analyzed in triplicate. The obtained MS/MS spectra from each sample were searched against the streptococci protein database and a specific SMU_118c protein, {"type":"entrez-protein","attrs":{"text":"Q8DWE3","term_id":"81588414","term_text":"Q8DWE3"}}Q8DWE3 (Swiss Prot and TrEMBL, Swiss Institute of Bioinformatics, Geneva, Switzerland, http://ca.expasy.org/sprot/), using SEQUEST algorithm in Proteome Discoverer 1.3 software (Thermo Scientific, San Jose, CA, USA).

For quantitative proteome analysis, three MS raw files from each group (control and experimental; total of 12 MS raw files) were analyzed using SIEVE software (Version 2.0 Thermo Scientific, San Jose, CA, USA) [35]. For the alignment step, a single MS raw file belonging to the control group (0 mM) was selected as the benchmark and the other files were adjusted to generate the best correlation to this reference file. After alignment, the feature detection and integration process was performed using MS-level data. For statistical analyses of protein abundance, peak integrations were summarized into protein-level annotation in SIEVE using a weighted average of intensities of LC-ESIMS/MS of a protein by run.

A statistical model based on an ANOVA framework with Tukey’s post hoc test was carried out. Relative abundance of SMU_118c protein from different bisHPPP concentration groups was considered significantly different from the control group (0 mM) when the values observed were >1.5 for increased and <0.5 for decreased abundance with a p-value cut-off of < 0.05 [35].