Data on proteome of Mycoplasma hominis cultivated with arginine or thymidine as a carbon source

Mycoplasma hominis is an opportunistic bacterium that can cause acute and chronic infections of the urogenital tract. This bacterium, like all other Mycoplasma species, is characterized by the reduced genome size, and, consequently, reduction of the main metabolic pathways. M. hominis cells cannot effectively use glucose as a carbon and energy source. Therefore, the main pathway of energy metabolism is the arginine dihydrolase pathway. However, several bacteria can use nucleosides as the sole energy source. Biochemical studies using Salmonella typhimurium have shown that three enzymes (thymidine phosphorylase, phosphopentose mutase and deoxyribose-phosphate aldolase) are involved in the thymidine catabolic pathway. All these enzymes are present in M. hominis. For understanding changes in the energy metabolism of M. hominis we performed shotgun proteome analysis of M. hominis cells in liquid medium with arginine or thymidine as a carbon source. LC-MS analysis was performed with an Ultimate 3000 Nano LC System (Thermo Fisher Scientific) coupled to a Q Exactive HF benchtop Orbitrap mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). Data are available via ProteomeXchange with identifier PXD018714 (https://www.ebi.ac.uk/pride/archive/projects/PXD018714).


a b s t r a c t
Mycoplasma hominis is an opportunistic bacterium that can cause acute and chronic infections of the urogenital tract. This bacterium, like all other Mycoplasma species, is characterized by the reduced genome size, and, consequently, reduction of the main metabolic pathways. M. hominis cells cannot effectively use glucose as a carbon and energy source. Therefore, the main pathway of energy metabolism is the arginine dihydrolase pathway. However, several bacteria can use nucleosides as the sole energy source. Biochemical studies using Salmonella typhimurium have shown that three enzymes (thymidine phosphorylase, phosphopentose mutase and deoxyribose-phosphate aldolase) are involved in the thymidine catabolic pathway. All these enzymes are present in M. hominis . For understanding changes in the energy metabolism of M. hominis we performed shotgun proteome analysis of M. hominis cells in liquid medium with arginine or thymidine as a carbon source. LC-MS analysis was performed with an Ultimate 30 0 0 Nano LC System (Thermo Fisher Scientific) coupled to a Q Exactive HF benchtop Orbitrap mass spectrometer (Thermo Fisher Scientific) via a nanoelectro-spray source (Thermo Fisher Scientific

Value of the data
• This dataset provides proteome data for M. hominis cells growing in culture with arginine or thymidine as carbon source. • These data can be interesting for the investigation of interaction with the environment of opportunistic bacteria M. hominis . • These data can be interesting for the investigation of metabolism of M. hominis and another mycoplasma species that can be a model of a minimal cell.

Data description
Mycoplasma hominis is a human opportunistic bacterium that can cause acute and chronic infections of the urogenital tract [1] . Like all other Mycoplasma species, it is characterized by the reduced genome size (about 550 ORFs), and, consequently, reduction of the main metabolic pathways. M. hominis cells cannot effectively use glucose as a carbon and energy source. Therefore, the main pathway of energy metabolism is the arginine dihydrolase pathway, which includes arginine deiminase, ornithine carbamoyltransferase and carbamate kinase [2] . However, M. hominis cells can utilize nucleosides. In thymidine catabolic pathway the thymidine phosphorylase, phosphopentose mutase and deoxyribose-phosphate aldolase are involved [3] .
We performed shotgun proteome analysis of M. hominis cells in liquid medium with arginine or thymidine as a carbon and energy source. LC-MS analysis was performed with an Ultimate 30 0 0 Nano LC System (Thermo Fisher Scientific) coupled to a Q Exactive HF benchtop Orbitrap mass spectrometer (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). Protein identification and label-free quantification were made by PEAKS software. The  data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the identifier PXD018714.
Totally, 466 proteins were identified in both cases of M. hominis culturing with arginine or thymidine as carbon source (table S1). Obtained datasets show good reproducibility between biological replicas as demonstrated by the heatmap ( Fig. 1 ). Range of differences in the protein changes is shown on volcano plot ( Fig. 2 ). Proteins, significantly changed between aforementioned conditions (fold change > 1.5, t -test with Benjamini-Hochberg correction, p < 0.05), are presented in Table 1 . All the above-mentioned enzymes from the metabolic pathways of arginine and thymidine utilization have been identified. When growing on thymidine, the only thymidine phosphorylase abundance was increased by 1.8 times, the abundance of other enzymes did not significantly change. Table 1 Significantly changed proteins for M. hominis growing in different growth conditions. Log2FC -logarithm of fold change ratio for growth with thymidine to growth with arginine.

Cell cultivation
M. hominis H34 strain was grown on Brain Heart Infusion (DIFCO, USA) supplemented with 10% horse serum (Biolot, Russia), 1% yeast extract (Helicon, Russia), penicillin (Sintez, Russia) with a final concentration 500 units/ml with the addition of 1% arginine or thymidine as a carbon source. The culture was grown at 37 °C till log-phase for 48 h with arginine or 96 h with thymidine carbon source.

Protein extraction
Aliquots (10 ml) of log-phase growing cells of M. hominis H34 were collected by centrifugation at 12,0 0 0 g at 4 °C for 10 min. Then cells were washed twice by addition of 1 ml cold PBS buffer and centrifugation at 12,0 0 0 g at 4 °C for 10 min, 10 μl of 10% sodium deoxycholate (DCNa) and 0.5 μl nuclease mix (GE Healthcare, USA) was added to the cell pellet. After incubation for 1 hour at 4 °C, the sample was resuspended in 100 μl 100 mM Tris-HCl buffer (pH 8.0) containing 0.1% DCNa, 8 M urea and 2.5 mM EDTA. After incubation for 20 min the sample was centrifuged at 16,0 0 0 g for 10 min at 4 °C to remove intact cells and debris. The supernatant was collected, and protein concentration was measured using BCA Assay Kit (Sigma-Aldrich, USA).

Protein preparation to shotgun proteomic
Disulfide bonds were reduced in supernatant (containing 200 μg of total protein) by the addition of Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma-Aldrich, USA) to a final concentration of 5 mM and reaction was incubated for 60 min at 37 °C. To alkylate free cysteines, chloroacetamide (Sigma-Aldrich, USA) was added to a final concentration of 30 mM and placed at room temperature in the dark for 30 min. The step of adding TCEP was repeated. Then the sample was diluted 6-fold with 50 mM Tris-HCl, pH 8.0 with 0.01% DCNa. Trypsin Gold (Promega, USA) was added for a final trypsin:protein ratio of 1:50 (w/w) and incubated at 37 °C overnight. To stop trypsinolysis and degrade the acid-labile DCNa, trifluoroacetic acid (TFA) was added to the final concentration of 0.5% (v/v) (the pH should be less than 2.0), incubated at 37 °C for 45 min and the samples were centrifuged at 14,0 0 0 g for 10 min to remove the DCNa. Peptide extract was desalted using a Discovery DSC-18 Tube (Supelco, USA) according to the manufacturer protocol. Peptides were eluted with 1 ml of 75% acetonitrile in water containing 0.1% TFA, dried in an Acid-Resistant CentriVap Benchtop Vacuum concentrator (Labconco, USA) and resuspended in 3% acetonitrile in water containing 0.1% TFA to the final concentration of 5 μg/ μl.

LC-MS analysis
LC-MS analysis was carried out on an Ultimate 30 0 0 RSLC nano HPLC system connected to a QExactive Plus mass spectrometer (Thermo Fisher Scientific, USA). Samples were loaded to a home-made trap column 20 ×0.1 mm, packed with Inertsil ODS3 3 μm sorbent (GL Sciences, Japan), in the loading buffer (2% ACN, 98% H 2 O, 0.1% TFA) at 10 μl/min flow and separated at RT in a home-packed fused-silica column 500 ×0.1 mm packed with Reprosil PUR C18AQ 1.9 (Dr. Maisch, Germany) into the emitter prepared with P20 0 0 Laser Puller (Sutter, USA) [4] . Samples were eluted with a linear gradient of 80% ACN, 19. MS data were collected in DDA mode. MS1 parameters were as follows: 70 K resolution, 350-20 0 0 scan range, max injection time 50 ms, AGC target 3 × 10 6 . Ions were isolated with 1.4 m/z window and 0.2 m/z offset targeting 10 highest intensity peaks of + 2 to + 6 charge, 8 × 10 3 minimum AGC, preferred peptide match and isotope exclusion. Dynamic exclusion was set to 40 s. MS2 fragmentation was carried out in HCD mode at 17,5 K resolution with 27% NCE. Ions were accumulated for max 45 ms with target AGC 1 × 10 5 .

Protein identification and quantitative analysis
Identification and label-free quantification analysis were performed with PEAKS software [5] with default settings. The data were searched against M. hominis ATCC 23,114 NCBI database