Quantification of Bacterial Proteins using PRM

Referred to the proteomic results, six related proteins, lipoteichoic acid synthase (Q2G093), signal peptidase I (Q2FZT7), teichoic acids export ATP-binding protein TagH (Q2G2L1), lipoprotein (Q2G0V0), UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase (Q2FWH4), and D-alanine-D-alanine ligase (Q2FWH3), were selected for further verifiable quantification for three samples from the azalomycin F group or the blank one, using the parallel reaction monitoring (PRM) technology. According to the similar procedure to proteome analysis, the peptides from tryptic hydrolysis were dissolved in 0.1% (v/v) formic acid and separated on an EASY-nLC 1000 UPLC system with a reversed phase analytical column. The gradient elution consisted of an increase from 7% to 25% solvent B in 40 min, 22% to 35% in 12 min, 35% to 80% in 4 min, and then holding at 80% for the last 4 min. The analysis process was achieved at a constant flow rate of 0.4 μL/min. The separated peptides were subjected to an NSI source followed by MS/MS analysis in a Q ExactiveTM Plus (Thermo Fisher Scientific, San Jose, CA, USA). The electrospray voltage applied was 2.0 kV. The full scan of m/z ranged from 400 to 1000, and intact peptides were detected in the Orbitrap at a resolution of 70,000. The peptides were then selected for MS/MS analysis using an NCE setting as 27, and the fragments were detected in the Orbitrap at a resolution of 17,500. The data-dependent procedure alternated between one MS scan followed by 20 MS/MS scans. The AGC was set at 3e⁶ for full MS and 1e⁵ for MS/MS, respectively. The maximum IT was set at 50 ms for full MS and 180 ms for MS/MS. The isolation window for MS/MS was set at 1.6 m/z.
The resulting MS data were processed using Skyline (v.3.6). Peptide parameters were set as trypsin (KR|P) for the enzyme, zero for the max missed cleavage, 7 to 25 residues for the peptide length, and alkylation on Cys for fixed modification. Transition parameters were set as 2 and 3 for precursor charges, 1 for ion charges, and b and y for ion types. The product ions were set from ion 3 to the last ion. The mass tolerance of ion match was set as 0.02 Da.