Accurate identification of proteogenomic characterization in Mycolicibacterium smegmatis MC2155 by 14N and 15N double-labeling mass spectra

ABSTRACT: To achieve deep coverage of M. smegmatis proteome, 240 μg proteins were reduced with 5 mM of dithiothreitol (DTT), alkylated with 20 mM of iodoacetamide (IAA). The alkylated proteins were separated by a 10% SDS-PAGE for 8 cm, and stained with Coomassie Blue G250. Gel lane was excised into 13 fractions based on the molecular wight (MW) and protein abundance, and digested with Ac-Trypsin (Wu et al., 2016) (12.5 ng/μL) at 37 °C for 12-24 h. The extracted peptides were desalted with a homemade C18 StageTip (Zhai et al., 2013), dried and dissolved in loading buffer (1% acetonitrile, ACN and 1% formic acid, FA) for MS analysis.The dissolved peptides (500 ng) were analyzed as described previously. Briefly, the liquid chromatography tandem mass spectrometry (LC-MS/MS) consisted of an EASY-nLC 1200 system (Thermo Fisher Scientific, San Jose, CA, United States) equipped with a self-packed capillary column (75 μm i.d. × 15 cm, 3 μm C18 reversed-phase fused silica) coupled to an Orbitrap Fusion Lumos (Thermo Fisher Scientific). For full MS scans, the automatic gain control (AGC) was 5.0 × 105, the scan ranged from 300 to 1,400 m/z at a resolution of 1.2 × 105 and a maximum injection time (MIT) of 50 ms. For the MS2 scan, only spectra with a charge state of 2-6 were selected for fragmentation by higher energy collision-induced dissociation (HCD) with a normalized collision energy of 32%, AGC of 1 × 105, and MIT of 35 ms. The dynamic exclusion was set to 30 s.

ORGANISM(S): Mycolicibacterium Smegmatis Mc2 155

SUBMITTER: Yao Zhang  

PROVIDER: PXD037081 | iProX | Thu Sep 29 00:00:00 BST 2022

REPOSITORIES: iProX