A Unique Ribosome Signature Reveals bacterium Translation Initiation Sites

ABSTRACT: Overnight stationary cultures of wild type S. Typhimurium (Salmonella enterica serovar Thyphimurium - strain SL1344) grown in LB media at 37°C with agitation (200 rpm) were diluted at 1:200 in LB and grown until they reached and OD600 of 0.5 (i.e., logarithmic (Log) phase grown cells). Bacterial cells were collected by centrifugation (6000 × g, 5 min) at 4°C, flash frozen in liquid nitrogen and cryogenically pulverized using liquid nitrogen cooled pestle and mortar. The frozen pellet of a 50 ml culture was re-suspended and thawed in 1 ml ice-cold lysis buffer (50 mm NH4HCO3 (pH 7.9) supplemented with a complete protease inhibitor cocktail tablet (Roche Diagnostics GmbH, Mannheim, Germany) and subjected to mechanical disruption by two repetitive freeze-thaw and sonication cycles (i.e. 2 minutes of sonication on ice for 20-s bursts at output level 4 with a 40% duty cycle (Branson Sonifier 250; Ultrasonic Convertor)). The lysate was cleared by centrifugation for 15 min at 16,000 × g and the protein concentration measured using the protein assay kit (Bio-Rad) according to the manufacturer's instructions. The lysate was added Gu.HCl (4M f.c.) and subjected to N-terminal COFRADIC analysis. Free amines were blocked at the protein level making use of an N-hydroxysuccinimide ester of (stable isotopic encoded) acetate (i.e. NHS esters of 13C2D3 acetate), which allows distinguishing in vivo and in vitro blocked N-terminal peptides. The modified protein sample was digested overnight with sequencing-grade modified trypsin (1/100 (w/w trypsin /substrate)) at 37°C and subsequent steps of the N-terminal COFRADIC procedure were performed.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Salmonella Enterica Subsp. Enterica Serovar Typhimurium Str. Sl1344

SUBMITTER: Petra Van Damme  

LAB HEAD: Petra Van Damme

PROVIDER: PXD005579 | Pride | 2017-08-09

REPOSITORIES: pride