Top-down liquid chromatography-coupled mass spectrometry (LC–MS) is an innovative approach that allows undigested proteins to be analyzed in the intact state. Although this emerging proteomics method offers significant advantages, technical limitations of the approach have previously restricted its use to single proteins or simple protein mixtures. In this paper, Ansong et al. (2013)utilized a top-down approach to characterize the proteome of Salmonella enterica serotype Typhimurium.1
To accomplish this, the researchers used single-dimension, ultra-high-pressure LC and a Velos Orbitrap mass spectrometer (Thermo Scientific) to identify 563 proteins and 1,665 proteoforms derived from post-translational modifications (PTMs). They also noted that salmonellae possess the ability to differentially express one PTM as an oxidative stress response. This PTM, S-thiolation, occurs when proteins with free thiols form mixed disulfides with low mass thiols like glutathione and cysteine in processes labeled S-glutathionylation and S-cysteinylation, respectively. In general, S-thiolation inhibits the oxidation of cysteine residues and thereby protects and regulates thiol-containing proteins.
Although S-glutathionylation is relatively well studied, Ansong et al. provide the first evidence of S-cysteinylation in a gram-negative bacteria (salmonellae). Further, the researchers note that while salmonellae prefer S-glutathionylation under basal conditions, the bacterial pathogen preferentially utilizes S-cysteinylation for thiol protection and environmental sensing when subjected to infection-like conditions. In order to verify these results, the team used bottom-up data to quantify glutathionylated and cysteinylated peptides. The finding that both artificial and natural infections result in upregulation of cysteine biosynthesis and downregulation of glutathione biosynthesis enzymes may offer an explanation for the differential selection of S-cysteinylation under stressful conditions. The researchers further note that the emerging top-down approach itself may play a large role in detecting these results, since previous methods actually altered the cell’s natural physiology and function.
The researchers assert that their discovery — that salmonellae contain a PTM acting as a switch to downregulate the synthesis and utilization of an energetically draining moiety (glutathione) in favor of a more efficient alternative (cysteine) when cellular conditions become less ideal — is an area ripe for further study.
1. Ansong, C., et al. (2013) “Top-down proteomics reveals a unique protein S-thiolation switch in Salmonella Typhimurium in response to infection-like conditions,” Proceedings of the National Academy of Sciences of the United States of America, 110(25) (pp. 10153–8).
Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.