A Bioanalytical Method for Quantification of Thioredoxins in Bacillus anthracis by Digestion with Immobilized Pepsin and LC–MS/MS and On-line LC/LC–MS/MS
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We describe a method for the quantification of proteins in a biological matrix through digestion with pepsin. Pepsin is a gastric protease that efficiently cleaves proteins in an acidic environment. In this study, it has been used to generate peptides used for the quantification of physiologically relevant thioredoxin proteins in a lysate of Bacillus anthracis—the causative agent of anthrax. Carefully selected signature peptides for proteins that were digested with pepsin were immobilized on agarose gel. Filtered samples were analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS) and by two-dimensional liquid chromatography tandem mass spectrometry (LC/LC–MS/MS) when additional selectivity was needed. Some important incubation parameters were adjusted to get the highest possible peptide yield. Escherichia coli was used as a surrogate matrix for the method development. The method was validated at a low nM range for selectivity, accuracy and precision. Validation showed that signature peptides were selective for the proteins, and that the method accuracy varied between 89 and 115 % with a precision of less than 12 %. The results from using pepsin in analyzing samples from Bacillus anthracis were similar to those previously obtained using western blot, and they validate pepsin as a suitable protease to generate signature peptides in a complex biological matrix as an alternative to trypsin.
KeywordsOn-line two-dimensional chromatography Mass spectrometry Quantification of protein Bacterial lysate Hydrolysis with immobilized pepsin
The authors wish to gratefully acknowledge support from The Knut and Alice Wallenberg Foundation, Swedish Orphane Biovitrum (SOBI) and Department of Environmental Science and Analytical Chemistry at Stockholm University. Tomas N Gustafsson wishes to acknowledge generous financial support from the County Council of Norrbotten (NLL-393301 and NLL-393021) and Karolinska Institutet.
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Conflict of interest
The authors declares that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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