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A Bioanalytical Met...
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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- Saleh, Aljona (författare)
- Stockholms universitet,Institutionen för analytisk kemi,Department of Analytical Chemistry, Stockholm University, SE-106 91, Stockholm, Sweden
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- Edlund, Per-Olof (författare)
- Department of Drug Design and Development (DD&D), SOBI, SE-112 76, Stockholm, Sweden
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- Gustafsson, Tomas N. (författare)
- Umeå universitet,Klinisk bakteriologi,Department of Clinical Microbiology, Clinical Bacteriology and Sunderby Research Unit, Umeå University, SE-901 85, Umeå, Sweden; Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77, Stockholm, Sweden
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- Sahlin, Margareta (författare)
- Stockholms universitet,Institutionen för biokemi och biofysik,Department of Biochemistry and Biophysics, Stockholm University, SE-106 91, Stockholm, Sweden
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- Sjöberg, Britt-Marie (författare)
- Stockholms universitet,Institutionen för biokemi och biofysik,Department of Biochemistry and Biophysics, Stockholm University, SE-106 91, Stockholm, Sweden
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- Granelli, Ingrid (författare)
- Stockholms universitet,Institutionen för analytisk kemi,Department of Analytical Chemistry, Stockholm University, SE-106 91, Stockholm, Sweden
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(creator_code:org_t)
- 2016-02-26
- 2016
- Engelska.
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Ingår i: Chromatographia. - : Springer Science and Business Media LLC. - 0009-5893 .- 1612-1112. ; 79:7-8, s. 383-393
- Relaterad länk:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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https://urn.kb.se/re...
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http://kipublication...
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Abstract
Ämnesord
Stäng
- 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.
Ämnesord
- NATURVETENSKAP -- Biologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences (hsv//eng)
- NATURVETENSKAP -- Kemi (hsv//swe)
- NATURAL SCIENCES -- Chemical Sciences (hsv//eng)
- MEDICIN OCH HÄLSOVETENSKAP -- Medicinsk bioteknologi -- Medicinsk bioteknologi (hsv//swe)
- MEDICAL AND HEALTH SCIENCES -- Medical Biotechnology -- Medical Biotechnology (hsv//eng)
- NATURVETENSKAP -- Kemi -- Analytisk kemi (hsv//swe)
- NATURAL SCIENCES -- Chemical Sciences -- Analytical Chemistry (hsv//eng)
Nyckelord
- On-line two-dimensional chromatography
- Mass spectrometry
- Quantification of protein
- Bacterial lysate
- Hydrolysis with immobilized pepsin
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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