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  • 1.
    Saleh, Aljona
    et al.
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Bruno, Oscar
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Granelli, Ingrid
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Edlund, Per-Olof
    Digestion of Enolase and Carbonic Anhydrase as Model Proteins for Therapeutic Proteins in Blood Plasma with Immobilized Thermolysin and Quantification of Some of the Peptides by LC/LC-MS/MS2014In: Chromatographia, ISSN 0009-5893, E-ISSN 1612-1112, Vol. 77, no 1-2, p. 59-74Article in journal (Refereed)
    Abstract [en]

    There is a need for fast method development in the early drug discovery phase of therapeutic proteins. Thermolysin has not been used for quantification of proteins in blood plasma earlier. It is a thermostable protease which permits the use of high temperatures for fast hydrolysis of proteins. Model proteins were digested with immobilized thermolysin on agarose gel. Protein-specific peptides were selected for quantitation and quantified based on stable isotope dilution. Protein digests of blood plasma were cleaned and separated using an automated LC/LC-MS/MS system. Essential digestion parameters that influence thermolysin hydrolytic activity were optimized for high peptide yield. The validated methods were selective, linear, precise and accurate with a limit of detection of 2 nM for both proteins. The proposed strategy for method development could be valuable for quantification of proteins in blood plasma samples. The study underscores and discusses important features of the enzymatic digestion and chromatographic separation.

  • 2.
    Saleh, Aljona
    et al.
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Edlund, Per-Olof
    Gustafsson, Tomas N.
    Sahlin, Margareta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöberg, Britt-Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Granelli, Ingrid
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    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/MS2016In: Chromatographia, ISSN 0009-5893, E-ISSN 1612-1112, Vol. 79, no 7-8, p. 383-393Article in journal (Refereed)
    Abstract [en]

    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.

  • 3. Sroka-Markovic, Janina
    et al.
    Johansson, Linda
    Andersson, Magnus B. O.
    Granelli, Ingrid
    Stockholm University, Faculty of Science, Department of Analytical Chemistry.
    Development of an HPLC Method for Determination of Related Impurities in Prilocaine Substance2012In: Chromatographia, ISSN 0009-5893, E-ISSN 1612-1112, Vol. 75, no 7-8, p. 329-336Article in journal (Refereed)
    Abstract [en]

    Development of a reversed phase high performance liquid chromatographic method for determination of six related impurities in prilocaine substance is reported. The test of related impurities in European Pharmacopoeia (Ph. Eur.) cannot meet the demands with the chromatographic parameters given, therefore different types of chromatographic systems and eight columns have been evaluated in the present study. A new method with a Hypercarb column was developed and validated. This method fulfils the demands in the Ph. Eur., and the validation shows that the method is selective, reproducible, linear, accurate and robust with sufficient limits of detection (0.001-0.004% of 2.5 mg prilocaine mL(-1)) and quantification (0.002-0.009% of 2.5 mg prilocaine mL(-1)).

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