Sherry Gregory (Presenter)
Thermo Fisher Scientific
Authorship: P Humphryes J Jones, V Barattini, S Gregory
Thermo Fisher Scientific, Chromatography Consumables, Runcorn, UK
| Short Abstract Currently overnight in-solution trypsin digestion of proteins is used during peptide mapping; however this protocol requires a number of steps, which can differ between laboratories, making method transfer and data analysis between user groups problematic. Additionally, due to the number of steps required, in-solution digestion can increase the potential for user error. As a result, this methodology often leads to variations in the chromatographic profile and complicates the adoption of robust, generic workflows. Here we describe a workflow including novel, rapid and precise digestion of Cytochrome C, followed by micro-elution solid phase extraction (SPE) clean-up and analysis with next generation UHPLC and high resolution mass spectrometry detection (UHPLC-HRMS). |
Long Abstract
Introduction and Objectives
Reproducibility is a fundamental requirement of quantitative peptide mapping workflows as it enables users to confidently assign data differences to the sample, and not the methodological conditions used. Currently overnight in-solution trypsin digestion of proteins is used during peptide mapping; however this protocol requires a number of steps, which can differ between laboratories, making method transfer and data analysis between user groups problematic. Additionally, due to the number of steps required, in-solution digestion can increase the potential for user error. As a result, this methodology often leads to variations in the chromatographic profile and complicates the adoption of robust, generic workflows. Here we describe a workflow including novel, rapid and precise digestion of Cytochrome C, followed by micro-elution solid phase extraction (SPE) clean-up and analysis with next-generation UHPLC and high resolution mass spectrometry detection (UHPLC-HRMS).
Methods
Four well characterized peptides, derived from cytochrome C, were used for assessment of the novel digestion procedure. Four exogenous peptides were also spiked in post digest, which allowed determination of the reproducibility of the digestion, and an independent assessment of the clean-up procedures.
Results and Discussion
Recovery and precision were assessed and compared between SPE and filtration; greater levels of recovery and precision were observed with SPE. Some selectivity differences between the two techniques are discussed, as well as reproducibility of both the digestion and clean-up method. Comparison of the different sample preparation techniques determined that when speed and method simplicity is required size exclusion filter plates were optimal, however when higher accuracy and precision are desirable SPE is more appropriate. The workflow described allows for the introduction of fast, generic, and robust analytical methods suitable for a high throughput, environment.
Conclusions
Excellent levels of recovery and precision were observed giving a high throughput and reproducible workflow that can be applied to non-targeted, semi-targeted or targeted quantitative environments.
References & Acknowledgements:
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