Abstract
The identification of proteins in samples of moderate to complex composition is primarily done by bottom-up approaches. Therefore, proteins are enzymatically digested, mostly by trypsin, and the resulting peptides are then separated prior to their transfer to a mass spectrometer. The following protocol portrays a bottom-up method, which was optimized for the application of CZE-ESI-TOF MS. Protein denaturation is achieved by addition of 2,2,2-trifluoroethanol (TFE) and heat treatment. Afterwards, disulfide bonds are reduced with tris-(2-carboxyethyl)phosphine (TCEP) and subsequently alkylated with iodoacetamide (IAA). The tryptic digest is performed in an ammonium bicarbonate buffer at pH 8.0. The digested protein sample is then concentrated in-capillary by transient capillary isotachophoresis (tCITP) with subsequent CZE separation of tryptic peptides in an acidic background electrolyte. Hyphenation to a time-of-flight (TOF) mass spectrometer is carried out by a triple-tube coaxial sheath flow interface, which uses electrospray ionization (ESI). Peptide identification is done by peptide mass fingerprinting (PMF). The protocol is outlined exemplarily for a model protein, i.e., bovine β-lactoglobulin A.
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Acknowledgments
This work was done within the Christian Doppler Laboratory for Innovative Tools for Biosimilar Characterization. The financial support by the Austrian Federal Ministry for Digital and Economic Affairs, the National Foundation of Research, Technology, and Development, and a Start-up Grant of the Province of Salzburg is gratefully acknowledged. Dr. Lorenz Stock (former member of the CD-laboratory) is gratefully acknowledged for his scientific and technical support.
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Segl, M., Stutz, H. (2022). Bottom-Up Analysis of Proteins by Peptide Mass Fingerprinting with tCITP-CZE-ESI-TOF MS After Tryptic Digest. In: Neusüß, C., Jooß, K. (eds) Capillary Electrophoresis-Mass Spectrometry . Methods in Molecular Biology, vol 2531. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2493-7_7
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