Abstract
Peptide mass fingerprinting (PMF) by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides a simple and direct means to unequivocally confirm identity of recombinant proteins based on predicted peptide profiles. Many universities or research institutions now carry mass spectrometry instrumentation as part of their core bioanalytical facilities or provide public service to outside investigators. This chapter provides methods we have used to generate routinely high quality samples for MALDI-TOF MS analysis. Following resolution of protein preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we easily process sets of 12 samples manually for MS analysis. Target bands are alkylated and digested in-gel with trypsin, followed by extraction of peptides and desalting with a C18 adsorbent resin (e.g., a “ZipTips”). Acquisition of PMF data on MALDI-TOF mass spectrometers is fast, and with on-site instrumentation, the entire process can be completed within 2 days.
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Acknowledgments
This work was supported by funding from the DOE Mid-South/Southeast Biofuels Consortium (DE-FG36-08BO88036) and the NSF EPSCoR, RII: Arkansas ASSET Initiative Plant Protein Production Center (#EPS-0701890).
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Savary, B.J., Vasu, P. (2012). Routine Identity Confirmation of Recombinant Proteins by MALDI-TOF Mass Spectrometry. In: Lorence, A. (eds) Recombinant Gene Expression. Methods in Molecular Biology, vol 824. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-433-9_2
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DOI: https://doi.org/10.1007/978-1-61779-433-9_2
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