Digestion of Native Proteins for Proteomics Using a Thermocycler

Abstract

Efficient protein digestion is a critical step for successful mass spectrometry analysis. Here we describe simultaneous tryptic digestion and gradual unfolding of native proteins by application of a temperature gradient using a single cycle of 5 min or less in a PCR thermocycler. Chemicals typically used for chromatographic techniques did not affect the digestion efficiency. Tryptic digestion was performed in a small volume (3 μL) with 1.5 μg of trypsin without denaturing agents. This rapid procedure yielded more peptides than conventional methods utilizing chemical denaturation for 18 proteins out of 20. Samples were directly spotted on the MALDI-TOF target plate, without additional purification, thus reducing losses on reversed phase resins.Mass spectrometry (MS) has become an indispensable technique in protein research. 1-4 Usually, denatured proteins are digested with a proteinase and the peptides can then be analyzed by MS. The mass spectrum is compared with the peptide mass fingerprints in databases for protein identification. 1-4 Such mass mapping requires purified proteins. Therefore, this technique is usually used in conjunction with protein fractionation methods such as liquid chromatography (LC) or one-and two-dimensional gel electrophoresis (1DE/2DE). Today multidimensional chromatography and analysis techniques based on computer-controlled HPLC systems are being adopted by industry and academia. 1,5-8 MDCA generally processes proteins in their native conformations rather than denatured as in SDS gels. Native proteins are also isolated by bimolecular interaction analysis instruments, such as BIAcore, which utilize the biological specificity of native proteins for other proteins or DNA for isolation of proteins from complex mixtures. 9-12 Subsequent chemical denaturation protocols 12,13 have been applied to the output of these separation techniques followed by conventional overnight trypsin digestion, with subsequent solid-phase capture and elution of samples for MS analysis.The use of direct protease digestion of native proteins from liquid chromatography or BIAcore would be a great advantage for further analysis by MS. Automation of the integrative LC-MS analysis is currently difficult mainly due to the complicated process of protein digestion. Some proteins are susceptible to proteinases and can be digested under standard conditions (37 °C, neutral pH, etc.), whereas most proteins are not digested effectively. To achieve efficient digestion, those proteins require a pretreatment step that denatures the protein prior to proteinase digestion. Proteins are usually unfolded using chemical denaturants. 13-15 Denaturation at 90 °C has been reported as an alternative to chemical denaturation of proteins. 15 Such denatured proteins are generally aggregated, forming large clumps of protein(s). The process of digestion of such aggregates is very slow (typically overnight digestion is required).*To whom correspondence should be addressed. E-mail: ot16@leicester.ac.uk. It is in the bounds of possibilit...

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