It’s time to analyze your protein and you are trying to decide where to begin. You are asking questions like: Which protease do I choose? How much enzyme should I use in my digest? How long should I perform my digest?
Unfortunately, there is no one-size fits all answer to this type of question other than… “well it depends.” All protease digests will be a balance between denaturing the protein sample to allow access to cleavage sites, optimizing conditions for the protease to function, and compatibility with your workflow and downstream applications. We provide general guidelines that work for most samples, but frequently you will need to optimize the conditions need for your specific sample and application.
Here, I use the example of a trypsin digest for downstream mass spectrometry to highlight key questions to ask and factors that can be optimized for any digest. Continue reading
Asp-N, Sequencing Grade, is an endoproteinase that hydrolyzes peptide bonds on the N-terminal side of aspartic and cysteic acid residues: Asp and Cys. Asp-N activity is optimal in the pH range of 4.0–9.0. This sequencing grade enzyme can be used alone or in combination with trypsin or other proteases to produce protein digests for peptide mapping applications or protein identification by peptide mass fingerprinting or MS/MS spectral matching. It is suitable for in-solution or in-gel digestion reactions.
The following references illustrate the use of Asp-N in recent publications:
Protein sequence coverage
- Jakobsson, M et al. (2013) Identification and characterization of a novel Human Methyltransferase modulating Hsp70 protein function through lysine methylation. J. Biol. Chem. 288, 27752–63.
- Carroll, J. et. al. (2013) Post-translational modifications near the quinone binding site of mammalian complex I. J. Biol. Chem. 288, 24799–08.
- Siguier, B. et al. (2014) First structural insights into α-L-Arabinofuranosidases from the two GH62 Glycoside hydrolase subfamilies. J. Biol. Chem. 289, 5261–73.
- Vakhrushev, S. et al. (2013) Enhanced mass spectrometric mapping of the human GalNAc-type O-glycoproteome with SimpleCells. Mol. Cell. Prot. 12, 932–44.
- Berk, J. et al. (2013) . O-Linked β-N- Acetylglucosamine (O-GlcNAc) Regulates emerin binding to autointegration Factor (BAF) in a chromatin and Lamin B-enriched “Niche”. J. Biol. Chem. 288, 30192–09.
- Roux, P. and Thibault, P. (2013) The Coming of Age of phosphoproteomics –from Large Data sets to Inference of protein Functions. Mol. Cell. Prot. 12, 3453–64.
PNGase F (Cat.# V4831) is a recombinant glycosidase cloned from Elizabethkingia meningoseptica and overexpressed in E. coli, with a molecular weight of 36kD.
PNGase F catalyzes the cleavage of N-linked oligosaccharides between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and
complex oligosaccharides from N-linked glycoproteins. PNGase F will not remove oligosaccharides containing alpha-(1,3)-linked core fucose,
commonly found on plant glycoproteins.
Determining whether a protein is in fact glycosylated is the initial step in glycoprotein analysis. Polyacrylamide gel electrophoresis in the
presence of sodium dodecyl sulfate (SDS-PAGE) has become the method of choice as the final step prior to mass spec analysis. Glycosylated proteins often migrate as diffused bands by SDS-PAGE. A marked decrease in band width and change in migration position after treatment with PNGase F is considered evidence of N-linked glycosylation.
Gel based data are often correlated with information obtained from mass spec analysis. Asn-linked type glycans can be cleaved enzymatically by PNGase F yielding intact oligosaccharides and a slightly modified protein in which Asn residues at the site of de-N-glycosylation are converted to Asp, by converting the previously carbohydrate-linked asparagine into an aspartic acid, a monoisotopic mass shift of 0.9840Da is observed. The deglycosylated peptides are then analyzed by tandem mass spectrometry (MS/MS), and software algorithms are used to correlate the experimental fragmentation spectra with theoretical tandem mass spectra generated from peptides in a protein database.
Arg-C (clostripain), Sequencing Grade (Cat.# V1881), is a specific endoproteinase isolated from the soil bacterium Clostridium histolyticum. It preferentially cleaves at the C-terminal side of arginine (R) residues. Unlike trypsin, Arg-C efficiently cleaves arginine sites followed by proline (P). This difference is important because every twentieth arginine is followed by proline. To illustrate this benefit, Arg-C was evaluated for protein analysis in two different experiments. In the first experiment, we studied the use of Arg-C for proteomic analysis. Yeast provides an excellent model proteome because its genome is well annotated. Yeast extract was digested in two parallel reactions, using trypsin in the first reaction and Arg-C in the second, using a conventional protocol consistent with LC-MS/MS analysis. As expected the trypsin digestion resulted in a high number of peptide and protein identifications (Figure 1). However, many peptides remained elusive. The parallel Arg-C digestion complemented the trypsin digestion by recovering an additional 2,653 peptides and providing a 37.4% increase in the number of identified peptides. Digesting with Arg-C also resulted in an increase in the number of identified proteins. In fact, 138 new proteins were identified in Arg-C digest compared to the parallel trypsin digest, offering a 13.4% increase in the overall number of identified proteins.
Figure 1. Side-by-side analysis of trypsin-digested and Arg-C digested yeast proteins.
In a second experiment, the ability of Arg-C to analyze individual proteins was analyzed, selecting human histone H4 as a model protein. Like other histones, this protein is heavily modified post translational modifications (PTMs) that alter histone structure and regulate interaction with transcription factors. As a result, histone PTMs are implicated in gene regulation and associated with multiple disorders. Technical challenges, however, impede histone PTM analysis. Histone PTMs are complex and some, such as acetylation and methylation, prevent trypsin digestion, as shown by our data. In this experiment, trypsin digestion of histone H4 identified several PTMs (Figure 2). However, certain PTMs were missing. By digesting histone H4 with Arg-C, we were able to identify the missing PTMs including mono-, dimethylated and acetylated lysine and arginine residues. We speculate that the PTMs in human histone H4, which modified arginine and lysine residues, rendered trypsin unsuitable for preparing the corresponding histone regions for mass spectrometry. The problem was rectified by replacing trypsin with Arg-C.
Figure 2. Identification of histone h4 PTMs after Arg-C digestion.
One of the approaches to identify proteins by mass spectrometry includes the separation of proteins by gel electrophoresis or liquid chromatography. Subsequently the proteins are cleaved with sequence-specific endoproteases. Following digestion the generated peptides are investigated by determination of molecular masses or specific sequence. For protein identification the experimentally obtained masses/sequences are compared with theoretical masses/sequences compiled in various databases.
Nonspecific proteases such as pepsin, proteinase K, elastase and thermolysin can offer an alternative to traditional sequence-specific proteases for certain applications. The following references illustrate the use of nonspecific proteinases for the mass spec analysis of proteins:
Papasotiriou, D. et al. (2010) Peptide mass fingerprinting after less specific in-gel proteolysis using MALDI-LTQ-Orbitrap and 4-chloro-alpha-cyanocinnamic acid. J. Proteome. Res. 9, 2619–29. This reference demonstrates the use of either chymotrypsin, elastase, trypsin or proteinase K in combination with matrix CHCA for increase peptide identification and sequence coverage using MALDI.
Neue, K. et al. (2011) Elucidation of glycoprotein structures by unspecific proteolysis and direct nanoESI mass spectrometric analysis of ZIC-HILIC-enriched glycopeptides. J. Proteome. Res. 10, 2248–60. Notes use of thermolysin or elastase in combination with ZIC-HILIC enrichment as alternative method for the characterization of glycopeptides.
Baeumlisberger, D et al. (2011) Simple dual-spotting procedure enhances nLC-MALDI MS/MS analysis of digests with less specific enzymes. J. Proteome. Res. 10, 2889–94. Data noted that samples digested with elastase followed by nLC separation and subsequent alternative spotting on both MALDI-LTQ-Orbitrap and MALDL-TOF/TOF instruments resulted in 32% additional peptides.