Improving Protein Digestion for Mass Spec Analysis

The latest Promega Webinar covered trypsin and protein analysis for mass spectrometry. Here we summarize some of the discussion from this webinar.

Guest Blog by Lynn Litterer, Promega Technical Services Scientist

You are studying your favorite biological system and fascinating questions about how it works. Chances are good that some of those questions involve protein interaction with other proteins and molecules in the cell. Where and when proteins are active is often regulated by post-translational modification. Mass spectrometry (mass spec, MS) is a powerful tool to identify protein sequence and modifications. Strategic use of mass spec tools can substantially improve the analysis of your samples.

Why is trypsin the first choice for protein sequencing?
Trypsin has several useful features.

  1. It cleaves proteins into peptides with an average size of 700-1500 Daltons, which is in the ideal range for MS.
  2. It is highly specific, cutting at the carboxyl side of arginine and lysine residues.
  3. The C-terminal arginine and lysine peptides are charged, making them detectable by MS.
  4. Trypsin is highly active and tolerant of many additives.
  5. Trypsin can be modified by methylation of lysines to prevent self-digestion at these sites.

Why are some of the predicted trypsin sites in my protein not digested?
Protein folding may prevent access of trypsin to some potential cleavage sites. This is a common problem with hydrophobic proteins. You can improve digestion by denaturing or solubilizing your protein. ProteaseMAX™ (catalog #V2071) is a MS-compatible surfactant which relaxes protein structure resulting in much faster and more efficient digestion of difficult proteins. A combination of Lys-C (catalog #V1671) and trypsin often gives better cleavage at Lys than trypsin alone. Lys-C is more tolerant of denaturing agents than trypsin. Denaturing agents can be used for Lys-C digestion; then the sample can be diluted for trypsin digestion. Trypsin is active in 0.1% SDS, 1M urea, 1 M guanidine hydrochloride or 10% acetonitrile. (The reference for this information is Proteolytic Enzymes. A practical approach. By R.J. Beynon and J.S. Bond. IRL Press. 1989) Note that SDS is not compatible with MS or HPLC (LC-MS/MS) instruments.

How can I improve the sequence coverage of my protein?

  1. Use a more complex instrument such as LC-MS/MS. Two peptides with different sequences such as DRVYVHPF and KRPPGFSPF may have the same mass (in this case, 1032.2 Da). The second round of MS breaks the peptides into smaller fragments which have clearly distinct masses.
  2. Use several proteases individually on separate aliquots of your sample. Asp-N (catalog #V1621) cuts at the N-terminal side of Asp and Cys residues. Glu-C (catalog #V1651) cuts at the C-terminal side of Asp and Glu residues. Chymotrypsin (catalog #V1061) digests hydrophobic proteins at aromatic residues (Tyr, Phe and Trp) and may also cleave at Leu and Met.
  3. Make sure that the peptide database you are using for reference contains the sequences from your target species. If you have a C. elegans sample and the database defaults to human proteins, you may miss the identification of some peptides because of lack of homology.

How can I identify post-translational modifications in my protein?
Simple modifications such as methylation, phosphorylation or acetylation will show up as an increased mass in LC-MS/MS analysis. Make sure to select the appropriate options in your analysis software to calculate the possible masses of modified amino acids.
More complex modifications such as glycosylation can vary in mass. You can use specific glycosidases to remove all or some of the sugar residues, allowing identification of the location and type of modification by mass.

  1. Peptide:N-glycosidase F (PNGase F, Cat. #V4831, coming soon) cleaves between Asn and N-acetylglucosamine (GlcNAc) unless the GlcNAc has a fucose side group. This removes the entire glycan from all types of N-linked glycoproteins. The Asn is converted to an Asp in the reaction, leading to a 1 Da change in mass.
  2. Endoglycosidase H (Endo H, Cat.# V4871, coming soon) cleaves between the first and second GlcNAc residues in high mannose and hybrid type glycans in N-linked glycoproteins. It does not cut complex-type glycans. After cleavage there is one GlcNAc remaining on the Asn adding 203Da to the peptide mass.
  3. Protein Deglycosylation Mixture (catalog #V4931, coming soon) contains a mixture of glycosidases that remove both N-linked and O-linked glycans.

Choose your protein purification and enrichment procedure to be compatible with the modification you hope to detect. For example, include phosphatase inhibitors if you are trying to measure phosphorylation of a protein.

Mass spec analysis can give you a great deal of information about your proteins. Using different enzymes on replicates from a sample can expand your data from simple protein identification based on a few peptides to more extensive sequence information and identification of post-translational modifications.

www.promega.com/webinars provides a schedule of upcoming webinars. In addition there are links to previous webinars that allow you to either view the recording or download a pdf of the presentation. There is also a pdf of additional material available for each past webinar.

To register for a webinar, use the “registration” link at www.promega.com/webinars This allows you to not only view the webinar, but also to participate in the live chat. Need a reminder? You can also sign-up for monthly invitations to webinars at the webinars page (see link above). Note: Live chat is only available for live webinars, not links to recorded webinars.

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Promega products are used by life scientists who are asking fundamental questions about biological processes and by scientists who are applying scientific knowledge to diagnose and treat diseases, discover new therapeutics, and use genetics and DNA testing for human identification. Originally, founded in 1978 in Madison, Wisconsin, USA, Promega has branches in 16 countries and more than global distributors serving 100 countries.

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