These Thermo Scientific Pierce Proteases are small, convenient packages of individual, purified highly-specific proteases for use in various combinations to obtain better sequence coverage and protein identification for mass spectrometry.
Improvement in sequence coverage and overall protein characterization can result from improving peptide chromatographic or ionization properties. The masses of these larger peptides can be accurately measured at high charge states on popular high-resolution mass spectrometers, reducing the database search time and increasing the sequence coverage and confidence. Finally, larger peptides can be fragmented effectively by new fragmentation techniques like electron transfer dissociation (ETD) or electron capture dissociation (ECD) that leave post-translational modifications, like phosphorylation, intact.
- Specific – proteases offer specific cleavage at multiple sites, enabling exploration of primary protein structure
- Increased sequence coverage – Better protein characterization results from overlapping peptides with complementary chromatographic, ionization and fragmentation properties
- Compatible – protocols are designed for MS applications
- Robust – streamlined data processing and analysis improves interpretation and confidence
- In-gel digestion of cell lysates – multiple individual protease digestions improves the number and confidence of peptide and protein sequence identifications.
- CID- and ETD-based fragmentation – powerful for indicating specific post-translational modifications, like phosphorylation and glycosylation.
- Green stretches – quantitative and high confidence sequencing results are obtained only when multiple protease and fragmentation combinations are used.
- Pairwise combinations – search results from two protease or fragmentation methods reveal complementary results. For example, trypsin digestion of Erk1 produces 87% coverage with CID, but when combined with LysC results, the total coverage increased to 93%.
Effective protein characterization and identification by mass spectrometry (MS) begins with protein digestion, thus generating peptide fragments. Trypsin is the protease of choice for accomplishing this task; however, digestion with alternative proteases, such as GluC, LysC, AspN or chymotrypsin, which cleave at different sites can improve sequence coverage and generate overlapping peptides. For example, LysC cleavage after lysine residues generates larger peptides than those generated by trypsin alone, and chymotrypsin cleavage of hydrophobic regions provides complementary peptides to trypsin. These larger or more hydrophobic peptides interact more strongly with reverse-phase traps and columns and can improve detection of peptides with hydrophilic modifications like phosphorylation or glycosylation.
Table 1. Cleavage sites of various proteases.
||Carboxyl side of arginine and lysine residues
||Carboxyl side of lysine residues
||Carboxyl side of glutamate
||Amino side of aspartate residues
||Carboxyl side of tyrosine, phenylalanine, tryptophan and leucine
Table 2. Percent sequence coverage for Erk1. Results were obtained by digestion with individual proteases, MS/MS analysis with CID or ETD fragmentation methods, and pair-wise combination of search results in Proteome Discoverer MultiConsensus Reports for Erk1.
||Sequence Coverage (%)
|Trypsin alone + LysC alone
|Trypsin alone + GluC alone
- Biringer, R.G., et al.(2006). Brief Func Genom and Prot 5:144-153.
- Schlosser, A., et al.(2005). Anal Chem 77:5243-5250.
- Wu, S-L., et al.(2005). J Proteome Res 4:1155-1170.
Pierce Trypsin, MS Grade
In-Gel Tryptic Digestion Kit
In-Solution Tryptic Digestion and Guanidination Kit
Standard-grade Proteases (including Protease Agarose Resins)
Note: Trypsin (Part No. 90055) has been replaced by Pierce Trypsin, MS Grade.