rChymoSelect™ MS Grade Protease
Recombinant Chymotrypsin for Peptide Mapping and Proteomics
- High specificity for Tyr, Phe and Leu
- Eliminates non-specific cleavages
- Free from trypsin contamination
- Resistant to autoproteolysis for cleaner peptide maps
- Consistent lot-to-lot cleavage performance
- Does not require calcium for stability
- Engineered and optimized for peptide-mapping workflows
Catalog Number:
Size
Catalog Number: VB8100
Catalog Number: VB8200
Chymotrypsin Optimized for Mass Spec: Cleavage Specificity, Stability, Reproducibility
Chymotrypsin has long served as a critical orthogonal protease to trypsin in peptide mapping and proteomics. However, traditional chymotrypsins purified from bovine pancreas have the following shortcomings: high non-specific cleavages, susceptibility to autoproteolysis, calcium dependency and inconsistent lot-to-lot performance. These deficiencies complicate mass spec workflows, especially in regulated biotherapeutic characterization.
Our novel recombinant chymotrypsin, called rChymoSelect™ MS Grade Protease, addresses all these pain points and sets a new benchmark for performance. This chymotrypsin belongs to a different class of chymotrypsin-like proteases than bovine chymotrypsin. Expressed in Pichia pastoris and purified to >98% homogeneity, it is engineered for high specificity, stability and reproducibility. It selectively cleaves at the C-terminus of Tyrosine, Phenylalanine and Leucine while avoiding non-specific cleavage at residues such as Alanine, Methionine, Histidine, Threonine, Valine, Serine, Glycine and others. rChymoSelect™ MS Grade Protease does not cleave after Tryptophan, enabling accurate analysis of tryptophan oxidation, a critical post-translational modification in biotherapeutic proteins.
Unlike conventional chymotrypsin, rChymoSelect™ MS Grade Protease is resistant to autoproteolysis, independent of calcium for stability, and free of trypsin contamination. rChymoSelect™ MS Grade Protease is not just a better chymotrypsin; it’s the first recombinant chymotrypsin designed specifically for mass spec-based peptide mapping and proteomics, delivering unmatched quality of peptide maps and consistency across every digest.
rChymoSelect™ MS Grade Protease vs. Traditional Chymotrypsin
rChymoSelect™ MS Grade Protease reduces non-specific cleavage, improving peptide analysis. Panel A. UV chromatograms comparing traditional chymotrypsin and rChymoSelect™ MS Grade Protease digestion of a model substrate. Traditional chymotrypsin produces a complex peptide mixture with numerous semi- and non-specific cleavage products (blue arrows). In contrast, rChymoSelect™ MS Grade Protease yields a simplified peptide profile with increased peak intensity for specific cleavages. Panel B. MS-based quantification of cleavage specificity shows that traditional chymotrypsin cleaves at multiple unintended sites. rChymoSelect™ MS Grade Protease demonstrates high fidelity for phenylalanine (F), tyrosine (Y) and leucine (L) while avoiding cleavage at tryptophan.
rChymoSelect™ MS Grade Protease exhibits resistance to autoproteolysis under standard digestion conditions. UPLC chromatograms show that traditional chymotrypsin generates numerous autoproteolytic fragments (blue arrows) following a 2-hour incubation at 25°C in 50mM Tris-HCl (pH 7.5). In contrast, rChymoSelect™ MS Grade Protease remains intact under the same conditions, demonstrating enhanced resistance to autoproteolysis.
rChymoSelect™ MS Grade Protease enables simpler analysis of W311 oxidation in panitumumab by avoiding cleavage after tryptophan. Mass spectrometry analysis of peptides containing W311 (tryptophan at position 311 in the heavy chain of panitumumab) following digestion with either traditional chymotrypsin or rChymoSelect™ MS Grade Protease. Traditional chymotrypsin produces multiple overlapping peptides containing W311, complicating quantitation and retention time assignment. In contrast, rChymoSelect™ MS Grade Protease generates cleaner fragmentation patterns, simplifying data interpretation and enhancing quantification accuracy for W311 oxidation.
rChymoSelect™ MS Grade Protease extends sequence coverage in regions not covered by trypsin. Trypsin cleaves at lysine (K) and arginine (R), which sometimes can produce peptides that are either too short (when cleavage sites are clustered) or too long (in R/K-scarce, aromatic-rich regions), both of which may escape detection in MS analysis. For example, in the panitumumab heavy chain, trypsin digestion results in discontinuous sequence coverage across residues 317–347. In contrast, rChymoSelect™ MS Grade Protease efficiently cleaves at phenylalanine (F), tyrosine (Y) and leucine (L), generating a well-sized, contiguous peptide in this region, complementing tryptic digestion and enhancing overall sequence coverage.
Key Differences Between Traditional Bovine Chymotrypsin and Recombinant rChymoSelect™ MS Grade Protease.
|
Feature |
Traditional Chymotrypsin (Bovine) |
rChymoSelect™ MS Grade Protease |
|
Source |
Bovine pancreas (animal-derived) |
Pichia pastoris (recombinant) |
|
Cleavage Specificity |
Tyr, Phe, Trp + non-specific sites |
Tyr, Phe, Leu, (Met*) |
|
Trp Cleavage |
Yes |
No |
|
Autoproteolysis |
High |
None** |
|
Calcium Dependency |
Yes |
No |
| Trypsin Contamination |
Possible |
None |
| Lot-to-Lot Reproducibility |
Moderate |
High |
| MS Compatibility |
Variable, lower peptide quality |
Optimized for clean peptide maps |
*Protein-dependent.
**Under recommended digestion conditions.
Protocols
Frequently Asked Questions
What is rChymoSelect™ MS Grade Protease?
rChymoSelect™ MS Grade Protease is a recombinant chymotrypsin engineered specifically for mass spectrometry peptide mapping and bottom-up proteomics. Expressed in Pichia pastoris and purified to greater than 98% homogeneity, it is supplied lyophilized and cleaves specifically at tyrosine, phenylalanine and leucine. It is designed to replace bovine pancreatic chymotrypsin in workflows that require high specificity and lot-to-lot consistency.
What are the cleavage sites of rChymoSelect™ MS Grade Protease?
rChymoSelect cleaves at the C-terminus of tyrosine (Y), phenylalanine (F) and leucine (L). Cleavage at methionine may also occur and is protein-dependent. It does not cleave after tryptophan, and it avoids the non-specific cleavage at alanine, histidine, threonine, valine, serine and glycine that is commonly observed with bovine chymotrypsin.
Does rChymoSelect cleave after tryptophan?
No. rChymoSelect does not cleave after tryptophan, unlike traditional bovine chymotrypsin. Each tryptophan therefore stays intact within a single peptide, which makes tryptophan oxidation directly quantifiable rather than distributed across multiple overlapping peptides. This is the primary reason to select rChymoSelect for oxidation monitoring in monoclonal antibodies and other biotherapeutic proteins.
Why use rChymoSelect as an orthogonal protease to trypsin?
Trypsin cleaves at lysine and arginine, producing peptides that are too short where those residues cluster and too long in aromatic-rich, lysine- and arginine-scarce regions; both can escape MS detection. rChymoSelect cleaves at aromatic and hydrophobic residues instead. In the panitumumab heavy chain, trypsin gave discontinuous coverage across residues 317 to 347, while rChymoSelect generated a single contiguous peptide across that region.
How does rChymoSelect improve tryptophan oxidation analysis?
Because rChymoSelect does not cleave after tryptophan, oxidation sites sit within single, well-defined peptides. Traditional chymotrypsin produces multiple overlapping W311-containing peptides in panitumumab, which complicates quantitation and retention time assignment. rChymoSelect generates cleaner fragmentation patterns, simplifying data interpretation and improving quantification accuracy for this critical product quality attribute.
Is rChymoSelect resistant to autoproteolysis?
Yes. After 2 hours at 25°C in 50mM Tris-HCl (pH 7.5), traditional chymotrypsin generated numerous autoproteolytic fragments while rChymoSelect remained intact. Autoproteolytic fragments contaminate peptide maps and consume enzyme activity during digestion. rChymoSelect is also calcium-independent and free of trypsin contamination, removing two further sources of variability in bovine-derived preparations.
Is rChymoSelect suitable for regulated biotherapeutic characterization?
Yes, for research use. The non-specific cleavage, autoproteolysis, calcium dependency and lot-to-lot variability of bovine chymotrypsin are precisely the deficiencies that complicate regulated biotherapeutic characterization, and rChymoSelect addresses each of them. Note that the current Limited Use Label License permits research use only; contact Promega for supply and licensing terms covering diagnostic, therapeutic or commercial use.
In what format and sizes is rChymoSelect supplied?
rChymoSelect™ MS Grade Protease is supplied as a lyophilized recombinant protease in 25µg and 100µg sizes. The lyophilized format supports long-term stability and allows reconstitution at the concentration your digestion workflow requires. The 25µg size suits method development and low-volume peptide mapping; the 100µg size supports routine or higher-throughput digestion.
Which protease should I pair with trypsin to extend sequence coverage?
Choose based on the residue composition of the region trypsin misses. rChymoSelect is the right choice for aromatic and hydrophobic-rich regions that are scarce in lysine and arginine. For acidic regions, use rAsp-N (N-terminal of aspartate) or Glu-C (C-terminal of glutamate). Lys-C and rLys-C cleave at lysine, and Arg-C Ultra cleaves at arginine, including Arg-Pro.
Specifications
Catalog Number:
What's in the box?
| Item | Part # | Size |
|---|---|---|
rChymoSelect™ MS Grade Protease (lyophilized) |
VB810A | 1 × 25ÎĽg |
SDS
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Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
Anti-HiBiT Antibody Limited Use Label License
NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.
This product and its derivatives may not be further transferred by the researcher and the purchased quantity of the product may be used only by the researcher, and then only for (1) research use, which may include drug discovery and development research; and (2) use in provision of services, that result in transfer of information or data only. No other commercial use of this product or derivatives is allowed. “Commercial use” means any and all uses of this product or derivatives by a party in exchange for money or other consideration, including, but not limited to, (1) use in further product manufacture; and (2) resale of the product or its derivatives, whether or not such product or derivatives are resold for use in research. Researcher may not attempt to reverse engineer this product by any method including, without limitation, any method of sequencing any portion of the product.
With respect to any uses outside this label license, including, without limitation, any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.
What's in the box?
| Item | Part # | Size |
|---|---|---|
rChymoSelect™ MS Grade Protease (lyophilized) |
VB820A | 1 × 100ÎĽg |
SDS
Search for SDSCertificate of Analysis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Storage Conditions
Anti-HiBiT Antibody Limited Use Label License
NOT FOR MEDICAL DIAGNOSTIC USE. FOR IN VITRO USE ONLY. BY USE OF THIS PRODUCT, RESEARCHER AGREES TO BE BOUND BY THE TERMS OF THIS LIMITED USE LABEL LICENSE. If researcher is not willing to accept the terms of this label license, and the product is unused, Promega will accept return of the unused product and provide researcher with a full refund.
This product and its derivatives may not be further transferred by the researcher and the purchased quantity of the product may be used only by the researcher, and then only for (1) research use, which may include drug discovery and development research; and (2) use in provision of services, that result in transfer of information or data only. No other commercial use of this product or derivatives is allowed. “Commercial use” means any and all uses of this product or derivatives by a party in exchange for money or other consideration, including, but not limited to, (1) use in further product manufacture; and (2) resale of the product or its derivatives, whether or not such product or derivatives are resold for use in research. Researcher may not attempt to reverse engineer this product by any method including, without limitation, any method of sequencing any portion of the product.
With respect to any uses outside this label license, including, without limitation, any diagnostic, therapeutic, prophylactic or commercial uses, please contact Promega for supply and licensing information. PROMEGA MAKES NO REPRESENTATIONS OR WARRANTIES OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING FOR MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE WITH REGARDS TO THIS PRODUCT. The terms of this label license shall be governed under the laws of the State of Wisconsin, USA.
Resources
Featured Resource
Maximizing rChymoSelect Performance for Biotherapeutic Protein Peptide Mapping
rChymoSelect™ MS Grade Protease is a novel, non-bovine chymotrypsin offering enhanced peptide mapping performance, which enables broader sequence coverage and more confident identification of post-translational modifications such as tryptophan oxidation. This poster discusses how we optimized the reaction conditions to maximize the performance of rChymoSelect for peptide mapping of biotherapeutic proteins.
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