Proteins are the core of biological activities, and studying their structure and function is critical for fields such as proteomics, structural biology, and biopharmaceuticals. To obtain high-quality peptides suitable for mass spectrometry analysis, protein samples must undergo precise enzymatic digestion, among which trypsin has become the most commonly used protease due to its high specificity.
Trypsin specifically hydrolyzes the peptide bonds at the carboxyl side of lysine and arginine residues. Unmodified trypsin is prone to autolysis, generating fragments that may interfere with protein sequencing or high-performance liquid chromatography (HPLC) peptide analysis. Moreover, autolysis may produce pseudotrypsin, which has been shown to exhibit chymotrypsin-like substrate specificity. Mass spectrometry-grade modified trypsin is porcine trypsin subjected to reductive methylation, which enables resistance to proteolytic digestion. In enzyme stability tests, no autolysis was observed for the modified trypsin, and its activity retention was superior to that of regular trypsin.
Product Overview
The sequencing grade trypsin provided by MtoZ Biolabs is a high-purity trypsin specifically optimized for protein mass spectrometry analysis (LC-MS/MS). It is suitable for applications such as protein digestion, proteomics, antibody analysis, and post-translational modification studies.
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Product Specification |
Trypsin Lyophilized Powder (100 μg) |
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Reconstitution Buffer（0.5 mL） |
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Molecular Weight |
23 kDa |
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Storage Conditions |
Lyophilized powder at -20°C Reconstituted solution at -80°C |
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Optimal pH |
7-9 |
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Shelf Life |
24 months at -20°C |
Protocol
1. Trypsin Reconstitution
(1) It is recommended to reconstitute lyophilized trypsin in 50 mM acetic acid (HAc) or 1 mM hydrochloric acid (HCl) to ensure stable enzyme activity.
(2) After reconstitution, aliquot and store at -80°C for long-term use. Avoid repeated freeze-thaw cycles to prevent loss of enzyme activity.
2. Digestion Conditions
(1) Applicable pH range: 7.0-9.0
(2) Recommended ratio: 1:100-1:25 (w/w)
(3) Temperature: 37°C, typically for 4-16 hours (specific time can be adjusted based on experimental needs).
3. Digestion Termination
To terminate digestion and prevent overdigestion, the following methods can be used:
(1) Lower the temperature: store the sample at -20°C or lower.
(2) Add termination reagents: such as 0.1% trifluoroacetic acid (TFA) at final concentration.
Features and Benefits
1. High Purity
Chromatographic purity >95% at 280 nm detection wavelength, ensuring highly specific digestion.
2. Low Autolysis
Optimized enzyme preparation process significantly reduces trypsin-derived peptide fragments, enhancing mass spectrometry data quality.
3. High Stability
Enzyme activity remains stable under recommended storage conditions, ensuring experimental reproducibility and data comparability.
4. Strong Batch-to-Batch Consistency
Strict quality control ensures batch-to-batch enzyme activity variation is less than 5%, suitable for long-term research projects.
5. Broad Compatibility
Applicable to various samples including cell lysates, tissue extracts, and recombinant proteins; compatible with mass spectrometry platforms such as MALDI-TOF, Q-TOF, and Orbitrap.
6. High Specificity
Specifically cleaves at the carboxyl side of lysine (K) and arginine (R) residues, generating highly uniform peptides favorable for subsequent database matching and quantitative analysis.
Applications
1. Proteomics
(1) Protein identification (LC-MS/MS)
(2) Post-translational modification studies (phosphorylation, acetylation, glycosylation, etc.)
(3) Protein quantification analysis (TMT, iTRAQ, Label-Free)
2. Structural Biology
(1) Sample preparation for X-ray crystallography
(2) Cryo-EM structure analysis
(3) Protein-protein interaction validation (e.g., XL-MS)
3. Biopharmaceuticals
(1) Monoclonal antibody (mAb) structure analysis
(2) Antibody drug consistency and heterogeneity studies
(3) Protein drug stability and degradation pathway analysis
MtoZ Biolabs' sequencing grade trypsin features a high-purity, low-autolysis optimized design, specifically developed for high-precision research in proteomics, structural biology, and biopharmaceuticals. Its high specificity, low background noise, and mass spectrometry compatibility ensure the accuracy and reproducibility of protein digestion, making it an ideal choice for high-quality protein research.
FAQs
Q1: What Is the Difference between Sequencing Grade Trypsin and Regular Trypsin?
A1: Sequencing grade trypsin is TPCK-treated to remove chymotrypsin activity, reducing nonspecific cleavage and ensuring high specificity and low background noise during digestion, making it suitable for mass spectrometry (LC-MS/MS) analysis. Regular trypsin may contain higher levels of autolysis products, affecting the reliability of protein identification and data quality.
Q2: Is Pre-activation Required?
A2: No pre-activation is needed. MtoZ Biolabs' sequencing grade trypsin is optimized for direct use upon reconstitution without additional processing.
Q3: Is It Suitable for Membrane Protein Samples?
A3: Yes, it is suitable for membrane protein samples. It is recommended to perform pretreatment using SDS, urea, or organic solvents to enhance Trypsin digestion efficiency in hydrophobic environments.
Q4: How Should Trypsin Be Stored after Reconstitution?
A4: After reconstitution, it is recommended to aliquot and store at -80°C for long-term preservation, avoiding repeated freeze-thaw cycles that can reduce enzymatic activity. For short-term use, it can be stored at -20°C, but repeated freeze-thawing should still be avoided to maintain optimal digestion performance.
Q5: How Can Trypsin Autolysis Be Prevented?
A5: Adding Ca²⁺ (e.g., 1 mM CaCl₂) can improve trypsin stability and reduce autolysis. It is also important to use the recommended buffer systems and avoid extreme pH conditions to ensure high digestion efficiency.
Q6: Is It Compatible with iTRAQ/TMT Labeling Experiments?
A6: It is fully compatible with iTRAQ and TMT labeling experiments. To ensure optimal experimental results, it is recommended to perform desalting before the labeling reaction to remove excess reagents and prevent interference with downstream experiments.
