Protein is a central functional molecule in cellular life activities, involved in numerous biological processes such as cellular metabolism, signal transduction, immune regulation, and disease progression. The study of protein structure and function is essential in fields such as biomedicine, proteomics, and drug development. Mass spectrometry (MS) is one of the most advanced techniques for protein analysis. However, prior to MS analysis, protein samples typically need to undergo digestion to break them down into detectable peptides, thereby improving detection sensitivity and accuracy.
Conventional protein digestion methods each have their own advantages and disadvantages. In-gel digestion is suitable for complex protein samples but tends to be labor-intensive. In contrast, in-solution digestion has become the preferred method for MS sample preparation due to its high protein recovery rate and operational simplicity. The in-solution tryptic digestion kits can streamline the digestion process, enhance protein cleavage efficiency, and provide high-quality peptides for subsequent mass spectrometry analysis, protein identification, and post-translational modification (PTM) studies.
Product Overview
The in-solution tryptic digestion kits utilize high-purity trypsin in combination with optimized buffers for protein reduction, alkylation, and enzymatic digestion, ensuring efficient protein degradation and generation of high-quality peptides suitable for mass spectrometry analysis. It is compatible with a wide range of protein sample types, including cell lysates, plasma proteins, tissue extracts, and recombinant proteins. The kit is widely applicable in protein identification, quantitative proteomics, and post-translational modification analyses such as phosphorylation and glycosylation.
Product Details
|
Product Details |
Size |
Storage Conditions |
|
DTT（1M） |
1 mL |
-20℃ |
|
IAM（1M） |
1 mL |
|
|
Trypsin |
25 μg/vial; 10 vials |
|
|
Trypsin Dilution Buffer |
3 mL |
4℃ |
Protocol
The in-solution tryptic digestion kits provide an optimized solution-based digestion protocol that enables rapid protein digestion with a simple and efficient workflow. It is suitable for various laboratory settings and experimental scales. The following are the recommended standardized steps; researchers may adjust the digestion time according to experimental needs.
1. Protein Sample Preparation
Take an appropriate amount of protein sample (ensure the protein concentration meets experimental requirements) and dissolve it in the provided buffer.
Note: If the protein sample contains interfering substances such as SDS or salts, impurities can be removed by ultrafiltration, acetone precipitation, or dialysis.
2. Reduction and Alkylation
Take 100 μL of protein solution as an example. The reagents listed below can be scaled proportionally:
(1) Reduction: Accurately add 1 μL of 1 M DTT solution to the sample using a pipette to achieve a final DTT concentration of 10 mmol/L. Incubate in a 56°C water bath for 1 hour.
(2) Accurately add 2 μL of 1 M IAM solution to the sample using a pipette to achieve a final IAM concentration of 20 mmol/L. React at room temperature in the dark for 40 minutes.
(3) Accurately add 1 μL of 1 M DTT solution to the sample using a pipette to achieve a final DTT concentration of 10 mmol/L to neutralize unreacted IAM.
3. Trypsin Digestion
(1) Add trypsin at a trypsin-to-protein ratio of 1:50 (w/w) (enzyme concentration: 0.25 μg/μL).
Gently mix to avoid vigorous shaking that may inactivate the enzyme.
(2) Incubate at 37°C in a constant temperature incubator for 3-4 hours or overnight (12-16 hours for complete digestion) to ensure thorough digestion of proteins into peptides.
(3) To stop digestion, add 1% formic acid or 0.5% TFA to inhibit protease activity.
4. Peptide Purification and Detection (Optional)
(1) Use C18 solid-phase extraction (SPE) to purify peptides, remove salts and impurities, and improve mass spectrometry sensitivity.
(2) Peptides can be directly used for LC-MS/MS mass spectrometry analysis, proteomics research, and protein function validation.
5. Peptide Storage
(1) Short-term storage (1-2 weeks): Store at -20°C.
(2) Long-term storage (>2 weeks): Store at -80°C in ultra-low temperature conditions. Avoid repeated freeze-thaw cycles to prevent degradation.
Product Notes
(1) Ensure that the protein sample is free of SDS, salts, or other interfering substances.
(2) Excessive trypsin may cause nonspecific digestion; optimize the trypsin-to-protein ratio based on experimental needs.
(3) Avoid high-temperature incubation that may lead to protein denaturation and trypsin inactivation.
(4) If not analyzed immediately after digestion termination, store at low temperature (-20°C) to prevent degradation.
Features and Benefits
1. High Digestion Efficiency
Utilizes high-purity trypsin with strong specificity to avoid nonspecific degradation and improve digestion efficiency. The optimized digestion system ensures thorough protein unfolding and enhances enzymatic cleavage performance.
2. Applicable to Various Sample Types
Suitable for cell lysates, tissue-extracted proteins, plasma proteins, recombinant proteins, and other sample types. Compatible with different protein concentrations by adjusting digestion parameters to fit diverse experimental protocols.
3. High Protein Recovery and Data Stability
Solution digestion effectively avoids protein loss caused by in-gel digestion, improving protein recovery. The in-solution tryptic digestion kits have been optimized and tested to ensure high reproducibility and stability across different batches.
4. Ready-to-Use Reagents
Ready-to-use reagents with no need for complex instruments. Simple operation reduces experimental variability and ensures data reproducibility.
Applications
1. Post-Extraction Digestion of Cellular or Tissue Proteins
Applicable for digestion of samples such as cell lysates, tissue homogenates, and serum/plasma proteins.
2. Protein Identification
Suitable for LC-MS/MS analysis, enabling the digestion of complex protein samples into appropriate peptides to enhance sensitivity and accuracy of protein identification.
3. Post-Translational Modifications (PTMs) Analysis
Applicable for studying phosphorylation, acetylation, methylation, glycosylation, and other PTMs. When combined with enrichment strategies, it improves the detection of modified peptides.
4. Protein Quantitative Analysis
Compatible with proteomic quantification strategies such as Label-Free, TMT, and iTRAQ, allowing comparative analysis of protein expression under different experimental conditions.
FAQs
Q1: What Sample Types Are Compatible with the Kits?
A1: The in-solution tryptic digestion kits are suitable for cell lysates, tissue proteins, recombinant proteins, and serum/plasma proteins.
Q2: How Can Digestion Efficiency Be Optimized?
A2: Digestion efficiency can be improved by adjusting the Trypsin-to-protein ratio (1:50 to 1:100), extending the incubation time (3-16 hours), or maintaining incubation at 37°C.
Q3: Are the Resulting Peptides Suitable for Mass Spectrometry Analysis?
A3: Yes. The kit’s optimized buffer system does not interfere with LC-MS/MS analysis and enables the generation of high-quality peptides.
Q4: How Should the Digestion Reaction Be Terminated?
A4: Add 1% formic acid (FA) or trifluoroacetic acid (TFA) to inhibit trypsin activity and prevent peptide degradation.
