Protein Hydrolysis Service

Proteins found in nature vary in size from 5 kDa to 400 kDa. The protein hydrolysis process cuts proteins into shorter segments, namely peptides. Protein hydrolysis is a crucial step before peptide mass spectrometry identification analysis, providing a foundation for successful protein identification and characterization, as well as biomarker discovery. Although mass spectrometry (MS) can study intact proteins, smaller peptides are easier to identify proteins and improve protein coverage because protein coverage may decrease due to solubility and heterogeneity. Therefore, the most common proteomics methods often utilize site-specific cleavage sites to produce smaller peptide fragments. Smaller peptides are easier to separate and characterize using high performance liquid chromatography (HPLC) and HPLC-MS.

 

Services at MtoZ Biolabs

MtoZ Biolabs offers two methods for protein hydrolysis: (1) in-gel digestion (protein hydrolysis in polyacrylamide gel matrix); (2) in-solution digestion (protein precipitation in chloroform/methanol, then redissolved in urea). Proteins separated by 1D or 2D gel electrophoresis can be hydrolyzed directly in the gel. In-gel digestion is effective and reproducible but more labor-intensive and time-consuming. Compared to in-gel digestion, the in-solution digestion method requires less effort and less time, but proteins hydrolyzed in solution may have low resolubility, possibly leading to some sample loss.

 

Analysis Workflow

MtoZ Biolabs can provide both in-gel digestion and in-solution digestion services. The workflow of protein hydrolysis includes:

1. Prepare Lysis Solution

2. Enzymatic Digestion in Solution or Gel

3. Denature Proteins using Denaturants (e.g., Urea and Guanidine)

4. Reduce Disulfide Bonds using DTT

5. Alkylate Cysteine Residues with Iodoacetic Acid or Iodoacetamide

6. Remove Reagents and Exchange Buffer

7. Denature Overnight (~18 Hours) with Trypsin or other Proteases in Ammonium Bicarbonate Buffer at an Appropriate PH and Temperature

8. Stop Digestion by Adding Acidic Substances

9. Enrich/Elute Proteins

 



Figure 1. Protein Hydrolysis Workflow

 

Sample Submission Requirements

MtoZ Biolabs can handle various sample types, including but not limited to:

1. Proteins from Natural Sources

2. Recombinant Fusion Proteins and Tagged Proteins

3. Different Subtypes of Antibodies

4. Membrane Proteins and Others

 

Service Advantages

1. Flexibility

We offer both in-gel and in-solution digestion strategies.

 

2. High Coverage and High Accuracy

Our hydrolysis service can greatly improve the accuracy and coverage of mass spectrometry analysis.

 

3. Wide Applications

Facilitates protein identification and characterization, discovers disease biomarkers, and helps understand biological processes.

 

4. Comprehensive Proteomics Services

We offer comprehensive proteomics services covering the entire process of proteomics analysis, providing one-stop solutions for your omics needs.