Proteins found in nature vary in size from 5 kDa to greater than 400 kDa. Protein digestion presents the process to cut proteins into shorter fragments, known as peptides. It allows for the identification and characterization of proteins according to their properties. Protein digestion is a crucial step prior to mass spectrometry (MS) analysis of peptides for successful protein identification and characterization, biomarker discovery, and systems biology. Although it is possible for MS to study intact proteins, the smaller peptides facilitate protein identification and improve the coverage of proteins that might be reduced due to solubility and heterogeneity. Therefore, the most common proteomic approaches utilize site-specific digestion to generate smaller fragments. Peptides are easier for separation and characterization by high performance liquid chromatography (HPLC) and HLPC-coupled MS.
Figure 1. The general workflow of MS analysis.
Overview of Protein Digestion Service
There are two methods for protein digestion at Creative Proteomics, (1) in-gel digestion (protein digestion in polyacrylamide gel matrix); (2) in-solution digestion (protein precipitation in chloroform/methanol followed by resolubilization and digestion in urea). These strategies are designed to solubilize hydrophobic proteins, digest protein with trypsin or other proteases, and remove SDS. The in-gel digestion methodology has become routine for proteins separated by 1-D or 2-D electrophoresis. Although robust, effective, and reproducible, in-gel digestion is laborious and time-consuming. The in-solution digest method requires less time, but it introduces sample losses due to low re-solubilization of aggregated proteins.
Workflow of Protein Digestion
At Creative Proteomics, we provide both in-gel and in-solution protein digestion services for protein analysis. The workflow of protein digestion consists of the following steps:
1. Lysate preparation. Lysis, fractionation, depletion, enrichment, and dialysis.
2. In-solution or in-gel digestion.
a. Protein denaturation using chaotropic agents such as urea and guanidine;
b. Reduction of disulfide bridges using DTT;
c. Alkylation of the cysteines by iodoacetic acid or iodoacetamide;
d. Remove regents and exchange buffer;
e. Overnight denaturation with trypsin or other proteases in an ammonium bicarbonate buffer at suitable pH and temperature for about 18 h;
f. Stop the digestion by the addition of (formic) acid.
3. Protein enrichment/cleanup.
We also provide the high-throughput, automated protein digestion service with 96-well formats.
Figure 2. The workflow of protein digestion.
We accept a range of samples, such as:
- Native proteins from natural sources
- Fusion and tagged proteins from recombinant sources
- Antibodies of different isotypes
- Membrane proteins
Please submit at least 100 ug of protein. For proteins in solution, the concentration should be greater than 1 mg/mL.
Advantages of Protein Digestion
- Flexibility. We provide both in-gel and in-solution protein digestion strategies.
- Accuracy and improved coverage. Our digestion service can greatly improve the accuracy and coverage of MS analysis.
- Multiple applications. Facilitate protein identification and characterization, discover disease biomarkers, and contribute to the understanding of biological processes.
- Complete proteomics services. We provide complete proteomics services that cover the whole process of proteomics analysis.
Creative Proteomics provides in-gel and in-solution protein digestion services for successful protein and proteome analysis. As every project has different requirements, please contact our specialists to discuss your specific needs.
- Switzar L, Giera M, Niessen W M A. Protein digestion: an overview of the available techniques and recent developments. Journal of proteome research, 2013, 12(3): 1067-1077.