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A Pivotal Stage in Proteomic Sample Preparation: Enzymatic Digestion

Proteomics is an analytical technique that examines protein expression, structures, functions, and interactions in a particular cell, tissue, body fluid, or organism. Protein composition and abundance are currently analyzed to identify disease markers or treatment mechanisms, as all changes in proteomes indicate pathological or biological processes [1].

 

Research Methodologies in Proteomics

The proteomic analysis method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as the mainstream approach for proteomic profiling, superseding the traditional gel - based methods. This is primarily due to its high-throughput capability, enabling the analysis of thousands of peptides and proteins within a relatively short time frame [2].

Building upon this, the proteomic analysis strategies are further categorized into the bottom-up and top-down approaches. Currently, the bottom-up strategy has gained widespread adoption. This is because peptides are generally easier to separate and identify compared to intact proteins[3].

 

Figure 1. The contrast between Bottom-up and Top-down[2].
Table 1. Comparison of the analysis objects and applicable scopes of Bottom-up and Top-down.
Sample Preparation in Bottom - Up Proteomics

The proteomic workflow based on LC-MS/MS consists of three main steps: sample preparation, protein/peptide separation, and data analysis[4].  Sample preparation is a crucial stage that significantly impacts the efficiency of proteomic research.

The typical sample preparation process in bottom-up proteomics generally encompasses protein extraction, reduction/alkylation, enzymatic digestion, fractionation, and desalting. After desalting, the enriched samples are introduced into the LC - MS/MS system for analysis. Among these steps, protein extraction and enzymatic digestion are the two most critical ones. They have a direct bearing on the accuracy of protein quantification as well as the subsequent mass spectrometry analysis. Below, we will delve into these two steps in detail.

Figure 2. Workflow of Bottom-up proteomics analysis[4].

1）Protein Extraction

The first step in a bottom-up proteomics workflow is to obtain a mixture of proteins from biological samples. This process encompasses several stages, such as sample pretreatment, enzyme inhibition, homogenization, protein extraction/precipitation, and protein fractionation. It's worth noting that the specific procedures vary depending on the type of sample.

Table 2. Methods for extracting proteins from different samples.

2）Enzymatic Digestion

Enzymatic digestion is the process of breaking down proteins into peptide segments under the action of proteases for subsequent mass spectrometry detection. Before enzymatic digestion, it is usually necessary to first add reducing agents (such as DTT, TCEP, etc.). These agents open disulfide bonds. Then, alkylating agents like iodoacetamide (IAA) or chloroacetamide (CAA) are used to block free thiol groups. This further disrupts the secondary structure of proteins and improves the efficiency of enzymatic digestion. There are numerous types of proteases that can be used in the enzymatic digestion step. Chymotrypsin, trypsin, Endoprotease Lys-C, Endoprotease Glu-C, Endoprotease Asp-N, etc., are all suitable for this process.

Table 3. Selection of Common Proteases.

After enzymatic digestion is completed, SPE cartridges are usually used to purify and enrich the peptide mixture. The enriched peptides are then freeze - dried and used for subsequent HPLC - MS/MS analysis.

Activity Verification

HY-E70196 Endoproteinase Asp-N. Endoprotease Asp-N is a kind of metalloproteinase that can specifically cleave the N-terminal side of aspartyl and sulfoalanine residues.

Figure 3. Activity of Asp-N.

Activity：1800 U/mg. Unit definition: One unit is defined as the amount of enzyme that 1 μg of Asp-N protease diluted 50-fold reacted with 0.072 μmol of antimicrobial peptide A protein. Reaction conditions: 50 μL of 20 mmol Tris (pH=8.0), dry bath incubation at 37°C for 1 hour, followed by analysis using HPLC.

Figure 4. Mass spectrometry analysis of specific peptide fragments.

Asp-N: Glucagon=1:50. 37℃, pH=8.0, 120 min. Analysis of specific peptide segments using ESI-MS/MS mass spectrometry with 100% specificity.

In this issue, we introduced the sample preparation for mass - spectrometry - based proteomics. Among all the steps, the enzymatic digestion step is of great importance. MCE's highly active proteases can effectively enhance the efficiency and accuracy of subsequent mass spectrometry analysis, enabling a deeper understanding of protein functions and interactions.

Product Recommendation

Trypsin (MS grade)

Trypsin MS grade is a serine protease enzyme, and hydrolyzes proteins at the carboxyl side of the Lysine or Arginine.

Endoproteinase Lys-C

Endoproteinase Lys-C is a protease that cleaves proteins on the C-terminal side of lysine residues and is commonly used for protein sequencing.

Endoproteinase Glu-C

Endoproteinase GluC (V8 protease) is a serine proteinase. Endoproteinase GluC is able to hydrolyze some serpins and all classes of mammalian immunoglobulins.

Endoproteinase Asp-N

Endoproteinase Asp-N (Asp-N) is a metalloprotease that can specifically cleave the N-terminal side of aspartyl and cysteic acid residues.

Carboxypeptidase B (MS grade)

Carboxypeptidase B (MS grade) is a peptide exonuclease that can specifically degrade peptide chains.