Protein Mass Spectrometry Analysis Experimental Tips for Success

Mass spectrometry analysis is a commonly used analysis method in basic and advanced drug research. It can identify compounds in simple mixtures and analyze more complex sample groups (such as proteomics). Its applications are very wide-ranging.

 

Proteome and Proteomics

A proteome covers all the proteins in a biological body, while proteomics studies the dynamic changes in the proteome under conditions such as environmental influences, internal changes (such as cell differentiation or death), cell types, cell compartments or the cell cycle.

 

Mass spectrometry provides a powerful tool for proteomics research. It can reveal changes in the abundance, isomers, and modifications (such as ubiquitination, ubiquitination-like, phosphorylation) of proteins. Traditional methods of studying interactions between specific proteins often involve systematic mutations and hybridization, which may introduce unnatural mutations or hybrid products, affecting the accuracy and reliability of the results, and the experimental process is complex and time-consuming. Mass spectrometry solves these problems well, and its sensitivity and accuracy have been greatly improved.

 

With its unique advantages, the mass spectrometry method has opened new ways to answer many biological questions. However, because of its high sensitivity and technical complexity, we must be very careful and rigorous in sample preparation and data analysis.

 

Before Starting a Mass Spectrometry Experiment, the Following Issues Need to Be Considered

1. Question 1

What biological question does my experiment want to solve, and how likely is mass spectrometry to help answer it?

 

2. Question 2

What type of sample am I using?

 

3. Question 3

How abundant is the protein of interest and how difficult is it to detect?

 

4. Question 4

How stable are the protein modifications, and how can protein degradation be prevented?

 

5. Question 5

How to avoid sample contamination that interferes with detection (such as keratin, polymer)?

 

6. Question 6

What controls should be included in the experiment?

 

7. Question 7

What kind of enzyme and digestion type should I choose to get perfectly sized peptide fragments for detection?

 

8. Question 8

What type of software should I use for analysis?

 

Basic Parameters for Mass Spectrometry Data Analysis

1. Intensity

The intensity directly reflects the abundance of a single peptide fragment, i.e., the number of times it is detected by a mass spectrometer. This value depends not only on the concentration of the original protein but also on the size of the peptide fragment and its "flight" ability. It is worth noting that not all peptides can "fly" in a mass spectrometer. If peptides cannot be effectively ionized or if the ions are too unstable and break apart, they may not be accurately detected by the mass spectrometer.

 

2. Number of Peptide Fragments

The number of peptide fragments here refers to the total number of different peptide fragments detected from the same protein. If the number of peptide fragments is low, it may mean that the protein itself is of low abundance, or that the size of the peptide fragments is not suitable for the current detection conditions (too small or too big). In this case, we can consider adjusting the digestion time of the peptide fragments, or try using different enzymes to optimize the detection effect of the peptide fragments.

 

3. Coverage Intensity

Coverage intensity is directly related to the number of peptide fragments and refers to the proportion of protein covered by the detected peptide fragments. In simpler samples, a good coverage level should be between 40% and 80% (mainly when dealing with purified proteins), depending mainly on the number of enzyme recognition sites and the length of the peptide fragments after digestion. In more complex proteomic samples, the coverage range of proteins is usually between 1% and 10%.

 

4. P-Value/Q-Value/Score

The results of peptide fragment identification need to be validated by statistical significance analysis, and the specific method depends on the software tool used. Usually, this validation is expressed through P-value, Q-value, or score, and these indicators should be less than 0.05 to ensure the reliability of the results.

 

(1) P-Value

Represents the possibility that the identification of the peptide fragment is a "false positive", i.e., the probability that it is actually not the peptide fragment but is erroneously identified.

 

(2) Q-Value

A further adjustment of the P-value, which takes into account the FDR (False Discovery Rate), i.e., the probability that the signal appears significant but is actually not significant.

 

(3) Score (in Mascot Software)

Represents the possibility that the identification of the peptide fragment is a random event, i.e., the probability that the identification result is not based on the real peptide fragment characteristics but is due to random factors.

 

By considering these indicators comprehensively, we can more accurately verify the reliability of the peptide fragment identification.

 

5 Key Tips

1. Tip 1

Before the experiment starts, you need to check the abundance, regulation mechanism, and expression spectrum of the target protein. For proteomic datasets, you also need to pay special attention to the regulation of the cell cycle and the characteristics of the cell line. For example, cancer cell lines often have interruptions in transcriptional regulation or signal pathways, leading to their uncontrolled growth.

 

2. Tip 2

During the experiment, it is recommended to use pipette tips with filters, disposable pipettes, and High Performance Liquid Chromatography (HPLC) grade water. At the same time, all plastics and solutions need to be autoclaved, and detergents should be avoided when cleaning glassware, to avoid introducing unnecessary impurities.

 

3. Tip 3

Check the compatibility of all buffer components, including protease inhibitors, detergents, Ethylenediaminetetraacetic acid (EDTA) and reducing agents. In addition, the salt concentration and pH of the buffer should be carefully checked to ensure they meet experimental requirements.

 

4. Tip 4

To ensure the stability of the protein sample, the protein sample should be stored in a low-temperature environment. It should be kept at 4°C during daily work to avoid repeated freeze-thaw cycles; and stored under -20°C to -80°C conditions for long-term storage.

 

5. Tip 5

To ensure the accuracy of the experimental results, each step of the experiment should be validated after it is completed. Common validation methods include Western blot and Coomassie Brilliant Blue staining.