Answers to Questions Related to Protein Post-Translational Modifications

1. What should be noted when analyzing phosphorylation sites in proteins?
Coverage: The higher the coverage, the greater the likelihood of detecting and identifying peptides with modification groups.
Occupancy of modification sites: If the percentage of modified proteins is low, the chance of detecting modified peptides decreases. If the percentage is relatively high (>30%), it helps in identifying modification sites.

2. How can the coverage be increased to locate phosphorylation or methylation sites?
Firstly, note that due to the uniqueness of protein sequences, each protein has different localization potential under high sequence coverage.
The coverage of proteins can be increased by the following means:
a) Having a relatively large quantity of protein (preferably >5μg) for mass spectrometry analysis;
b) Ensuring the protein is of high purity;
c) Analyzing using multiple mass spectrometry instruments/techniques;
d) Enrichment of phosphorylated proteins using TiO2 columns

3. Does glycosylation inhibit the identification of protein samples?
In cases where the purity and concentration of proteins are very high, mass spectrometry-based protein identification remains the best choice. However, carbohydrate chains (CHO) can disrupt mass spectrometry-based identification through the following mechanisms:
a) Carbohydrate chains can block trypsin digestion;
b) Variable molecular weights of carbohydrate chains can reduce the detection of glycopeptides;
c) Solution: Hydrolyze N-linked carbohydrate chains with N-glycosidase (such as PNGaseF) before gel loading and mass spectrometry analysis.

4. I want to perform a preliminary analysis of potential phosphorylation sites in proteins. What is your company's most cost-effective related service? What are the limitations? Do you have any recommendations?
Our most cost-effective preliminary analysis is using the MALDI-TOF/TOF method to analyze pure proteins from gels or high-purity preparations.
Limitations: If the protein is large, impure, or has low yield (<500 ng), the coverage will be low.
Recommendation: Providing high-quality samples can significantly reduce the cost of preliminary analysis. (Please consult for details)