Protein Post-translational Modification Analysis

Up to now, scientists suggested that human proteome presents significantly more complexity than the human genome. While the genome comprises 20,000-25,000 genes, the proteome is estimated at over 1 million proteins. The increase in proteomic diversity is further increased by protein post-translational modifications (PTMs).

PTMs refer to the covalent and generally enzymatic modification of proteins during or after protein biosynthesis. It denotes changes in the polypeptide chain as a result of adding distinct chemical moieties to amino acid residues. PTMs are the foundation of governing intricate cellular process, such as cell division, growth, differentiation, signaling and regulation. Also, PTMs are involved in many cellular processes including the maintenance of protein structure and integrity, regulation of metabolism & defense processes, cellular recognition and morphology alternation. Consequently, analysis of protein post-translational modifications, including the modification categories and modified sites, is particularly crucial for the study of cell biology and disease diagnostics and prevention.

For more information on protein post-translational modification, please click the article Overview of Post-translational Modification Analysis

With years' experience in advanced experiment equipment, Creative Proteomics can provide a variety of PTM services to assist your scientific research, including:

Workflow of our PTMs analysis service

• Digestion of proteins into small fragments
• Protein separation and analysis using LC/MS/MS
• Database search
• PTM mapping
• Full protein annotation

Advantages of our PTMs analysis:

• Newest version of mass spectrometer: Q ExactiveTM MS/MS, with the highest resolution.
• Wide and full coverage range: at least 3 enzymes will be used to ensure wide and full coverage.
• Professional data analysis: constantly upgraded bioinformatics analysis systems.

FAQ-Post Translational Modification

What are the concerns about mapping a phosphorylation site in my protein?

You will have two major concerns:

Coverage: Can the mass spectrometry technique(s) deliver high sequence coverage? With higher coverage, there is a greater statistical chance to detect and identify the peptide containing a modification group.

Occupancy of the modification site: What percent of the protein preparation is modified? If it is low, then the chance of detecting the modified peptide decreases. If the occupancy is high (>30%), then this works in the favor of identifying a modification site.

How can coverage be increased for mapping phosphorylation or methylation sites?

First, note that, due to its unique sequences, every protein has a different potential to be mapped at high sequence coverage.

Coverage for all proteins increases by:

a) Starting with a higher amount of protein (>5 μg of protein is desirable) for MS analysis
b) Making sure that the protein is pure
c) Using multiple MS instruments/techniques for analysis will increase coverage
d) Enriching the phosphopeptides using a TiO2 column

Will glycosylation sites inhibit the identification of sample protein?

If purity and concentration of the protein is high, then in most cases MS-based protein identification is still favorable.

Attached carbohydrate (CHO) chains, however, can impair MS-based identification of specific peptides through the following mechanism:

a) CHO chains can block trypsin cleavage
b) The variable mass of attached CHO chains can reduce glycopeptide detection.
c) Options: Cleave N-linked CHO chains with N-glycanase (PNGaseF) prior to gel loading and MS analysis

I want a preliminary analysis for potential phosphorylation sites in my protein. What is your lowest cost service? What are the limitations? What are your suggestions?

A lowest-cost preliminary analysis is a method which uses MALDI-TOF/TOF on a pure protein from a gel or a highly pure preparation. Limitations: The coverage of the protein will be low, if the protein is very large, or if it is impure, or if it is in low yield (<500 ng).

Suggestions: For a preliminary, low cost analysis, provide only a high quality sample (this would be a single band on a gel that is in the 1 μg range).