Q&As in Protein Sequencing Experiment for Beginners

It is inevitable that beginners may encounter various troubles while doing protein sequencing experiments in the lab.

Protein Samples are of High Purity. Is There Any Quick Solution for Protein Sequencing?

Peptide mass fingerprinting (PMF) can be used to rapidly determine protein sequencing. Because the sequence of each protein is specific, the quality of the polypeptide produced by the specific enzyme cleavage is also certain. Therefore, this property can be used to determine the peptide spectrum of the protein produced by the enzyme digestion, ie, to determine the peptide spectrum of the protein. The map is then aligned with the database's theoretical peptide map to infer the protein sequence.

Which Mass Spectrometry Technique is Protein Peptide Mapping Based on?

The mass spectrometer for mass determination of peptide mixtures is MALDI-TOF-MS (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) with high sensitivity and simple peaks. Each peak represents a peptide fragment. When MALDI-TOF-MS is used to analyze the peptide mixture, it is able to tolerate appropriate buffers and salts, and almost every peptide sheet produces only singly-charged ions. Therefore, MALDI-TOF is the preferred method for analyzing PMF.

The Advantages and Disadvantages of Peptide Mapping Identification?

The advantage of analyzing the protein sequence by measuring the peptide map of the protein is that the technique only uses mass spectrometry to analyze the protein. The determination is rapid, and because of the specificity of the protein sequence, the peptide mapping method can be used to infer more reliable and accurate proteins. However, as peptide mapping has to compare the data with the database information, it can’t accurately analyze proteins that are not included in the database.

What are the High Accuracy Protein Sequencing Methods?

Although the peptide map generated by proteolysis is theoretically specific, there are many factors affecting the enzymatic cleavage of proteins and the quality of peptides. Therefore, peptide mapping is a relatively crude method for protein sequencing. Methods that are more accurate for protein determination include Edman degradation sequencing, tandem mass spectrometry sequencing, etc.

If the Protein Samples Contain Multiple Proteins, is There a Way to Sequence All Proteins at One Time?

MS/MS mass spectrometry can detect multiple proteins in a single protein band. It only needs to cut the protein gel and send it to be sequenced. During this process, the tool used to cut the gel should be clean and pollution-free. Cutting off the miscellaneous proteins should also be avoided.The mass spectrometry identification company will cut the protein in the gel sample, and determine the peptide sequence, then compare the peptide sequence with the known protein database, so as to obtain the ID of the protein in the gel sample and determine the sequence of the protein.

Can Edman Degradation Sequence All Proteins?

The advantage of Edman degradation sequencing is that it can sequence unknown proteins. At present, this technology is almost perfect, but Edman also has certain limitations. For example, Edman degradation sequencing can’t solve the problem of N-terminal cyclization and closed sequencing of proteins. Besides, modified proteins can’t give an accurate signal, which, to a certain extent, limits the application of Edman degradation.

Prime Jones is a senior researcher from MtoZ Biolabs. She is specialized in the field of proteomics study.