Part II: 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.  

For Unknown Proteins, How to Accurately Analyze Protein Sequences If the N-terminus of the Protein is Cyclized or the N-terminus Has Unknown Modifications?

For proteins of unknown sequence, especially engineered recombinant proteins, in vitro synthesized proteins, and engineered antibodies, or proteins whose gene sequences have not been studied, their information is often not included in protein databases.To achieve accurate analysis of such protein sequences, de novo protein sequencing must be used. De novo protein sequencing is an analytical method based on mass spectrometry. Similar to the traditional method of mass spectrometry, the de novo sequencing method also cuts the protein into small polypeptide fragments first, and separates the polypeptide fragments through high performance liquid chromatography, then determines the sequence of the polypeptide fragments through two stages of mass spectrometry. Compared with traditional mass spectrometry sequencing, de novo sequencing is a precise algorithm to ensure that the identified polypeptide sequence can deduce protein sequences with high accuracy, and can realize protein de novo determination without relying on database comparison.

de novo sequencing


How to Verify the Results of Protein Sequencing?

If a protein sequence is successfully identified, Western blot is a good method for verification. Because of the specificity and sensitivity of the Western blot method, the protein identified by Western blot can determine the result of protein identification with 100% accuracy, and exclude the false positives identified by mass spectrometry. A monoclonal antibody prepared against a sequence of the above protein can be selected for detection, and the accuracy of protein sequencing can be determined by comparing the results of Western blot with the predicted molecular weight of the identified protein sequence.

protein identification
How to Predict Protein Structure When the Protein Sequence is Already Known?

Protein structure prediction, which predicts its folding, secondary, tertiary, and quaternary protein structure from the protein primary structure. Currently, there are two commonly used protein structure prediction methods, homologous modeling and folding recognition.  

Homologous modeling predicts the structure of the protein through the known homologous or the structure of the protein from the same family. Folding recognition firstly summarizes the known protein folding method as a template for the target protein folding, and then predicts the matching target protein folding process according to the database.

The above are some of the questions that so many beginners may care about in terms of protein sequencing, but the problems they may encounter in practice may be different.

Can Edman Degradation Sequencing Determine Unknown Proteins Containing More Than 130 Amino Acids?

The 130 amino acid sequence is relatively long and has exceeded the limits of Edman sequencing of proteins. It is recommended to use de novo protein sequencing to determine the protein sequence. De novo protein sequencing does not require the use of any protein database and can sequence any protein, including mutations, bioengineered non-native proteins, etc.

In the Experiment, a Target Protein is Identified by Western Blot. The Molecular Weight is About 29KD. Is It possible to Run a One-way SDS-PAGE Directly and Then Cut the Gel for PMF?

It is entirely feasible to use SDS-PAGE separation and then cut the protein gel for PMF. However, the PMF comparison is a peptide map, and there is a certain false positive probability, so the requirement for protein purity is strict. Therefore, it is recommended that the SDS gel be ran away to ensure that the cut-out protein gel is not affected by other protein gels. In addition, when cutting the gel, be careful not to be contaminated by keratin and other miscellaneous proteins. PMF identification efficiency is higher than N terminal sequencing, and the price is lower. However, if the amount and the purity of the protein are enough, the PMF results will be equally credible.

N terminal sequencing


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