Q&As About Protein Sequencing
Protein research often involves protein identification and the study of protein sequence. Newcomers to protein sequencing often encounter a variety of problems. This article is going to answer some of the common questions about this topic.
If the Protein to be Sequenced Has a Small Molecular weight, How to Prepare Protein Samples?
If the protein to be studied has a relatively small molecular weight, the protein can be separated using a higher concentration of SDS-PAGE gel, then the protein gel can be Coomassie-stained, and the protein band corresponding to the size of the target protein can be cut off. The protein strips cut can be sent for mass spectrometry.
Can I Cut SDS Protein Bands and Sent Them for Mass Spectrometry Sequencing?
Yes. SDS-separated protein bands can be directly cut and sent for mass spectrometry identification. In order to improve the accuracy of identification, when running the gel, it is necessary to select suitable gel concentration for the separation of the target protein band.
What are the Requirements for Shipping Samples?
Protein strips and dry powders are relatively stable and can be shipped in ice packs. Protein solutions require dry ice delivery. I recommend that you freeze the samples and transport them. The lyophilized samples are very stable. Frozen samples need to avoid repeated freezing and thawing of protein samples to avoid protein degradation.
Why Digest Proteins into Polypeptide Fragments Before Mass Spectrometry Sequencing?
The greater the group of protein digested proteins, the lower the accuracy of mass spectrometry detection. Therefore, before the mass spectrometry detection, the protein must be digested into small molecule polypeptides to improve the accuracy of mass spectrometry detection. In general, a 6-20 amino acid polypeptide is suitable for mass spectrometry detection.
How to Improve the Assay Efficiency of Modified Peptide Sequences in Protein Sequencing?
Under current conditions, it is difficult for MS to give 100% protein sequence, and some peptides are often lost. Protein phosphorylation modification also inhibits trypsin digestion, and the content of phosphorylated peptide is higher than that of non-phosphorylated peptide. With much less content, the mass spectrometer responses to the phosphopeptides of the mass spectrometer may be suppressed. Therefore, try to reduce the content of non-phosphorylated peptides to as low as possible. Methods for reducing non-phosphorylated peptides include fractionation, IMAC (Solid Phase Metal Affinity Chromatography), and antibody binding. The molecular weight of the peptide was determined by MALDI-TOF-MS, and the presence or absence of phosphorylation was determined based on whether the molecular weight of the obtained peptide was greater than the predicted molecular weight by 80 Da or an integral multiple thereof.
The Results of the N-terminal Amino Acid Sequence Determination of the Protein Have two Residues Which Proceed Two Amino Acids Edman Degradation at the Same Time. What are the Possible Reasons of This Result?
Two amino acid residues are detected in the Edman degradation reaction, which may caused by protein impurities and contamination with miscellaneous proteins. If one of the amino acids is glycine, it may be led by incomplete removal of glycine from the remaining buffer in the protein band.
Prime Jones is a senior researcher from MtoZ Biolabs. She is specialized in the field of proteomics study.