Is there a requirement for protein activity for protein sequencing?
Answer: Protein sequencing is not required to maintain the activity of the protein because the function of the protein does not need to be studied.
Can the bands on SDS gel be cut off and sent to mass spectrometry for sequencing?
Answer: Yes. To improve the accuracy of the identification, you need to choose the appropriate gel concentration for the separation of the target protein strips and try to run the protein strips near the target protein.
Can SDS protein bands be too thin for protein identification?
Answer: In general, a naked-eye visible protein band is sufficient for sequencing protein bands stained with KOMAS and SYPRO Ruby.
What if the cut SDS protein strip may contain more than one protein band?
Answer: If the cut protein strip contains more than one protein band, it can be analyzed for protein identification, and then the data can be compared to infer the protein sequence. In fact, most of the protein strips cut after SDS-PAGE gel separation contain more than one protein, and the identification of these complex proteins can be done by chromatography-mass spectrometry tandem identification.
Can the results of Western blot be used for protein sequencing?
Answer: Protein sequencing is possible after Western blotting. You can compare the western blotting results with SDS-PAGE gel, find out the position of the corresponding protein bands on the SDS-PAGE gel, and then cut off the protein gel for mass spectrometry. If the amount of protein is large enough and the purity is high enough, the protein strip on PVDF membrane can be cut off directly for N-terminal sequencing.
What should I pay attention to during protein sample preparation?
Answer: The sensitivity of protein sample identification by mass spectrometry is high. Trace amounts of foreign contaminating proteins introduced during the process are also very likely to be identified by mass spectrometry, which can have a significant impact on the accuracy of protein sequencing results. Therefore, during sample preparation for mass spectrometry, clean and contaminant-free containers should be used; reagents with purity level for mass spectrometry identification should be used; freshly prepared solutions should be used, and gloves and headgear should be worn to avoid contamination of samples by keratin.
What are the requirements for the preparation of various types of samples for protein sequencing?
Answer: Usually, for SDS-PAGE gel separated protein samples, the protein bands stained with KOMAS and SYPRO Ruby are well compatible with mass spectrometry identification. However, for silver-stained proteins glutaraldehyde must not be used as a fixative, which will affect the subsequent mass spectrometry analysis. Also, both protein dry powder and proteins are capable of sample sequencing. If the concentration of the protein solution is low. During the sample preparation process, it is necessary to minimize the use of surfactants such as SDS and pay attention to reducing the salt concentration, which can improve the accuracy of protein identification accordingly.
Can Edman degradation sequencing determine unknown proteins containing approximately 130+ amino acids?
Answer: The 130 amino acid sequence is relatively long and is beyond the limits of protein Edman sequencing. It is recommended that protein sequences be determined using de novo sequencing. Protein de novo sequencing does not require any protein database and can sequence any protein, including mutations, biologically modified unnatural proteins, etc.
Is there a fast method for protein sequencing with high purity protein samples?
Answer: Rapid determination of protein sequencing can be achieved by identifying the peptide mass fingerprinting (PMF) of the protein. The sequence of each protein is specific, and the quality of the peptide produced by a specific enzyme cleavage is also certain. Therefore, this property can be exploited to infer the sequence of a protein by measuring the mass profile of the peptide produced after protein digestion and then comparing it with the theoretical peptide profile of the digested protein in the database.
