When can I choose peptide spectroscopy for identification?
Answer: The biggest advantage of the method of protein sequence analysis by measuring protein peptide spectrogram is that the protein is analyzed by mass spectrometry only, and the determination is faster. Moreover, because of the specificity of the protein sequence, peptide profiling can infer proteins with a high degree of confidence. However, peptide profiling relies on database information comparison. For proteins that are not contained in the database, peptide mapping is not able to perform accurate analysis.
If a protein sample contains multiple proteins, is there a method to sequence all proteins at once?
Answer: MS/MS mass spectrometry can detect multiple proteins in a single protein strip. Simply cut off the protein gel and send it for sequencing. In this process, it is ensured that the cutter used to cut the gel is clean and free of contamination, while avoiding cutting off any stray proteins next to it.
Which mass spectrometry technique is the basis for protein peptide profiling?
Answer: The most efficient mass spectrometer to determine the mass number of a peptide mixture is Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS). The sensitivity is high and the spectral peaks are simple. Each spectral peak represents a peptide.
There are more than 8 peptides on the map. Why are these 8 selected?
Answer: The number of peptides submitted for retrieval is selected based on the signal intensity of the peptides, while some of the peaks are filtered during the software processing, such as the autolytic peak of trypsin.
Why is the result of KOMAS staining better than that of silver staining?
Answer: The main reason is that the sensitivity of silver staining is higher and the amount of protein obtained is less. And the silver staining has to be desilvered during the processing of the sample, which increases the number of elutions and the loss of the sample is larger. Noise and keratin contamination have a greater impact on the target protein and have an impact on the final database search.
Why are there fewer peaks in my PMFs? Do these fragment counts and peaks meet the requirements for a minimal database search? Does it affect the search results?
Answer: Because we use trypsin to degrade the protein, which is a more specific enzyme that only hydrolyzes the C-terminus of arginine and lysine. It is possible that your sample has too many or too few sites for enzymatic degradation. It is difficult to distinguish the peak with the matrix. If the enzymatic digestion sites are low, the molecular weight of the peptide fragment is high, and the sample inside the gel stays inside the gel, which is not reflected in the mass spectrometry graph. Solution samples can be collected, but the larger the molecular weight the more inaccurate the retrieved results. Theoretically the more peaks in the plot the better, if they all match then the protein is more certain and the opposite is less. Usually you should get at least 4 peptides.
How to understand the significance of these fragment peak sizes? What standard should the number of these enzymatic fragments and their peaks generally meet to be considered better?
Answer: The significance of the size of the fragment peak horizontal coordinate is the mass size of the peptide fragment that is enzymatically cleaved down. The vertical coordinate indicates the intensity of the relative signal of the collected peptide fragment. The left side is relative and the right side is absolute. The higher the absolute, the better the resistance to interference. The accuracy of the mass spectrometry is determined by the instrument, and our instruments have an accuracy of 10 ppm or less.
