MS technology has an essential role in protein identification and other experiments; however, the current application is not particularly extensive. Therefore I collected some frequently ask questions in the field and offered some solutions, hoping to better help you understand the technology.
Questions 1: What is the difference between MS and tandem mass spectrometry? When should I do MS, and when should I apply tandem mass spectrometry?
Answer: a mass spectrometry approach mainly refers to peptide fingerprinting (PMF), namely to precisely measure the molecular weight of enzyme fragment with the help of a mass spectrometer and compares the results to library to achieve identification of proteins; while the tandem mass spectrometry is on the basis of MS and further select the peptide fragments of and then make in-depth analysis and comparison—in this way, it get the sequence of the peptides identified with the results of the PMF, finally enabling identification of proteins. Tandem mass spectrometry can gain part of a short peptide sequence, with higher reliability. With the magazine data requirements are increasingly stringent, protein identification with Tandem mass spectrometry is a big trend, and even if the current level of mass spectrometry results do also need to choose some PMF results from tandem mass spectrometry verification.
Question 2: what if the species studied is not model organism?
A: You can refer to the closest relationship model organism to make a successful identification of proteins, but if the mass spectra identification results is great without proof of a new protein, then technology such as de-novo can be used.
Question 3: How to evaluate the quality of mass spectrometry?
A: In general, if PMF scores more than 60 points (P <0.05), it is a successful identification; for tandem mass spectrometry, scoring more than 60 points, or though no more than 60 minutes, but there are at least a score of over 30 points for peptides, it is also a successful identification.
Question 4: what’s the purpose of some special mass spectrometry?
A: some special uses include mass spectrometry analysis of the phosphorylation sites, protein sequencing, mixing protein identification (shotgun technique) and the accurate molecular weight measurement, analysis of the position of the disulfide bond etc.
Question 5: What staining method is better?
A: The best stain method is Coomassie brilliant blue, then silver stain, but the success rate is lower and therefore recommending tandem mass spectrometry for protein identification to greatly improve the success rate of identification.
To be continued in the next blog entry…