14 FAQs in MS identification That You’d Better Know (Part Three)
Part One-see the previous entry here
Part Two-see the previous entry here
Question 11: under the setting of commercial mass spectrometry processes, what data does a company generally provide?
A: Mass spectrometry process usually includes three steps: proteolysis, MS data acquisition and database indexing. Identification of protein for each point, the general process will provide three types of data, including: spectra (PDF format, including 1 mass-spectrogram and 5-10 tandem mass-spectrogram), MS peak list (text format, including mass fragments information), and the indexing results (PDF or excel format within local library, or Mascot format withink Online search page).
Question 12: what factors affect the success of Mass spectrometry?
A: The mass spectrometry failure can be divided into two categories: one is ineffective mass spectrometry; another is no protein databases reference to compare with. The first category can be subdivided into: protein content is too light too low; the protein spot is too small and the make the operation unfavorable, mixture of proteins even after two-dimensional electrophoresis separations, operational errors in proteolysis and mass spectrometry, etc. In order to avoid these factors, we’d better pick darker protein spots with appropriately larger area (for some small but very clear spots, we could use larger pipette tip) and the operator is better to have rich experience in the field. Solution to the second category can be: find a larger scope of database, using the EST database, or build a database of local mass spectrometry based on a single species of protein database.
Question 13: What is the difference between tandem mass spectrometry and mass spectrometry sequencing?
A: Mass spectrometry often gets results using software to automatically retrieve and matching existing database, and MS sequencing deduces peptide sequence based on difference between mass peak and adjacent peak of the molecular weight.
Question 14: for an unknown protein, should we use protein sequencing or mass spectrometry?
A: the relatively simple method is mass spectrometry, and the price is not high. If cannot determine with mass spectrometry, you can combine with N-terminal sequencing up to 10 amino acids. The combined analysis is basically enough to determine the target protein, and the current proteome database is pretty powerful.
The END of this series. Thank you.