I have just started doing proteomics work and are trying different settings of sonication.
I performed 2D gel electrophoresis and take some spots for protein identification using MALDI-TOF/TOF.
Because the animal i work on did not have its genome sequenced. For MS identification, based on homology search, one of the protein i got is the "70 kDa heat shock protein" of my species, with a MASCOT score of 379 (100% C.I.), and 14 matched peptide. However, based on estimation, this spot has a much different observed MW and pI in the gel compared to the theoretical MW and pI of the matched protein from GenBank.
I would like to ask some questions about this:
(1) Does the MS result suggest that the protein is very likely a hsp70?
(2A) How to account for the discrepency between the observed MW/pI and the theoretical MW/pI?
(2B) Because this discrepency is present in many other identified spots, is it simply due to low sequence coverage of the analyzed peptide fragments in the database, which is common for species without a sequenced and annotated genome? But I also have a protein with high score 115 (100% C.I., 40% sequence coverage) also have such an discrepency in MW/pI.
(2C) Or finally does it implies that the sonication setting I used is too strong so it broke the polypeptide into smaller fragments, such that the spot i picked for MS is actually a fragment of the original, intact polypeptide?
I appreciate it very much for any suggestions. Many thanks.
Question about Sonication settings
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