I have run sucrose gradient fractionated (acetone precipitated) (HEK) cell lysed proteins on (6x8 cm dimension) 1D-SDS-PAGE. The gel was sliced and in-gel digested (with alkylation and reduction) . Digested peptides were analyzed Agilent (1200 series) Nano-LC and LTQ-Orbitrap (Discovery) systems. We have performed 110 minutes RP-gradient to separate peptides. A top 10 peaks selection based DDA (with CID) was used for acquisition.
When we analyzed the results using MASCOT and Swissprot, as anticipated we generated a list of proteins. But we were unable to find back about 12 signature proteins associated with specific biology question. These proteins are used as markers while performing westerns and antibody labeled imaging. The proteins might have gone undetected due to its low abundance and problem with dynamic range of the instrument.
Hence I planned to perform more targeted acquisition using a inclusion list (to be active while choosing the masses for MS/MS acquisition). I took the help of the tool MS-Digest . I performed the in-silico digest of candidate proteins. After picking +2 and +3 monoisotopic masses to be used, each protein is generating around 200 MI masses, but limit of number of parent ion inclusion list is about 500 masses per method. Hence it is problem to analyze all the candidate proteins in a single run.
My question is; I wish to scrutinize the list of peptides/masses to be used in parent ion inclusion list. I can think of some of criterion of peptides for selection such as hydrophobicity, property of fragmentation etc. I am seeking for a tool which does this selection in-silico and provides a scoring kind of gauge as a measure of probability of the peptide being analyzed. Can any one in the forum suggest with such tool published?