Hello, 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 parent mass inclusion list (to be active while choosing the masses for MS/MS acquisition). I found a paper describing PeptideSeive<http://tools.proteomecenter.org/wiki/index.php?title=Software:PeptideSieve>. This software seems really useful. I started the work of prediction of proteotypic peptide with this software. I did generate a list of peptides to be anticipated by physio-chemical properties of the sequence using the software. My questions are; 1) What does the scores in PeptideSeive<http://tools.proteomecenter.org/wiki/index.php?title=Software:PeptideSieve>output refers to? Is score near to one is best or it is other way round? 2) I have a list of predicted peptide sequences in a text/excel file. I need to calculate mono-isotopic(MI) masses and +2 and +3 charged m/z through the same. I have Thermo-Xcalibur which can do this for me. But there I have to copy paste each and every sequence and perform the work manually for around 100 odd peptides. Can any one share a ready made program/script (perl/python/any other) in which I can use text/excel as input and get a text file of calculated mono-isotopic masses and +2 and +3 charged m/z as a output (if it is .txt i can upload the same into my method file of parent mass inclusion list)? I kinda know the logic that the program must define all the MI of amino acids and should add-up the same when a sequence is given. But I just do not know any of the programming Can any one help me with this???? Thanks in advance -- You received this message because you are subscribed to the Google Groups "spctools-discuss" group. To view this discussion on the web visit https://groups.google.com/d/msg/spctools-discuss/-/i3f2F7LxYtUJ. To post to this group, send email to [email protected]. To unsubscribe from this group, send email to [email protected]. For more options, visit this group at http://groups.google.com/group/spctools-discuss?hl=en.
