Hi
Our lab has made a diseased mosue model and we are using it for biomarker discovery.
We have done mouse plasma proteome by ITRAq and lable free LC MS/MS commercially. However the bioniformatics output given by them is suboptimal and I am not convinced with the data. So we wish to reanalyse the data.
I am presenty looking for academic collaborators who would be interested in analysing our raw spectras for protein identification and determining the differentially abundant proteins.
Please inbox me if the project interests you. We can work it out in a collaborative mode.
Regards
Deepak
The project is interesting. We can work as a collaborative effort. Dr Bhamidipati Narasimha Murthy
There are several open source packages that can help with the statistical feature analysis (e.g., XCMS, MZmine). However, be sure that you are getting the most out of your raw LC/MS/MS data first, see: https://www.researchgate.net/publication/315874699_Mass_Spectral_Data_Processing. In terms of peptide sequencing this can make a big difference independent of the sequencing package that you are using, see: https://www.researchgate.net/publication/315813593_Improved_MSMS_Peptide_Identification_Using_Advanced_Peak_Detection_and_Deconvolution
Method Mass Spectral Data Processing
Poster Improved MS/MS Peptide Identification Using Advanced Peak De...
Dear Sir,
We done our ESI-MS of our novel recombinant protein sample against and found varying color (green,red,yellow) of peptide when matching to our known protein sequence (isolated and purified from plant source ms spectra have identified 7 peptide). I have found all the peptide matching in our ESI-MS of recombinant protein (0ut of7 peptide we have found 3 green 1 yellow and 3 red peptide). please clarify should i consider it my recombinant protein is have same sequence as native plant protein. Secondly what could be the reason of getting varying colour peptide.
Please clarify the significant of peptide deduced in sequence represented with varying color like green,Red and yellow. Could you please elaborate the percentage of similarity level of green, yellow and green peptides????
Since the original post says LC/MS/MS, I assume that you are looking at peptide sequence coverage, and by color coding, sequence agreement with the database sequence and/or false detection rate disambiguation.
1) You will never get complete sequence coverage.
2) The sequences in the public database are for "representatives" of the class. There are both known and unknown splice variants and post-translational modifications, which lead to partial and no sequence alignments, depending on the assumptions that you make in the software.
3) There are also common motifs in protein chemistry (e.g., ATP binding domains), which are shared by multiple proteins and not really predictive of any given protein. This is why we use false detection rate (FDR) estimates to weed these out.
Since you are using iTraq, you should focus on those peptides that are truly differentially-expressed and ignore the rest (except as noted below). These peptides could arise by mutation (e.g., the loss of a endoprotease cleavage site), splice variation (e.g., the loss of presence of a new intron in the mRNA coding for the protein), a difference in post-translational modification withing the sequence of the peptide (e.g., acetylation, phosphorylation or glycosylation), or simply up or down regulation of the parent protein. It is your job to track down through the LC/MS/MS spectra which of these alternatives explain the difference in abundance. This requires identifying which protein gives rise to the peptide that shows the difference. Here is where all the trouble begins since there is no way to truly automate this process. If you have corroborating peptides in the other spectra that show that the your differentially-expressed iTraq peptide is present in normal abundance, then you likely have an isotype issue. If all of the other peptides of that parent are of equal relative abundance to the peptide that caught your statistical eye, then you have an abundance shift in the protein, likely due to changes in regulation of the gene itself. If there are no other peptides identified to the same parent are found, then you must move on since you have no way to corroborate your finding. This later case can just be an aberration that requires more samples to verify, or may be a PTM or splice variant of another protein that just coincides in mass and sequence with a neighbor. If at least one of the other peptides from that parent is present in equal abundance, then you may have a spice variant or PTM and now you have more work to do to confirm what has changed.
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