Highlights of DC-PPMA
(i) A major facet of DC-PPMA is that the user can‘view’ the database (in the form of an excel file) that they will be using further for MS/MS based search or data analysis, viz., prior to database search, the user can know, what are the m/zvalues of precursor ions (of proteolytic/truncated peptide sequences) and what are the m/z values of fragment ions that will be involved in the search engine process. To the best of our knowledge, this particular facet is not available in any proprietary or online tool that are utilized for proteomic or peptidomic investigations. By viewing/knowing the database containing m/z values of precursor ions and fragment ions, it is possible to know better about ‘true negatives and false positives’ and the user obtains a better understanding about the process of matching between the experimental MS/MS data and the theoretically calculated values present in the database. Therefore, if DC-PPMA is used to construct appropriate ‘decoy database’ by choosing suitable protein sequences, then he/she can obtain better comprehension about false discovery rate (FDR). Thus, viewing and analysis of decoy and target databases can be very helpful in more reliable annotation of MS/MS spectra towards proper protein identification.
(ii) Another notable feature of DC-PPMA is that the Module 1 calculates the m/z values for ‘singly as well as multiply charged (protonated)’ precursor ions (viz., proteolytic peptides) and also calculates the m/z values for ‘singly and multiply charged fragment ions: a-, b-, c-, x-, y- and z- ions’. Consequently, in the ‘custom’ MS/MS database (created from Module 1), the m/z values of each fragment ion (e.g., b, y, c, z ions) and their respective ‘charge state’ are generated and shown in the excel sheets, which can be anticipated from MALDI and ESI MS/MS experiments. Therefore, DC-PPMA can be useful for ‘manual spectral annotations and interpretations’ for proteomic researchers and/or protein/peptide chemists, who use both ESI and MALDI MS/MS.
(iii) Additionally, DC-PPMA can be of immense utility for ‘targeted analyses’, particularly for multiple reaction monitoring (MRM ) based experiments, wherein it is essential to‘design suitable channels’ that should encompass the m/zvalues of precursor and pertinent fragment ions. In this context, the output from Module 1 can be used for ‘designing MRM method’ , because the custom database built by Module 1 would consist ofm/z values of singly as well as multiply protonated precursor and relevant fragment ions. Thus, DC-PPMA can be helpful for quantitative studies also.
(iv) Furthermore, peptide sequences, either single or multiple sequences can be uploaded in DC-PPMA. This particular feature can be useful for peptidomic investigations and also for de novosequencing exercises or assignments. Consequently, DC-PPMA can indeed prove to be worth for de novo sequencing of polypeptides and proteins as well. Additionally, it can be used for discovery based proteomic/peptidomic analysis also, if/when the transcriptome and/or the genome of their sample is also known.
(v) In addition, the custom databases generated by DC-PPMA can also be utilized further for planning about probability-based scoring algorithms or scoring schemes, so as to identify more peptides and proteins in a reliable fashion.