4 Replies Latest reply on Dec 28, 2018 5:41 AM by ayushi_jain

    Using bioconfirm software for protein identification

    ayushi_jain

      Hi

       

      I am currently working on proteome analysis of a cell line. I have done LC-MS/MS of the trypsin digested sample and I want to identify the proteins in it. How can I analyze my data using Agilent Bioconfirm B.07.00 ? The quick start guide of the software suggests that sequences from a fasta formatted database can be pasted in the sequence editor window. I tried it with 5-10 protein sequences from the fasta file of the cell proteome (downloaded from uniport) but that doesn't seem to be useful because it matches these protein sequences with the compound list but reports as if they are part of a single protein. What is the right way to identify proteins using Bioconfirm? Can I use Bioconfirm for online database search -such as uniport, swissprot for single or multiple taxonomies?

       

      Thanks

        • Re: Using bioconfirm software for protein identification
          carhaney

          ayushi_jain,  Bioconfirm is designed to confirm the identity of a protein that you believe to be present, such as a you would do with a recombinant protein.  It is not a search engine that you would use to discover the identity of unknown proteins.  To search a species proteome you would need a tool such as Agilent's Spectrum Mill, or third party software such as MASCOT or PEAKS

            • Re: Using bioconfirm software for protein identification
              ayushi_jain

              Hi Carhaney

               

              Thanks for reply.

              I have two more doubts, if you can help.

              1. Do you mean that bioconfirm can be used for matching compounds with only one protein at a time?

              2. Does any other feature of mass hunter qualitative analysis B.07.00 allow such database identification for peptides? I read about PCDLs but I doubt their application in case of peptides.

               

              Thanks

                • Re: Using bioconfirm software for protein identification
                  carhaney

                  In answer to your questions:

                  1) Bioconfirm can search multiple proteins and variable modifications.  However, the purpose of Bioconfirm is to confirm so it works best when only one or a few protein sequences are used.  With Bioconfirm B07, the search relies very heavily on the mass of the peptide rather than the MSMS sequence match so if you put in many proteins your false discovery rate will be high.  Bioconfirm is best, for example, if you've isolated a protein and want to get coverage information or look for specific modifications on a known or predicted structure.  If you are trying to identify unknown proteins present in a digest  Bioconfirm is NOT the correct tool.

                  2) You are correct, MassHunter qual is not designed to identify peptides using PCDLs.  If you wish to search SwissProt or NCBI FASTA databases of proteins you need other software.  There are many available, though most are not free.