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Protein Identification

eagle-i ID


Resource Type

  1. Material analysis service


  1. Fee for service
  2. Resource Description
    Protein Identification: The PSR provides identification(s) for protein or a mixture of proteins in either solution or gel phase using a bottom-up approach. Protein in solution or in gel can be digested and the peptides analyzed by nano LC-MS/MS. Peptide fragments generated by tandem MS are searched on MASCOT to internally sequence the protein. This method produces numerous sequences from low fmole of material and enables us to identify hundreds, even thousands, of proteins in a single run. Protein Identification includes these steps: Enzyme Digestions: Protease digestion breaks large proteins into small peptides that are more amenable to mass spectrometry analysis. Trypsin predominantly cleaves peptide chains at the carboxyl side of the amino acids lysine or arginine, generating positively charged peptides that fragment well by HCD. Thus, trypsin is the most common enzyme used for proteomics. However, chymotrypsin, Arg-C, Lys-C, Asp-N and many other enzymes can be used as needed. LC-MS/MS analysis: The digested peptides are separated by nano C18 reverse phase column before being sent to the mass spectrometer for further analysis. A Thermo orbitrap Fusion, Thermo QE Plus and Bruker timsTOF Pro is used for acquiring data. The length of LC gradients will be determined by the complexity of the final samples analyzed by MS/MS. Database Search on MASCOT: Collected data is searched on MASCOT against the latest version of Uniprot or NCBI database. Data can also be searched against databases provided by the researchers in fasta format. MASCOT results will be compiled using Scaffold so the users can view and compare the data more easily. 2D LC-MS/MS Analysis (optional): For complex mixtures, the PSR recommends performing pre-fractionation prior to LC-MS/MS analysis. In this approach, the complex mixtures of proteins are digested and fractionated on an online or offline fractionation column first to reduce the complexity of samples submitted to LC-MS/MS analysis. Each fraction is collected and then separated again by a separation column for LC-MS/MS analysis, separately; the results are then merged prior to downstream informatics analysis. As many as several thousands of proteins in one sample have been identified using this approach.
  3. Service Fee URL
  4. Contact
    Liwen Zhang
  5. Service Provided by
    The CCC Proteomics Shared Resource (PSR)
  6. Website(s)
  7. Website(s)
  8. Part of Collection
    Campus Chemical Instrument Center
  9. Part of Collection
    The Ohio State University Comprehensive Cancer Center Collection
  10. Part of Collection
    Dr. Vicki Wysocki Ohio Eminent Scholar Collection
Provenance Metadata About This Resource Record

    Copyright © 2016 by the President and Fellows of Harvard College
    The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016