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LC-MS/MS protein identification service

eagle-i ID


Resource Type

  1. Material analysis service


  1. Fee for service
  2. Resource Description
    "Purpose: Identification of proteins at high sensitivity from tryptic peptides (or peptides produced by alternative proteases), or identification of locations of chemical modifications or PTMs on purified proteins. Nano-capillary reverse-phase HPLC is interfaced directly with hybrid linear ion trap mass spectrometers (LC-MS/MS) for maximum sensitivity. Proteins are identified by comparing MS/MS spectra to sequence databases, typically using SEQUEST®. For reliable identifications, the exact protein sequence must be in the database. Highly homologous proteins sometimes can be identified if a substantial portion of the tryptic peptides have identical sequences. Requirements: An intact 1D or 2D gel (see #1 above for details and detection limits/expected results). Include an image of the gel with bands/spots of interest clearly marked and a completed "MS/MS Submission Form." Complete all entries on the submission form, including the species of sample origin, Molecular Weight, gel thickness, type, etc. Requires service in-gel protease digestion. Results: High-confidence (>99%) identifications are expected for proteins identified by two or more peptides, provided that the protein or a very close relative is in the sequence database used for the search. However, it should be noted that in a substantial number of cases, the MS/MS matches to peptide sequences for an identified protein actually only define a family of homologous proteins. That is, there may be insufficient sequence coverage to unambiguously define a single protein. Hence, in a multi-species search, the protein from an incorrect species may be listed or an isoform or splice variant may be listed that is homologous but not identical to the isoform present in the analyzed sample. The SEQUEST® output from the database search consists of a list in PDF file format of peptide sequences matched to protein(s). For complex protein mixtures, the list of peptide identifications may not be listed to reduce report length, but a full list of peptides can readily be provided. It should be noted that all identifications of peptides and proteins based on MS/MS data are identified based on correlation of observed peptide masses and fragmentation patterns with those predicted from sequences in the database. Even when data is stringently filtered, there is a very small but finite possibility that any individual peptide identification is incorrect."
  3. Service Fee URL
  4. Related Resource
    Thermo Fisher Hybrid LTQ-OrbitrapXL™ mass spectrometer with online Eksigent® Nano-capillary HPLC
  5. Related Resource
    Applied Biosystems® 5500 Q TRAP® Mass Spectrometer with online Waters NanoACQuity® Nano-capillary HPLC
  6. Service Provided by
    Proteomics and Metabolomics Facility (Wistar)
  7. Website(s)
  8. Related Technique
    Protein identification
  9. Related Technique
    Liquid chromatography-tandem mass spectrometry
  10. Related Technique
    Mass spectrometry data analysis
  11. Related Technique
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