Protein samples are digested with the appropriate enzyme and the resulting peptides are analyzed using on-line reverse phase HPLC coupled to an nanospray ionization source on an Orbitrap Velos mass spectrometer. The Mascot search engine is used to search the acquired MS/MS spectra and the resulting protein identifications are further validated using the Scaffold proteomics software. Within the Scaffold software supplementary statistical analysis is applied to further validate the peptide identifications and assist in data interpretation process. Your data will be sent to you as a Scaffold file. To view your file you will need to download the free Scaffold viewer here. With the viewer you will have access to all of the raw MS/MS spectra acquired for your sample as well as a report of all of the parameters used in the data analysis for potential publication.
There are multiple options for quantitative analysis of proteins and peptides, each with their own advantages and disadvantages. We encourage you to contact us directly to discuss the specifics of your project so that we can help you to determine the best approach.
- Isobaric Tagging – Isobaric tagging is based upon chemical isotope incorporation with tags of identical chemical structure and the same total mass. The labile portion of the tag that is released during fragmentation (i.e. reporter ions) will vary in mass and relative quantitation is based on the intensity of these reporter ions. Isobaric tagging approaches enable multiplexing. Tandem Mass Tag (TMT)
- Metabolic Labeling – Stable isotope labeling using amino acids in cell culture (SILAC) involved incorporation of isotopes through the use of media containing 13C or 15N labeled amino acids. Relative quantitation is based on the intensities of the light and heavy labeled peptides. (please note that we do not provide cell culture services but can support experimental design and proteomic analysis of SILAC experiments). SILAC
- Spectral Counting – Spectral Counting is based upon the assumption that the number of MS/MS spectra identified for a given protein is directly proportional to the abundance of that protein in the sample. Spectral Counting can be used to obtain relative quantitation of proteins in a complex sample without the requirement of labeling. (Anal. Chem. 2005, 77, 6218-6224.)
- Selected Reaction Monitoring (SRM) – SRM assays are a targeted approach for the quantitation of peptides. Depending on the experimental design, these assays can provide either relative or absolute quantitation. (Molecular Systems Biology. 4:222, 2008.)
Postranslational modifications are chemical changes to a protein that result in a change in mass that can be detected by a mass spectrometer. Some commonly studied modifications include phosphorylation, glycoclyation, acetylation, oxidation, and methlation. Please contact us directly to discuss the specific needs of your project.
In-gel digestions are most successful with Coomassie stained gels. In cases where higher sensitivity is required, silver stain or Sypro Ruby can also be used. When doing silver staining, make sure to use a mass-spec compatible staining kit. Silver stained gel spots should be submitted in 1% acetic acid. Coomassie or Sypro Ruby gel spots may be submitted dry (dehydrated) or in de-staining buffer (some sample loss may occur over time). Our standard in-gel and in-solution digestion protocols include reduction and alkylation of cysteine residues followed by enzymatic digestion with trypsin. Alternative enzyme digestions can be performed if needed, please contact us directly to discuss the available options and pricing. Following enzymatic digestion, samples are purified and concentrated using C18 tips or spin filters.