Protein samples are digested with the appropriate enzyme (generally trypsin) and the resulting peptides are analyzed using on-line reverse phase nanoflow HPLC coupled to an nanospray ionization source on an Orbitrap Eclipse mass spectrometer. The Proteome Discoverer 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 Proteome Discoverer, supplementary statistical analysis is available to assist in data interpretation process. Your data will be sent to you as a Proteome Discoverer file. To view your file you will need to download the free Proteome Discover 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
- Data Independent MS/MS acquisition – This is a data acquisition approach in which MS/MS data is acquired in a manner which is independent of the detected precursor masses, generally using wider isolation windows. The benefit of this approach is the speed of acquisition – faster speed enables sampling rates consistent with LC peak widths. We generally analyze this data using DIA-NN.
- 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.