Proteomics Services
Protein Identification - Quantitative Analysis - Protein Characterization - Protein Digestion -
Protein Identification
Gel Spots
Standard protein identification consists of MALDI-TOF/TOF mass spectrometry analysis of peptides resulting from the in-gel enzymatic digestion of the target protein. The MASCOT search engine is used to search the experimental mass spectrometry data against the sequence databases to obtain a protein identification. All protein identification experiments include both MS and MS/MS data acquisition. In general MS/MS analysis will be done on the 2-4 most abundant peaks in the MS spectrum. For highly abundant (i.e. Coomassie stained), single component samples of known origin (i.e. biological source is represented in sequence database), identification should be straight-forward and highly successful. However, if the sample is complex (containing more than one protein), in low abundance (i.e. silver stained), or from a source not contained in the database, the data analysis will become more involved. Extended analysis may include the collection of additional MS/MS spectra, optimization of the database search, and manual interpretation of the spectra.
Complex Samples
For the analysis of complex samples (i.e. containing more than 1 protein) it is necessary to perform a chromatographic separation of the peptides prior to MS analysis. Complex samples are analyzed using an on-line reverse phase capillary flow HPLC coupled to an electrospray ionization source on the LTQ linear ion trap mass spectrometer. The SEQUEST 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 we can apply supplementary statistical analysis to assist you in data interpretation. A free Scaffold viewer can be downloaded at: http://www.proteomesoftware.com/ . 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.
- Introduction to Scaffold
- Self-Paced Scaffold Tutorial
- Sample Scaffold Data for tutorial (must have free Scaffold Viewer installed to view data)
Quantitative Analysis (top)
Isotope Labeling
All types of isotope based quantitative analysis are supported by the available instrumentation, including ICAT, ITRAQ, and SILAC. With the exception of SILAC, sample preparation for the labeling experiments can be performed in the core facility. There are advantages and disadvantages to the various labeling options and we encourage you to contact us directly to discuss the specifics of your project to help you determine the best approach. Please note, due to the expense and labor involved in using labeling reagents, special set-up fees will be charged.
Difference Gel Electrophoresis (DIGE)
The DIGE workflow is supported through the facilty from the point of gel imaging to protein identification. Gels are imaged using a multiwavelength Typhoon imager. The Delta 2D software package is utilized to analyze the images and determine quantitative ratios. Relavent spots from subsequent preprative or post-stained gels can be robotically picked, digested, and spotted on MALDI targets using the Genomics Solutions ProPicII and ProPrep workstations (coming soon!). Standard protein identification of the spots is performed on the MALDI-TOF/TOF mass spectromer.
Protein Characterization (top)
Posttranslational Modifications
High resolution MS and MS/MS spectra are collected using the MALDI-TOF/TOF mass spectrometer. Extended analysis is performed to obtain information on specific features of interest, such as posttranslational modifications. Purification techniques such as immobilized metal affinity chromatography (IMAC) to isolate phosphorylated proteins or peptides may also be applied. Please contact us directly to discuss options that may be applicable to your project.
De-novo Sequencing of Peptides
High resolution MS/MS spectra are collected using the MALDI-TOF/TOF mass spectrometer. Customers will receive a peak list and spectrum for manual interpretation. Extended de-novo sequencing analysis will include manual and software evaluation as well as homology investigation via MS-BLAST searching.
Protein Digestion (top)
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 such as SilverQuest from Invitrogen. 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). Please refer to the sample submission page for specific instructions on how to process and submit your samples for analysis. Our standard digestion protocols include rededuction 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 either Millipore C18 Zip-tips or Waters C18 Sep-pak cartridges.
N-Terminal Sequencing (top)
Automated analysis by Edman degradation. Pure proteins can be submitted dry or in aqueous solution. Otherwise the best sample preparation is gel separation, electroblotting onto PVDF and staining with Coomassie Blue. Minimum recommended sample amount is 10 pmol. Contact us for details. Please note that many native proteins are N-terminally modified in which case Edman degradation will yield no sequence information.