Services

Intact MW Determination

The molecular weights of the proteins are determined after HPLC separation.  Please submit samples in solution (no gels), minimum of 10ul at 1uM.

Gel band LC-MS/MS

This service identifies proteins using the UniProt or other protein databases.  It is appropriate for samples containing a limited number of expected proteins.  Please attach an image of your gel with the bands of interest CLEARLY indicated with your submission (or email it to us).  Bring your gel submerged in water in a covered container.  We prefer to excise the gel bands ourselves.  If you have excised the gel band, submit the gel piece(s) in clean tubes.

Gel band PTM LC-MS/MS

Post-Translational Modification (PTM) Mapping

In order to detect post-translational modifications, larger sample amounts and longer run times maybe required. Enrichment may be required to detect modifications such as phosphorylation, acetylation, or methylation.  At a minimum a well staining Coomassie band is required.  We strongly recommend that you send as much sample as possible (i.e. multiple Coomassie stained bands, pooled).

Gel-free LC-MS/MS

Protein-Protein Interaction or Mixture Analysis

This analysis is intended for less complex protein mixtures (i.e. affinity-purified samples) and usually requires longer data collection times to distinguish multiple proteins.  We strongly recommend eluting IPs using acidic or basic conditions.  If this is not possible, arrange to have the proteins on beads brought to us and we will digest the proteins on the beads.  We also strongly recommend having an appropriate control to help you distinguish "true" protein interactors with non-specific interactors.

Gel-free PTM LC-MS/MS

Post-Translational Modification (PTM) Mapping

PTM mapping can be done without running the sample through a gel, however significantly larger quantities may be required.  The ratio of modified:unmodified protein in conjugation with the complexity of your sample will determine the success.  Often an alternative lysis protocol is necessary to preserve enough of the PTM for identification.  Enrichment may be required to detect modifications such as phosphorylation, acetylation, or methylation.  

Global gel-free LC-MS/MS

This is a simplified method to identify (and quantitate) proteins from a cell, cell compartment, tissue or whole organism.  A 4 hour gradient is utilized on a 50 cm long analytical column for great LC separation.  Typically ~3500 proteins can be confidently identified and quantitated.  If desired, SCX fractionation can be added upstream to increase the depth of proteome coverage.  Please submit frozen cell/tissue pellets.  We have optimized protocols to ensure good, reproducible data from as little as 50,000 mammalian cells.  We would like to have roughly 3ug of peptides to analyze by MS/MS.

Global PTM LC-MS/MS

This service identifies the post-translational modifications, such as phosphorylation and acetylation, in the whole set of expressed proteins in a given type of cell, tissue, or organism at a given time, under defined conditions.  If your interested in mapping the global changes in a particular PTM, this is the experiment of choice.  The same process as the global gel-free LC-MS/MS is employed with an additional PTM specific enrichment step.  As with any PTM mapping project, more starting material will be required.

Quantitative Proteomics

We offer several approaches for quantitative proteomics:

Label-Free

This method is utilized to determine the relative amount of proteins in two or more biological samples. The initial cost of this method maybe lower than other protein quantitation methods because the method does not require chemical tagging or isotopic labeling.  However, more biological replicates need to analyzed compared isotopic labeling techniques.  

Stable Isotope Labeled

The SILAC (stable isotope labeling with amino acids in culture) method is a protein quantitative technique which detects differences in protein abundance among biological samples. It uses in-culture non-radioactive isotopic labeling of amino acids. SILAC reagents are not supplied by the facility and are the responsibility of the researcher.

Isobaric Mass Tag Labeled

Labeling multiple samples from potentially different biological sources or conditions enables simultaneous protein identification and quantitative analysis. Tandem Mass Tag (TMT) and iTRAQ reagents are two popular labeling kits for this type of analysis. By combining up to ten different labeled samples, the costs associated with mass spectrometry instrument run times are drastically reduced.