Mass Spectrometry

The MS Core sample submission form may be downloaded here. Your Exp ID should be a simple, unique name for each sample (i.e., JJ1-1, JJ1-2, etc). It is helpful for our team to see an image of a gel for samples you submit and you may attach it to the sample submission form when submitting your samples.

We strongly recommend setting up a consultation with a member of the MS Core team before planning experiments that will be analyzed by MS. 

Frequently Asked Questions

Affinity Purification / Immunoprecipitation Sample to Identify Protein-Protein Interactions

How much material do I need?

The amount of starting material required for AP-MS varies depending on the level of protein expression and the method of purification. For a FLAG- or Strep- tagged protein that is transfected into 293T cells and is highly expressed, 1-2 15cm plates of cells at >80% confluence should be sufficient. An aliquot of your eluted sample should be run on a gel and visualized by silver-staining. Your bait band should be visible on the gel, and ideally it should be the most abundant band in the lane. 

How should I submit my sample(s)? 

Most importantly, we do not accept samples in buffers containing detergents (NP-40, SDS, Triton, etc).

If you are performing FLAG or Strep affinity purifications, we strongly recommend following the Krogan lab's protocols, which can be found here. This protocol elutes with purified FLAG peptide or desthiobiotin, respectively, in a buffer that contains 0.1% RapiGest. RapiGest is a MS-compatible detergent with properties similar to SDS that breaks down upon the addition of acid.

For immunoprecipitation with endogenous antibodies, we recommend an acidic elution with glycine. We have had success eluting with 0.2 M glycine pH 2.5 for 5-10 min, followed by neutralizing the pH by adding 1/10th volume of 0.1 M Tris pH 9.0. 

We do not recommend submitting gel slices for AP/IP samples, as they take much longer to analyze by MS and don't yield superior information. When at all possible we recommend designing an elution strategy that keeps the protein in solution for MS analysis.

Samples to Identify Post-Translational Modifications (PTMs) on Purified Proteins

How much material do I need?

For PTM analysis of a protein of interest, we require more material than for standard AP-MS experiments. We have the most success with proteins that are clearly visible on a Coommassie-stained gel. 

How should I submit my sample(s)?

Our standard procedure for PTM analysis of most proteins is to purify the protein, run it in a gel, stain with Coommassie, and cut out the band for your protein of interest and submit the band to us. We will then apply in-gel trypsin digestion and high resolution MS analysis on our LTQ Orbitrap Elite MS system. We recommend trying this method first and optimizing the experiment based on these results.

If the standard procedure does not work then some optimization may be necessary. If it is possible to elute pure, abundant amounts of your protein in solution, then this allows us to subject the protein to multiple different enzymes that may increase coverage of the protein sequence. 

Samples for Global Studies (Global Analyses of Protein Abundance or Post-Translational Modifications)

Most global analyses require specialized methods for quantification. You should consult with a member of our team before designing global proteomics experiments. 

How much material do I need?

For global analysis of protein abundance, we recommend providing a minimum of 100 ug of material (as measured by a Bradford assay). 

For global analysis of post-translational modifications, we recommend providing a minimum of 10 mg of material (as measured by a Bradford assay) for optimal results. Lower amounts may be provided, but coverage will be more limited to the most abundant proteins in the sample.

What post-translational modifications can I analyze?

We have experience performing global analyses of phosphorylation, ubiquitination, and acetylation. There are additional antibodies available for other PTMs that are amenable to the approaches used in the MS Core. 

How should I submit my sample(s)?

Most importantly, we do not accept samples in buffers containing detergents (NP-40, SDS, Triton, etc).

For most easy to lyse mammalian cells, you may provide a cell pellet that has been washed with PBS and snap frozen in liquid nitrogen. 

If you will be lysing cells or tissues yourself, we recommend lysing in a buffer containing 8 M urea, 0.1 M Tris pH 8.0, 150 mM NaCl, and protease inhibitors (we use Complete tablets from Roche). You should also sonicate your samples to shear DNA, and spin down the sample to remove insoluble material.