The Mass Spectrometry and Proteomics core facility is available to all University of Nebraska campuses' investigators.  Outside investigators are also welcome at a slightly higher rate.

MSPCF maintains and uses equipment for protein separation and imaging, as well as for sample preparation for mass spectrometry analysis. We are equipped with a wide range of bioinformatics tools for data mining and evaluation.

SPECIAL NOTE: If you are interested in submitting samples to the MSPCF, we strongly recommend that all persons involved in the project (PI, graduate students, post-doc, techs, etc.) schedule an initial consultation meeting with Dr. Vikas Kumar (Director), so we can assist you in choosing the right methods for your mass spectrometry experiment.  This can save you time and money.  In addition, we specialize in the development of customized projects outside of our routine analyses.  

To submit samples for analysis, please complete the online submission form.

Samples can be dropped off at DRC1 1027 or 1050. Please email ahead to schedule a time for that. 

Our operations flow chart:

Services

Protein Identification – Protein IDs

This is the most common request by our users. This is accomplished by proteolytic digestion, followed by LC-MS/MS. We usually perform all steps of the protocol for you but will need to discuss the best method and approach for your particular project. After MS/MS data is collected, spectra are being correlated against a specific database, that you must specify in Sample submission form. After the analysis of spectra is completed, we will send out the list of all proteins detected in your samples, with all additional information, including a method section that can be used in publications.

Protein Interactome Analysis

If you are sending us a pooldown (IP), we suggest eluting proteins from the beads before sending it to us. This can be done using either high pH (0.5 M ammonium-hydroxide/0.5 mM EDTA, pH 11.0) or low pH (0.2 M glycine, pH 3.0). You may also decide to boil your sample in a sample buffer, but this is not our preferred method. Please neutralize the sample to around pH 7.5 and bring it to us on dry ice. We will start from there. As of the protocol details, it is similar to Protein Identification. For this type of experiment, you will have to have at least 3 biological replicates, and some sort of a negative control (usually IgG). You are also encouraged to confirm your specific protein with Western blot, before we start the prep for MS.

Protein Post-translational Modifications (PTMs)

Mass spectrometry has a capability to detect and characterize most fixed PTMs that are stable in proteins. The greatest challenge in PTM detection is their occurrence and frequency, as most of them are rare and dynamic events. PTM projects have to be well researched and discussed, and almost always involve specific biochemical enrichment strategies. For the determination of one, or multiple PTMs, it is better to start with a single highly purified protein in SDS-PAGE gel slice. This is our recommendation, rather than a rule. The protocol will involve a careful selection of multiple enzymes (2 or 3 enzyme approach) to maximize peptide coverage for specific sites of interest.

Quantitative MS

Determination of a change in protein expression between two or more samples has become extremely popular, but also particularly challenging. With the right approach and bioinformatics tools, we can analyze differences in protein expression in many studying systems, for example between healthy and sick animals or cancer cell lines, wild type and knockout organisms, cell lines with different treatments etc. For this, we have two approaches: labeled or label-free quantitative MS methods.

There are several very well-established methods that utilize labelling of proteins or peptides for their quantitative MS/MS analyses, and the one mostly used in our core is TMT labelling (tandem mass tags). These isobaric small chemical tags have the same structure and covalently attach to the free amino acid termini of Lys residues on a peptide. During MS/MS analysis, where peptides are being isolated and fragmented, each tag produces a unique reporter ion signature that makes quantitation possible. Protein quantitation is done by comparing the intensities of reporter ions.

Other very popular quantitation method is label-free MS/MS. The method has shown good results with highly reproducible chromatography step. Data is generated using either Maxquant, Proteome Discoverer or Scaffold.

MW determination

Using either MALDI or LC-based mass spectrometry, we can determine the high accuracy molecular weight of the specific biomolecule. Your sample should be without salts and detergents.

Rates

For assistance in experimental design of mass spectrometry and proteomics based projects or to inquire about establishing a research-based collaboration, contact the Drs. Kumar or Lagundzin.