Protein Analysis and Identification by Mass Spectrometry

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Protein Analysis and Identification by Mass Spectrometry

The W.M. Keck Biomedical Mass Spectrometry Laboratory within the Biomolecular Research Facility identifies and analyzes proteins from gels and solution using current mass spectrometry techniques. There are techniques to compare samples which have different amounts of the same proteins.

To discuss projects, contact Dr. Nicholas Sherman 434.924.0070.

A poster shows some of the data obtained by mass spectrometry analyses. This poster is on display near room 1105 Jordan Hall.

Analyses available:

  • Protein identification and sequencing from gel or solution by either peptide mass fingerprinting or ESI-LC/MS/MS at the low femtomole level (Simple mixture  MS/MS service).
  • Analysis of protein mixtures such as tissue and media (Complex mixture MS/MS service)
  • analysis of samples prepared by Laser Capture Microdissection
  • Proteomics, comparison of proteins in tissue samples using chemical/isotopic labels or label free analysis
  • Absolute quantitation of proteins using labeled peptides and selective reaction monitoring (MRM service)
  • Identification of phosphorylation sites using titanium dioxide enrichment
  • Identification of post-translational modifications (e.g.  ubiquitination) (de novo sequencing service/PTM service)
  • De Novo (manual) sequence analysis of novel proteins to obtain sufficient sequence data for cloning
  • High resolution, high mass accuracy measurements of peptides and small proteins by ESI
  • Mass determination by MALDI mass measurements for confirming identity of peptides and proteins, either by staff or open access self service
  • Identification of interaction partners
  • Quantitative analysis of metabolites, peptides, biomarkers

Equipment

Our current mass spectrometers (see pictures)

  • Thermo Scientific LTQ Orbitrap Velos for high accuracy, high sensitivity analysis
  • Bruker Microflex for mass analysis by MALDI
  • Thermo Scientific TSQ Quantum Access MAX triple quadrupole instrument
Statement of resources for grant applications.
A brief description of the principles of the instruments are in a talk by Dr. Jim Farmar, Assistant Director of the laboratory.

Sample types analyzed

  • gel bands, stained with Coomassie, silver, fluorescent stains. Note that some silver stains are incompatible with mass spectrometry analysis (see the protocols suitable for sequencing). Gels stained with Coomassie, silver, Sypro, Pro-Q Diamond can be scanned through the Shared Instrumentation Core.
  • proteins in solution
  • tissue samples
  • media
  • Laser Capture Microdissection tissue samples

Consulting with Dr. Sherman before an experiment is strongly advised to ensure that the sample and experiment are compatible with the analysis.

Sample Submission and Data Retrieval

Enter samples for analysis in the GenoLogics LabLink system. Data will be posted in that system. New users may want to look at the getting started page

We have instructions and suggestions for preparing samples.

Acknowledgements

All investigators are asked to acknowledge the director, Dr. Nicholas E. Sherman, and our funding in any publications using our data.  The W.M. Keck Biomedical Mass Spectrometry Laboratory is funded by a grant from the University of Virginia Pratt Fund through the School of Medicine.

References

Papers describing the use of the techniques are:

Mandal, A., Naaby-Hansen, S., Wolkowicz, M.J., Klotz, K., Shetty, J., Retief, J.D., Coonrod, S.A., Kinter, M., Sherman, N., Cesar, F., Flickinger, C.J. and Herr, J.C. (1999) Biol. Reprod. 61 1184-1197

S.L. Hanna, N.E. Sherman, M.T. Kinter, J.B. Goldberg (2000) Microbiology 146 2495-2508

A recommended reference on the procedures is:

Protein Sequencing and Identification Using Tandem Mass Spectrometry M. Kinter and N.E. Sherman (Wiley InterScience, 2000) (Health Sciences library catalog QP551 .K495 2000)