Mass spectrometry analysis and identification of proteins

The Biomolecular Research Facility identifies and analyses proteins and peptides by mass spectrometry. For further information, contact Dr. Nicholas Sherman 434-924-0070.

Posters (PowerPoint and pdf) prepared in October 2007 show some of the data obtained by mass spectrometry analyses; these posters are on display near room 1105 Jordan Hall. Another poster (PowerPoint or pdf) shows the results of our current methodology for indentification of phosphorylation sites.

Analyses available:

  • Protein identification from gel or solution by either peptide mass fingerprinting or ESI-LC/MS/MS at the low femtomole level.
  • Analysis of protein mixtures such as tissue and media
  • comparison of tissue samples 
  • Identification of post-translational modifications (deamination, glycosylation, etc)
  • Identification of phosphorylation sites using titanium dioxide enrichment 
  • 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
  • Confirmation of peptide identity by MALDI mass measurements
  • Differential quantification by isotopic or iTRAQ labeling, or label free analysis.
  • Identification of binding partners
  • Biomarkers

Equipment 

Our current instruments 

  • Thermo Finnigan LTQ for standard analysis
  • Thermo Finnigan LTQ-FT for high accuracy measurements
  • Applied Biosystems QSTAR Elite which offers different fragmentations from the Thermo Finnigan ion trap instruments
  • Brukler Microflex for mass analysis by MALDI.

A brief description of the principles of the instruments are in a recent talk on mass spectrometry at the University of Virginia (Microsoft Power Point file)

Sample types analysed

  • gel bands, stained with Coomassie, silver, Sypro. 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 Protein Sciences Core.  
  • proteins in solution
  • tissue samples
  • media

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 

The W.M. Keck Biomedical Mass Spectrometry Lab at the University of Virginia has switched the way that sample analysis requests are made and how data is retrieved to the Proteus LIMS (Laboratory Information Management System).

Proteus has several advantages over the previous more manual methods used.

  1. Sample entry is performed on the web and is accessible there at any time. 
  2. Status of your project is updated as it progresses. 
  3. Results are posted to the project when completed (all in electronic format). 
  4. A special feature of Proteus is the ability to share information and data with anyone in the world (we can give access to part or all of your sample information and data to any other user(s) of your choosing). 
  5. Information/data is permanently on the LIMS so you can retrieve a copy of lost data, keep track of data when students graduate, etc. 

Using Proteus system

To login to submit samples or retrieve your data, go to:
http://128.143.19.247:8080/lablink/Login.do

If you have not used Proteus before, see the page on starting with Proteus.

Submitting samples 

Complete sample information in the Proteus system. We prefer to have the whole gel and a picture showing the bands or spots to be analyzed. If shipping the gel from outside the University of Virginia, we suggest sealing it in a bag placed between sheets of cardboard for protection. Send samples to:

Dr. Nicholas Sherman
Room 1101 Jordan Hall,
Department of Microbiology, Box 800734,
University of Virginia Health System,
1300 Jefferson Park Avenue
Charlottesville, VA 22908-0734
(phone 434-924-0070)

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 )