Resource Head & Manager
Michael Myers, Ph.D.
   
  Protein Chemistry Technicians
Daniel Perkowski
Lisa Lotte Schmidt
   
  2D Gel Technician
Gula Nourjanova
516.367.8462
nourjano@cshl.edu
   
   
Description
The Proteomics Shared Resource is a specialized technical facility that performs protein identification and characterization of post-translational modifications using mass spectrometry. Traditionally, most of the protein identification has been performed from proteins isolated by gel electrophoresis, but simple or complex mixtures can also be analyzed. The facility also carries out 2D gel electrophoresis or multi-dimensional liquid chromatography for analysis of complex mixtures. The Proteomics Shared Resource is continually exploring new protocols to improve the sensitivity, throughput, and scope of the analyses performed by the resource. Current equipment and protocols allow characterization of proteins in the 10-20 fmol range.

Equipment and Services:
Protein Digestion
Facility staff process submitted samples in preparation for mass spectrometry by washing (gel slices), treating with protease and desalting.
 
Protein Identification
MALDI-TOF mass spectrometry is used for purified proteins and usually provides a definitive match. In cases where no match is obtained, or in cases where there is evidence of more than one protein in the band, the sample is analyzed by LC-MS/MS.
 
LC-MS/MS is principally performed on the LTQ ion trap mass spectrometer. Nano-scale reverse phase chromatography is performed to maximize the sensitivity of this approach.
 
For complex mixtures, the sample is either analyzed by 1D LC-MS/MS or by 2D LC-MS/MS. The resulting fractions are then analyzed by nano-scale reverse phase chromatography, in a manner analogous to that used for gel-derived samples.
 
Characterization of Post-translational Modifications
Post-translational modifications can be readily detected by characteristic mass shifts.
 
Protein Characterization
Mass determination of whole proteins by MALDI-TOF mass spectrometry is used during crystallographic studies to verify the quality of a protein preparation prior to crystallization or to determine poorly structured regions that may hinder crystallization.
 
2-Dimensional Gel Electrophoresis
High-resolution two-dimensional gels are used to track qualitative and quantitative changes between samples or to determine if a protein has undergone post-translational modification. Following electrophoresis, non-radioactive samples are visualized by staining with silver, coomassie blue or Sypro Ruby Red stains; radioactively-labeled samples are visualized by autoradiography. The resulting images are analyzed using ImageMaster software from Amersham Biosciences.