Proteomics
Matrix Assisted Laser Desorption Time of Flight (MALDI-TOF) mass spectrometry offers a powerful technique for protein identification. Identification is achieved by first digesting the protein into peptide fragments using a proteolytic enzyme such as trypsin. Mass spec analysis generates a mass fingerprint or profile which is unique to a particular protein. Comparison of this peptide mass fingerprint with databases of known protein digest fingerprints provides the means of protein identification.
The SERI Proteomics Facility offers high throughput protein identification with robotic spot excision, trypsin digestion and MALDI-TOF mass spectrometry. The facility features the ProXcision spot cutting robot capable of picking from fluorescent SYPRO Ruby or Coomassie blue and Silver stained gels. Trypsin digestion is performed using a Multiprobe II liquid handling workstation utilizing Millipore zip plate technology. Peptide mass fingerprints (PMF) are obtained on a PerkinElmer prO-TOF 2000 MALDI-TOF MS. PMF are searched on an updated local copy of the NCBI protein database or Protein Information Resource NREF (Non-redundant REFerence) protein database using the ProFound search engine. Reports are created in word format and also copies of the raw data and peak list are generated.
The SERI Proteomics facility offers these services:
-
Differential display analysis with Nonlinear Dynamics 2D Evolution software.
-
Spot picking using the ProXcission spot picking robot from PerkinElmer.
-
In gel trypsin digest
-
MALDI-TOF MS peptide mass fingerprint analysis
-
Database searching for protein identification
-
Comprehensive report creation.
Important Considerations
-
MALDI-TOF works best on purified protein typically separated by 1D or 2D PAGE.
-
Protein identification by MALDI-TOF can generate both false positives and false negatives. It is therefore important to give careful consideration to the validity of the protein identification made.
-
Though MALDI-TOF is a very sensitive technique for protein identification it is important to consider the starting amount of protein available and whether it is sufficient for protein ID. As a rule of thumb, most Coomassie blue stained spots can be identified successfully. With the more sensitive dyes, such as SYPRO Ruby, obtaining protein ID from faintly stained spots is not very reliable.
Service Fees (pricing through 2006)
Differential Display Analysis: $45 per hour
Spot picking + Trypsin Digestion: $35 per spot
MALDI-TOF MS*: $40 per spot
*Includes database searching and report generation
Contact Information
Dr. Ian Rawe
20 Staniford Street, Boston, MA 02114
(781) 325 2439
This e-mail address is being protected from spam bots, you need JavaScript enabled to view it
