Third-Party Application (system support)
An Automated Method for Biotinylated Peptide Purification and MALDI Target Spotting for Analysis of Proteins by Mass Spectrometry
Kelly L. Foster, John G. Dapron, Jodi M. Zobrist, Layle K. Barbacci,
Tom C. Hassell, and William K. Kappel
Sigma-Aldrich Corp.
Biotechnology Division
P.O. Box 14508, Saint Louis, MO 63178
www.sigmaaldrich.com
This Biomek® application is an automated process designed to allow for characterization of biotinylated peptides by MALDI-MS. The peptides are purified from a mixture using Sigma's high capacity streptavidin coated plate and then spotted on a MALDI target. This method can be used to facilitate protein identification and characterization from a proteome of interest.
The method begins with denaturation, reduction and biotinylation of proteins through specific residues. The proteins are tryptically digested and subjected to fractionation by ion exchange HPLC. The effluent from the chromatography column is collected in the wells of a Streptavidin High Capacity (HC) plate that provides for selective capture of the biotinylated peptides. The plate is transferred to the Biomek Automated workstation for wash and elution of the peptides. The elutions are then spotted onto a multi-well MALDI target by the Biomek robot and analyzed by mass spectroscopy. Finally, database searching can be utilized to ascertain the identity of the proteins in the original mixture.
Biotinylation may be achieved specifically at cysteine or lysine residues, phosphorylation sites, or glycosylation sites depending on the reagent used. The method therefore allows for determination of protein structure as well as potential for information on posttranslational modifications. The key component of this capture method is Sigma's Streptavidin HC plate that offers a binding capacity >300 pmole/well. The multi-well plate format allows for quick fractionation and easy automation of washing and elution steps that would otherwise require more labor-intensive procedures.
Sigma-Aldrich offers a wide variety of coated plates, biotinylation reagents as well as solubilization reagents, proteases, and mass spectrometry tools for proteomics research.
Features
- Denaturation and biotinylation of a protein through specific residues
- Digestion of protein mixture
- Fractionation by ion-exchange HPLC
- Capture of biotinylated peptides from HPLC effluent in Streptavidin HC plates
- Wash and elution of peptides on Biomek Automated workstation
- MALDI target spotting with Biomek
- MS analysis of peptides with database searching for protein identification
* All trademarks are the property of their respective owners. Where applicable, the PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman-LaRoche, Ltd.
