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
| Q. | What is the binding capacity of the Streptavidin HC plates? |
| A. | The binding capacity of the Streptavidin HC plate is ³ 300 pmole biotin/well for a small molecule and approx. 4 µg/well for a biotinylated protein. |
| Q. | What are the kinetics of binding to the Streptavidin HC plate? |
| A. | Maximal binding is achieved on the Streptavidin HC plate with small biotinylated molecules in 1-2 hours. With biotinylated proteins, maximal binding is achieved in 4 hours. |
| Q. | What pH range can I use for binding to the Streptavidin HC plate? |
| A. | Binding may be accomplished to the Streptavidin HC plate in the range of pH 3-9. |
| Q. | What solvents and/or salts are compatible with binding to the Streptavidin HC plate? |
| A. | Binding of biotinylated peptides has been accomplished on the Streptavidin HC plate in as much as 25% acetonitrile and 0.5M ammonium chloride at pH 3 or 7. |
| Q. | What is the best way to elute biotinylated molecules from the plate? |
| A. | Elution may be achieved from the plate
with a variety of reagents depending on your analysis method. In
order of relative MALDI signal obtained, the reagents that may be
used are:
|
| Q. | How does the Streptavidin HC plate compare to monomeric avidin agarose? |
| A. | Similar results were obtained using the Streptavidin HC plates and the monomeric avidin agarose when binding at pH 7. This is to say that when binding from a mixture of biotinylated and non-biotinylated peptides, the same specificity and the same sequence coverage was obtained with both formats. When binding at pH 3, the Streptavidin HC plates offer superior sequence coverage to the monomeric avidin agarose. The Streptavidin plates also offer a high throughput format which is easily automated as compared to monomeric avidin agarose. The monomeric avidin agarose offers a higher relative biotin binding capacity. |
| Q. | Do I have to use the entire Streptavidin HC plate? |
| A. | No. You may cover unused wells with sealing tape (Sigma-Aldrich T2162). |
| Q. | How stable are the Streptavidin HC plates? |
| A. | The plates are stable for 4 years from the date of manufacture when stored unopened at 2-8°C. After opening it is recommended that the plate be used immediately. If only partial plates are used, it is recommended that the unused wells be immediately sealed with tape. The entire plate should be stored at 2-8°C and used within one week. |
| Q. | Are there other methods of digesting the proteins of interest besides trypsin? |
| A. | Sigma-Aldrich offers a wide variety of high purity proteases and cleavage reagents designed for protein analysis. Some of these include Endoproteinase Arg-C (P6056), Asp-N (P3303), Glu-C (P6181) or Lys-C (P3428), Chymotrypsin (C6423) and Cyanogen bromide (Fluka 16772). |
| Q. | Could I use this spotting protocol with other MALDI targets? |
| A. | For this method, the labware holder was modified for use with the Shimadzu Biotech Axima-CFR MALDI target. This was performed to allow for reproducibility of target placement and spotting accuracy. The platform holder could in theory by modified to accommodate any manufacturers multiwell type MALDI target. |
* 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.
