Question

Describe Surface-Enhanced Laser Desorption Ionization - Time of Flight (SELDI-TOF) mass spectrometry as an approach for the rapid detection and quantification of proteins.

Answer 1

Surface-enhanced laser desorption–ionization (SELDI) is a combination of solid-phase chromatography and time-of-flight mass spectrometry (TOF-MS). The binding of various protein classes is due the different coatings on the chips. ProteinChip arrays are available with different chromatographic properties, including reverse phase (H50), hydrophobic, hydrophilic, weak ion exchange (CM10/WCX2), and immobilized-metal affinity surfaces (IMAC3/IMAC30), or they are preactivated for the coupling of ligands. To reduce the sample complexity and to enrich target molecules depending on the chip surface each analysis involves an in-built fractionation step

Once the SELDI-TOF MS spectra have been collected sophisticated bioinformatics software tools are used for data processing which are based on genetic algorithms, decision trees, and a unified maximum separability algorithm. ProteinChip DataManager™, ProteinChip Biomarker Wizard™, and Biomarker Patterns™ are commonly used. The following generic protocol is used in SELDI-TOF MS.

The minimum sample requirement is 1–10 μg of the total protein per spot and 50–200 μl sample volume if a bioprocessor is used.

Assay Procedure as follows

1. Assemble arrays in a bioprocessor.

2. Precondition with appropriate buffer

3. Add 50–150 μl of sample to each spot and incubate for 30 min with gentle shaking

4. Wash three times with 150 μl binding buffer for 5 min with shaking

5. Wash twice with 150 μl double distilled water, 1 min per wash

      6. Remove the bioprocessor; invert and remove any remaining water

7. Remove the reservoir from the bioprocessor

8. Allow arrays to air dry

9. Add twice 1 μl of 50% saturated SPA or CHCA matrix solution to the spot surface and allow to air dry in between

10. The probes now are ready to be analyzed. If necessary, store the chips in a dark room at room temperature until processing

11. Place the ProteinChip arrays in the SELDI-TOF MS and irradiate with a pulsed UV nitrogen laser

12. Set the mass analysis range to be evaluated

13. Adjust the laser intensity and other parameters

14. Run instrument checks and calibrate the instrument

15. Save the calibration curves (apply to spectra prior to analysis)

16. Load cassettes with chips into the protein chip reader

17. Make test shots to optimize laser intensity and detector settings

18. Incorporate laser intensity and detector settings into a spot protocol

19. Assemble spot protocols into a chip protocol

20. Assemble chip protocols into a cassette protocol

21. Run the cassette protocol and collect data

22. Normalize the data to a total ion current with an external value of 1

23. View the spectra for high laser intensities for detection of high-molecular-weight species; set a cut-off mass range around 10 kD to exclude noise and leave the high-mass range unrestricted

24. View the spectra for low laser intensities for detection of low-molecular-weight species; set a cut-off for exclusion of matrix noise between 1500 and 2000 Da

25. Set noise definitions to desired settings

26. Perform manual or automatic peak picking

            27. Perform statistical analysis