TABLE 2.
Troubleshooting table.
| Step | Problem | Possible reason | Solution |
|---|---|---|---|
| 1 | Labeling reaction is not successful or efficiency too low | Protein samples may contain primary amine components that compete with labeling reagents | Perform complete dialysis of protein sample to make sure protein samples are free of primary amines bearing reagents. Prepare fresh regents |
| 2-4 | The “heavy” to “light” ratio of the end time point mixture is not 1:1 | Problem during labeling rection and mixing of “heavy” and “light” samples | Ensure precision during pipetting, resuspension, mixing and transferring of samples |
| Problem of not performing “Experimental time-point” and “Reference” labeling reactions under identical condition | Perform “Experimental time-point” and “Reference” labeling reactions side by side, and essentially, under identical conditions | ||
| Problem during mixing of “heavy” and “light” samples | Be sure to equally mix experimental time-point and reference labeling samples | ||
| 10 | No signal, or poor signal intensity | Possible protein sample loss during extraction and precipitation | Be sure to mix and vortex as directed before centrifugation to reach equilibrium of extraction. Using gel-loading tips, carefully remove the aqueous layer without touching the finer emulsion that is created between the layers, containing protein of interest |
| 21: A (x), B (xiv) and C (iv) | No signal, or poor signal intensity | Problem during desalting process | Ensure that all buffers needed for desalting are appropriately prepared, as directed |
| Ensure that the pH of the reconstituted peptide is below 2 by pipetting 1 μL onto a pH strip; if not add more TFA | |||
| Ensure that air is not drawn into the column-tip and that it does not dry during the process | |||
| 24 | No signal, or poor signal intensity | Sample-matrix crystallization process is not optimized or irregularities of spotting on MALDI plates | Optimize sample-matrix crystallization conditions (See ref. Cohen et. al.) |
| Always use high-quality, ultra pure reagents and freshly prepared matrix | |||
| Take extra care when spotting and spot as uniform as possible | |||
| 24 | No signal, or poor signal intensity | Possible problem of having low signal-to-noise of peptide ions on MS | Increase laser to obtain optimized signal |