Table I.
| Category | Western blotting | MRM proteomics |
|---|---|---|
| Principle | Protein separation based on protein 3-D structure or the length of the polypeptide using gel electrophoresis. Then, proteins are transferred to a membrane (e.g., nitrocellulose) and stained with specific antibodies | Selective quantification of surrogate peptide(s) in a digested protein sample by LC-MS/MS |
| Selectivity | Cannot distinguish between homologous proteins | Can differentiate proteins even when they differ by a single amino acid residue |
| Sensitivity | If good antibodies are available, this method is sensitive and can detect proteins up to picomole (per mg of total protein) level | If the best peptide is used, MRM method is comparable or better in sensitivity than Western blotting |
| Number of end points | Single end-point. Poor quality control | Multiple endpoints, e.g., multiple MRM transitions of more than one surrogate peptides (if available) |
| Reproducibility | Poorer reproducibility than MRM because of the cumbersome procedure | Highly reproducible. Most of the steps are automated |
| Reagent quality | Selective antibodies are difficult to synthesize | Well-characterized synthetic peptides are readily available |
| Reagent availability | Antibodies are not always available | Peptides can be commercially procured |
| Costs | Quantification of a single protein is more economical than the MRM method | Instrument cost is high. However, it can quantify multiple proteins at a time, reducing the overall cost |
| Key advantages | Technique and expertise are routinely available in many laboratories | Selective, multiplex quantification, robust, large dynamic range |
| Major limitations | Nonselective, low dynamic range | Cost of instrument and sophisticated method development |
LC-MS/MS liquid chromatography tandem mass spectrometry, MRM multiple reaction monitoring