August 2013

Protein quantification: by mass spectrometry or Western blotting?

In the past several years, a mass spectrometric method known as selected reaction monitoring or multiple reaction monitoring increasingly has proved to be adept at quantifying a targeted set of proteins in a complex sample. In a recent editorial in the journal Molecular & Cellular Proteomics, associate editor Ruedi Aebersold at the Swiss Federal Institute of Technology Zurich and co-editors Ralph A. Bradshaw and Alma Burlingame at the University of California, San Francisco, say that the time has come to let the protein-quantification data generated by SRM and MRM stand on their own and that they don’t need to be supported by data from the protein-quantification workhorse used in most molecular biology and biochemistry laboratories, Western blotting.
In Western blotting, a method that was developed more than three decades ago, proteins from a sample are separated on a gel and transferred onto a membrane. The presence of a certain protein is then determined by highlighting the band that contains it using putatively specific antibodies. The density of the highlighted band reflects the relative abundance of the protein in the sample.
SRM also was developed decades ago for the quantification of small molecules in complex samples. More recently, the method has been adapted for use in targeted quantitative proteomics. Much like Western blotting, the method detects and measures the amounts of known proteins in complex samples. Peptides derived by proteolysis of a protein sample are ionized in an electrospray ion source; the peptide ions uniquely associated with the targeted protein or proteins are isolated and fragmented. The signal intensity of fragment ions uniquely associated with the targeted peptide then is recorded over time, indicating the abundance of the peptide in the sample.
In their editorial, Aebersold, Burlingame and Bradshaw explain, “Authors who submit papers containing quantitative protein data generated by MS are frequently asked by reviewers to validate some of the values by Western blotting. We believe with the advances that have occurred that this request is now outdated, causing the unnecessary use of scarce resources and not achieving the main intent: objective cross-validation of results.”
The authors say they support the use of independent techniques to verify results, but in this case the quality of SRM data outstrips that of Western blotting. For example, the mass spectrometric method measures several peptides per quantified protein, thus generating several independent measurements, whereas in Western blotting only a single signal, the intensity of the detected band, is available.
However, the authors add that their argument against using Western blotting to validate protein-quantification data obtained by SRM “should not be construed as meaning the Western blots have no value and that the technique should be dropped from the arsenal of useful biological methods.” Rather, they say, the notion of Western blotting as the gold standard “for quantifying proteins in complex samples has to be seriously questioned, now that SRM assays for proteins can be developed and used with comparative ease.”

Rajendrani MukhopadhyayRajendrani Mukhopadhyay ( is the senior science writer and blogger for ASBMB. Follow her on Twitter (, and read her ASBMB Today blog, Wild Types.


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