Absolute quantitation of peptides and proteins using mass spectrometry: Is it a Fact or a Fiction?

Thu, October 15 2009, 12:00 AM
Posted By: Sciclips

Considerable efforts have been made to develop tools for the absolute quantitation of peptide/protein using mass spectrometry (MS). Proteomics researchers are so fascinated with the concept of absolute quantiatation and they are spending enormous amount of time for developing tools or optimizing methods for the absolute quantitation of proteins in a cell or tissue or organ. These experiments and technology development efforts not only take lot of time but also involve huge capital investment. The question is, whether it is worth spending so much money in these efforts? The arguments for justifying such studies are that it is necessary to understand the absolute quantity of a protein for quantitative proteomics. This will enable us to quantitate exact amount of toxins in food/environment, biomarker proteins and drug target proteins and so on. It looks very reasonable to measure the exact quantity of a disease biomarker or Staphylococcal enterotoxins in various foods.

Let us take an example of measuring Staphylococcal toxins in a meat sample. By using expensive isotopically labeled peptides or complex in vivo/in vitro expression system derived proteins/peptides or various software intense label-free methods, we can measure the exact amount of toxins present in the meat sample. Now, we need to see, is there any real practical applications of this information? Do we really need to know the exact concentration of toxins? What are the advantages of this method over antibody based ELISA approaches, which are more frequently used; less expensive and more robust?

Take another example, a disease biomarker, which is much more appealing than the toxin. The argument will be it is required to know the absolute quantity of a biomarker protein in a tissue or organ of cell. The question is, for what? Take a situation, where a laboratory has discovered a breast cancer biomarker protein. There upon, after adopting laborious absolute quantitation methods they determined the exact amount of the protein expressed in breast cancer cells/tissue. It is good to know the absolute concentration of the biomarker protein, but, does it helps in diagnosing or finding a cure for breast cancer? It may be useful in diagnosis, but, what would be the advantage of this method over immunological assays (e.g. ELISA)? Does knowing the absolute quantity helps in better diagnosis of breast cancer? Does the information on absolute quantity helps in discovering new drugs? It is worth to consider the following comments. Even the most sophisticated MS technology is limited by relatively poor analytical sensitivity, compared to most clinical immunoassays. Indeed, the Human Proteome Organizations (HUPO) own plasma proteome efforts have failed to demonstrate the ability to routinely measure analytes in the dynamic range that most clinically useful analytes are found. Until we can employ rapid up-front concentrating and fractionating techniques for MS measurements, this analytical sensitivity barrier will likely not be overcome whereby hundreds to thousands of biological samples can be analyzed by MS every day for a given protein analyte( Emanuel F. Petricoin III, George Mason University, Clinical Chemistry 55: 1427-1430, 2009)

We may need more and more interaction of cell biologists/ biochemists/geneticists with analytical chemists who are predominantly dominate the mass spectrometry/proteomics field. The foremost challenge in any absolute protein quantitation approaches using cells/ tissues or organs is the biology of the target protein/s. We need to consider the protein expression regulation, protein degradation, protein turnover and so on. Do the currently used absolute quantitation approaches really give actual concentration? The amount of target proteins may be different in various stages of cell division, environmental conditions, expression of other interacting partners etc. Without knowing all these bits and pieces, which may takes years to understand fully, what would be the benefit of knowing the absolute quantity of a target protein in a test sample? Does this number really reflect the absolute quantity? The concentration of a target protein in cell/tissue/organ may be a moving target. How do we quantitate the exact amount of these proteins that change all the time? We need to clarify the above questions before undertaking any major experiments on absolute quantitation. Another argument will be the capability MS method sin multiplex analysis where multiple proteins can be analyzed in a test sample. Though the MS supporters claim that multiplexing is not possible by immunoassays, the fact is bead based immunoassays can be used for multiplex analysis. Even if consider the fact that only MS based can use used for multiplex analysis, what would be real application of this technology?. Multiple biomarkers can be analyzed in a single experiment. This is true, but how many clinical assays are depend on multiple biomarker analysis? What are the risks if multiple biomarkers are used in diagnostic assays? Do we really need absolute quantitation of multiple biomarkers? May be there are applications in laboratory research. Still, it is not possible to quantitate absolute amount of multiple proteins in a sample considering the biology of these biomarker proteins. Ideally, it would be best to identify a biomarker protein or a drug target protein using various proteomics approaches. Once we have a biomarker, we should spend most of our time and money for validating biomarkers for diagnosis or drug discovery. Then, spend more time on developing a drug that would target the identified biomarker/protein, rather than spending our scientific energy and time for developing tools for measuring absolute quantity of this biomarker.

This does not mean that we should not develop methods for the absolute quantitation of proteins. We need to develop tools for this, however, we should not deviate our focus on applications of proteomics in clinical diagnosis and drug discovery. These are the areas that will benefit the society. We need to think about the right strategies and technologies that will meet these goals. Proteomics needs long-term strategy and we need to spend our time on developing technologies that will have immediate benefit for the enhancement of the field as well as future direct applications. Proteomics has tremendous opportunities and we should all think about developing strategies or technologies that will sustain this field by providing long-term benefits to the society.

Additional information

Absolute quantitation of proteins/peptides (PubMed)

Categories: Proteomics