Fast Breaking Comment by Forest M. White

Forest M. White answers a few questions about this month's fast breaking paper in the field of Biology & Biochemistry.

From •>>August 2003

Field: Biology & Biochemistry
Article Title: "Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae"
Authors: Ficarro, SB;McCleland, ML;Stukenberg, PT;Burke, DJ;Ross, MM;Shabanowitz, J;Hunt, DF;White, FM
Journal: NAT BIOTECHNOL
Volume: 20
Page: 301-305
Year: MAR 2002
* MDS Proteom, Charlottesville, VA 22901 USA.
* MDS Proteom, Charlottesville, VA 22901 USA.
* Univ Virginia, Dept Chem, Charlottesville, VA 22901 USA.
* Univ Virginia, Hlth Sci Ctr, Dept Biochem & Mol Genet, Charlottesville, VA 22908 USA.
* Univ Virginia, Hlth Sci Ctr, Dept Pathol, Charlottesville, VA 22908 USA.

Why do you think your paper is highly cited?

...the field of phosphoproteomics is fairly new and very hot at this time.

The paper describes a modification (derivatization of peptide carboxylate residues) to an existing methodology (immobilized metal affinity chromatography for isolation of phosphorylated species). In the paper we applied the modified methodology to the analysis of protein phosphorylation in Saccharomyces cerevisiae and identified several hundred phosphorylation sites, approximately an order of magnitude increase over previously published results. I think the paper is highly cited because the field of phosphoproteomics field is fairly new and very hot at this time. Recent publications describing the state-of-the-art in phosphoproteomics tend to cite the paper, other citations have come from publications in the general field of protein post-translational modification analysis by mass spectrometry. Click here to view a graphic depicting the analysis of phosphorylated peptides from S. cervisiae.

Does it describe a new discovery or a new methodology that's useful to others?

The paper actually describes a modification to a methodology that has been around for over 20 years. Hopefully the modified methodology will be useful to others attempting to map protein phosphorylation events by mass spectrometry.

Could you summarize the significance of your paper in layman's terms?

The modified methodology in the paper virtually eliminates non-specific binding to a particular type of affinity chromatography column. Previously, the high degree of non-specificity was a limiting factor in the broad application of this methodology to complex biological systems. Eliminating the non-specificity of this affinity chromatography method enabled us to identify several hundred protein phosphorylation sites in Saccharomyces cerevisiae. The method is not fully developed yet, but in the future I am hopeful that this will be the core technology of a method that allows us to map signal transduction pathways from complex biological samples.

How did you become involved in this research?

I first started working in this field as a post-doc in Don Hunt's group at the University of Virginia. Scott Ficarro, a first-year graduate student, and I were attempting to map protein phosphorylation events in human platelets. We quickly realized that the standard methodology was limited by non-specificity, and that the vast majority of the non-specifically bound peptides contained multiple acidic residues (glutamic or aspartic acid). Don Hunt suggested derivatization of the carboxylate groups to eliminate non-specific binding to the affinity column. Scott and I then went back into the lab and worked on implementing Don's suggestion. Several years of method development were spent prior to the application to S. cerevisiae, which was meant to be a proof-of-principle study to demonstrate the feasibility of the technology in a very complex biological sample.

Forest M. White, Ph.D.
Biological Engineering
Massachusetts Institute of Technology
Cambridge, MA, USA

ESI Special Topics, August 2003
Citing URL - http://www.esi-topics.com/fbp/2003/august03-ForestMWhite.html