When biochemists talk about protein quantification, they often refer to the use of colorimetric assays such as the Bradford and its rather mundane application for the calculation of specific enzyme activities.
The past decade, however, has witnessed a rapid expansion in both the methods and applications of quantitatively assessing protein levels. These include mass spectrometry (MS) of proteolytic fragments combined with liquid chromatography, isotopic labeling of peptides and isotope dilution mass spectrometry (IDMS). The availability of many extremely accurate measuring tools has enabled investigators to map out the relationship between disease processes, and protein abundance and distribution, as recently discussed at the “La Jolla Proteomics Conference.”
Jing Wei, Ph.D., group leader of the proteomics biomarker discovery program at Biogen Idec, presented her experiences in protein biomarker development at the meeting. The aim of the research in her group is to apply proteomic tools—peptide separation, MS-based identification and quantification techniques—to clinical questions such as searching for new patient stratification tools, developing therapeutic biomarkers, and understanding the mechanism of action of putative biomarkers.
The work flow of the Biogen Idec team begins with sample preparation, followed by its elucidation through online 3-D nano LC- MS/MS technology. Biological samples to be analyzed include tissues and cells, both frozen and formalin-fixed; serum-free culture medium; tumor interstitial fluid; and several types of body fluid. The accumulated data are analyzed and subjected to data mining, allowing for appropriate candidate selection.
For quantitation, Dr. Wei and her coworkers use iTRAQ™ isobaric tagging system (Applied Biosystems). This set of reagent labels free amines at the N-terminus and Lysine residues of peptides in the complex mixture and allows up to eight samples to be differentially tagged and mixed together prior to separation and MS analysis.
According to Dr. Wei, the iTRAQ quantitation technology is powerful and effective using ESI-CID-PQD MS/MS on LTQ or LTQ-Orbitrap instruments (Thermo Scientific), and able to measure subtle changes of protein levels in the proteome. Pulsed-Q Dissociation (PQD), a new fragmentation technique that eliminates the low mass cut-off for ion traps and allows quantitation with iTRAQ labeling reagents.
The group has developed discovery programs in oncology and neurology and so far has identified a number of marker candidates that have proceeded to MS-based verification and antibody-based validation stages.