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.
Protein Changes After Stroke
Zezong Gu, M.D., Ph.D., and his associates at both the "target=_University of Missouri-Columbia School of Medicine>University of Missouri-Columbia School of Medicine and the Burnham Institute, with Joseph Fox, Ph.D. and Gongyi Shi, Ph.D., at Bruker Daltonics have pursued the changes that occur in the proteins of the brain following stroke.
An important component of the process is abnormal proteolysis by matrix metalloproteinases which causes degradation of its substrates, an area of research in, which Dr. Gu has long focused.
Drs. Fox and Shi worked with Dr. Gu to utilize MALDI imaging mass spectrometry (MALDI-IMS) to determine both relative abundance and spatial distribution of the proteins in damaged tissues. The goal was to identify mass signatures correlated with abnormal proteolytic activity and apply this technology to the elaboration of the molecular profile after stroke.
Mice were subjected to cerebral ischemia followed by 24-hour reperfusion. The brains were processed and one set of sections was histologically stained while the adjacent tissue sections were analyzed via direct ionization of analytes from the tissue by the Ultraflex-III MALDI-TOF mass spectrometer. Selected ions were then displayed as pseudocolor images in which the color intensity corresponds to ion signal abundance.
In response to the ischemia, the brain showed localized swelling due to the traumatic injury. The overall anatomical brain structure was intact as evidenced by selective localization of a number of molecular ions. Interestingly, some molecular ions colocalize well with the injury area.
“Our studies have drawn us to the following conclusions,” Dr. Gu stated. “First, the MALDI-IMS tools are able to determine both the relative abundance and spatial distribution of the proteins and peptides in tissue traumatized by stroke. Second, multiple molecular ions colocalize within the injured area. Finally, these investigations point the way to identification of changes in brain architecture, proteins, and cell type that can act as targets for therapy.”
Ansgar Brock, Ph.D., senior researcher at the Genomics Institute of the Novartis Research Foundation, discussed his group’s work for the Joint Center for Structural Genomics using hydrogen-deuterium exchange.
In this process a covalently bonded hydrogen atom is replaced by a deuterium atom or vice versa. Mass analysis of the products yields information concerning solvent accessibility of various parts of the molecule. From these data, a description of the tertiary structure of the protein can be gleaned.
“Our goal is to better understand the three-dimensional solution structure of proteins and their dynamics,” Dr. Brock said.
As part of the hydrogen-deuterium exchange procedure, proteins are subjected to a fast exchange procedure, before being proteolytically fragmented for analysis by liquid chromatography MS. The mass shifts caused by the exchange of deuterium for hydrogen or vice versa allow investigators to infer aspects of the folding of proteins, as the exchange will preferentially take place on residues that are part of flexible regions and exposed to the solvent interface.
The technique is highly suitable to study protein-protein interactions. “No single technique is sufficient to provide a complete picture of protein structure and dynamics,” Dr. Brock stated. “This is why the hydrogen-deuterium exchange data produced by the genomics center are combined with results from the limited proteolysis experiments and analytical size-exclusion data to reinforce the picture.”