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The Frontier of Quantitative Proteomics
Achievement/Results
In the past decade, we have seen the successful rise of sequencing technology. It is now possible to accurately determine the sequence of any nucleic acid molecule in any organism we wish. This opens up almost limitless research opportunities and also establishes genomes, a basic layer from which most bioinformatic studies must take root. However, in order to apply scientific knowledge, we must be able to understand biological systems to the extent that we are able to manipulate them to our benefit. Thus far, it has proven difficult to link genotype to many phenotypes of interest (Makowsky R, Pajewski NM, Klimentidis YC et al. (2011) Beyond missing heritability: prediction of complex traits. PLoS Genetics, 7,1–9.)
Currently, biologists focus mostly on protein coding genes. It must be noted that each of these gene has the possibility of coding for several different proteins and each protein has the ability to acquire one or more post-translational modifications that effect the function or regulation of the protein, such as phosphorylation, acetylation or ubiquitination. These are 3 common examples but over 100 such modifications have been identified. This means that the number of different proteins present in a cell can easily be hundreds of times greater than the number of genes. On top of that, the genome sequence is static throughout the life of the cell while the proteome is dynamic and continually changing. Lastly, we must note that almost all chemical reactions that give rise to life are directly regulated by proteins. One could say that the proteome is “closer” to phenotype and without information on protein modifications and abundance, we are leaving an enormous gap in our understanding of biological systems.
IGERT student, Ryan Sartor is currently undertaking a project to develop assays that measure the abundance of both unmodified and modified proteins and can theoretically be applied to any tissue sample and therefore any organism. Known as multiple reaction monitoring (MRM), these assays have the ability to give absolute quantification of a protein of interest. This means the ability to report the precise number of molecules that are present in a given sample. In addition to studying the behavior of proteins and protein modifications throughout different processes, this will allow the determination of stochiometric protein/protein and protein/metabolite ratios. Absolute abundance measures will also facilitate direct comparison between any two datasets. In theory, assays can be developed using available technology that measure over 200 proteins in less than one hour.
Ryan is working on a set of 75 Arabidopsis proteins. The protocols for developing such assays are very incomplete and one of the goals is to determine that most efficient way to bring such assays into existence. Ryan is working with an Agilent 6410 triple quadrupole mass spectrometer and using an online HPLC 2-dimentional separation which makes use of both reverse-phase and strong cation exchange columns set up in tandem. The development process has turned into an iterative one where assays are developed for all 75 proteins, the results are accessed, improvements are made and the process is repeated. The first iteration was used to examine the fastest approach possible where the assay could be developed for ~100 proteins by one person in about 1 weeks. The assay would then run with a single reverse-phase column in 30 min. This method gave successful assays for 8% of the proteins. The second iteration focused on improving the separation on a 2D column with a runtime of about 2 hours and gave a 24% success rate. Ryan is currently working on improving both the separation and the signal intensity through more thorough methods using other types of mass spec data to assist in assay development.
Address Goals
Primary: The goal of an IGERT student Ryan Sartor’s project is to use quantitative protein measurements of both abundance and modifications as traits to be examined in a QTL experiment on the Arabidopsis Bay x Sha RIL population. The proteins of interest are ones that have been previously shown to be involved in defense. The hope is that loci may be discovered that effect defense related protein modifications or protein abundance, therefore providing novel information about the poorly understood molecular pathways involved in plant defense. If successful the same approach will be applied to Zea mays with the goal of applying this knowledge to improving crop defense.
Secondary: Many challenges exist in the MRM development. These assays require comprehensive knowledge of mass spec and HPLC instrumentation, as well as peptide and column chemistry, protein biology and computational analysis. However, MRM assays hold much promise in advancing the fields of proteomics and quantitative biology. A single example of why interdisciplinary, systems-level training is crucial for the next generation of scientists.
