| Abstract:
During the past decade mass spectrometry has become widely accepted as an essential tool to better understand protein function, facilitating both the identification and quantification of proteins in complex samples.
We, and others, have previously described a novel approach to mass spectrometry based protein identification [1-3] that facilitates the simultaneous acquisition of qualitative and quantitative information, in a data independent fashion without the use of isotope labelling.
We have used this approach to generate absolute quantification values for proteins contained in biological systems. We have extended this to study samples from a range of organisms, as well as to construct protein abundance curves for specific tissues, cell lysates and biofluids. An important aspect of this absolute quantitation procedure is that it allows sample loading onto a given analytical column to be determined and optimized, to ensure that ideal chromatographic and mass spectrometric performance is obtained. This results in the maximum number of peptide and proteins being determined from the sample, whilst maintaining maximum accuracy for quantitative measurements. This we believe is an aspect often overlooked in modern proteomics experiments. Absolute quantification also provides a mechanism to define the protein stoichiometry present within a sample. In this manner protein pathways and families can be discerned and compared, and the mechanism by which proteins interact can be probed.
It is a common proteomics experiment to use relative quantitation, to determine information about protein expression changes within an experiment. In many respects this can be considered as an isolated island of information that can only be compared within a given experiment. The possibility of performing absolute quantitation of proteins generates a bridge between data sets, allowing the comparison of protein amounts across experiments, instruments, organisms, and laboratories.
In this presentation we will focus on the absolute quantification of proteins from a variety of different biological samples using label-free LC-MS. We will show absolute quantitation data from cell lysates of E.coli, human cardiac tissue, a study of stroke tolerance in the mouse brain, and the monitoring of known Gaucher disease biomarkers from the plasma of patients undergoing therapy.
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