1. Field of the Invention
The present invention relates generally to LC/MS analysis of protein mixtures. More specifically, the present invention relates to absolute quantitation of proteins by LC/MS analysis of enzymatically digested proteins in simple or complex mixtures.
2. Background of the Invention
The study of proteins is crucial in a number fields including understanding and combating disease through identification of proteins, discovering disease biomarkers, studying protein involvement in specific metabolic pathways and identifying protein targets in drug discovery. An important technique that is often used in these studies is liquid chromatography combined with electrospray ionization mass spectrometry (ESI-LC/MS) to quantitate and identify peptides and proteins present in simple and complex mixtures.
One approach for quantifying peptides and proteins in simple and complex mixtures involves determining the corresponding relative abundance between two experimental conditions. During these experiments it is important to compare identical components between the two experiments in order to accurately determine relative ratios of peptides to particular protein(s). By doing so, multiple relative abundance values for each peptide to a given protein can be obtained to quantitatively characterize the differential expression of proteins between and among different physiological conditions.
Another approach to the quantitative study of proteins is to determine the absolute concentration of the peptides and/or proteins that result from enzymatic digestion of a given protein sample. In this approach, digestion of a protein sample using a protease such as trypsin produces many smaller polypeptides, each having a specific primary amino acid sequence. It is known that a given mole quantity of protein produces the same mole quantity for each tryptic peptide cleavage product if the proteolytic digest is allowed to proceed to completion. Thus, determining the mole quantity of tryptic peptide to a given protein allows determination of the mole quantity of the originating protein in the sample. Absolute quantitation of the protein can then be accomplished by determining the absolute quantity of the peptides to that protein(s) in the digest mixture.
Typically, absolute quantitation of proteins requires one or more external reference peptides that are used to generate a calibration response curve for specific polypeptides from a given protein (i.e., synthetic tryptic polypeptide product). The absolute quantitation of the given protein is determined from the observed signal response for the specific polypeptide in the sample relative to that generated in the calibration curve. If the absolute quantitation of a number of different proteins is to be determined, separate calibration curves are generated for each specific external reference peptide for each protein.
U.S. Patent Application No. 2004/0229283 to Gygi et al. (“Gygi”) describes a conventional technique for absolute quantitation of proteins in complex mixtures that uses a synthesized derivative peptide as a standard. A derivative peptide is a peptide that is chemically identical to a naturally occurring peptide of a given protein. The derivative peptide is introduced to a complex mixture. The mixture is analyzed using LC/MS resulting in ionization intensities for the derivative peptide. This intensity signal response is compared with an intensity calibration curve created using the introduced synthetic molecule to determine the amount of the derivative protein in the mixture. A disadvantage with using synthetic peptides is that extra steps are required to synthesize an authentic sample, and to later “spike” the synthetic standard prior to being able to determine the absolute quantity of the protein itself.
Another technique for absolute quantitation of proteins employs an S35-methionine or other types of radio label, whose specific activity is known. In this radio labeling techniques, an amino acid, such as S35-methionine, that is radio labeled is fed to a cell. As proteins are synthesized, the proteins incorporate the S35-methionine instead of methionine. Based on the extent of incorporation of the radio label, the absolute amount of the peptide or protein can be determined. A disadvantage with using radio labels is that in some instances, such as studies on humans or other organisms, radioactive feeding or doping is expensive and may be deleterious to the subject and therefore impractical. Consequently, determining absolute quantitation of proteins using radio label techniques is limited to expendable biological systems such as microbes and plants.
Other protein quantitation techniques provide relative quantitation of protein amounts between two samples. Relative quantitation provides information as to how specific protein abundances change due to a perturbation (environment-induced, drug-induced, disease-induced). But, such relative quantitation techniques do not provide the absolute quantity of a particular protein present in a sample.
Consequently, a technique for determining the absolute quantity of a protein in a sample that does not suffer from the disadvantages or requirements of the prior art is required.