The present invention (priority date 25 August 2000) makes use of unique isotopic tags (for example: 18O, 15N, 13C or 2H) of a specific biopolymer that can be exploited for determining the absolute concentration of the biopolymer in crude solutions. In preferred embodiments the biopolymer is either a polypeptide or a polynucleotide. Particularly, the invention provides a method for the determination and quantitation of biomolecules in crude mixtures by way of a separation technique in combination with mass spectroscopy. In one general embodiment, a target biomolecule is selected for analysis and an analog thereof is generated. Peak area integration of the labeled and non-labeled peptide pairs provides a direct and precise measurement for the amount of the target protein in the crude solution without the need of expensive Maldi TOF Mass Spec. This technology has broad utility for fermentation analysis, biomarkers and biologic drug studies, medical diagnostics from serum, tissue extracts or cell lysates -- anywhere precise quantitation of biopolymers is desired from a crude mixture. The redundancy of fragments pairs allows several measurements from a single sample to confirm accuracy and increase sensitivity. Addition of the calibrated standard analog to the crude mixture provides a self-correcting assessment of the extent of proteolytic digest and recovery during the entire method.
Accurate quantitation of proteins and peptides in crude solutions such as fermentation broths, cell lysates, or plasma and serum is challenging and requires robust analytical techniques with good sensitivity and specificity because of the complexity and extreme dynamic range that characterizes these solutions. Such quantitation has been typically achieved using enzyme-linked immunosorbent assays (ELISA) because of their high specificity, sensitivity and throughput. However, ELISAs rely on expensive, high quality antibodies that are not always obtainable, developing immunoassays is time-consuming - usually taking several months or longer, and have well-known deficiencies with signal to noise ratios. Alternatively, 2-D electrophoresis may be used for quantitation by recording the differences in staining patterns of proteins derived from two sample cell populations or tissues. Advances in 2-D gels has focused on higher resolution image analysis of staining intensity; however, 2D gel electrophoresis is labor intensive and can have shortcomings in data quality. A third technique utilizes High Performance Liquid Chromatography (HPLC). For HPLC, target proteins in crude mixtures are separated by various column types and quantified by their absorbencies, typically at 280nm. Drawbacks include low resolution power on crude mixtures and many glycoproteins, and lack of sufficient datapoints for accurate analysis. The present invention enables the direct, absolute quantitation of biopolymers, such as proteins or nucleotides, in crude solutions by incorporating unique tags using stable isotopes as internal standards for the target biopolymer. After fractionation with a protease or restriction enzyme, the resultant mixture is separated by chromatography and analyzed by mass spectroscopy. Peak area integration of the target biopolymer fragment along with its stably-labeled analog provides for direct measurement of the amount of the target biopolymer in the crude solution. more
Protein Quantification and Peptide Mapping Using 15N-Stable Isotope Labeling, Jack Cunniff, Sigrid Paech, Shauna Bowden, Alfred Gaertner, and Paul T. Jedrzejewski Proteome Digest, Fall Issue, 5-9, 2002
Tools for Protein Engineering of Alkaline Proteases, Sigrid Paech, Grant Ganshaw, Alfred Gaertner, Christian Paech Poster Abstract for Protein Society Meeting March29-April 2, 2003
Protein Determination of Subtilisin using a 15N-Labeled Internal Standard and Peptide Mapping with LC/MS, Christian Paech, Sigrid Paech, Grant Ganshaw 15th International Mass Spectrometry Conference, Barcelona, Spain, August 27-September 1, 2000 more
This technology is supported by 1 US patent and 1 European patent. more