The use of transgenic host species is a powerful tool for determining the influence of specific genes on phenotypic traits and for expressing advantageous traits in desired host species. A critical step for this methodology is determining the expression and concentration of transgenic polypeptides in the host species. As transgenic technology is widely used in many industries, methods for accomplishing this step that are simple, accurate and high-throughput must be established.
Conventional screening tools, such as RT-PCR and northern blot analysis (mRNA), are limited by a statistically poor correlation between mRNA abundance and corresponding polypeptide levels. Immunoassays, such as immunoblots, radioimmunoassays and enzyme-linked immunosorbent assays (ELISAs), have several major disadvantages. They rely heavily on high quality antibodies that are not always obtainable for weakly or non-immunoreactive antigens. Additionally, membrane and membrane-associated polypeptides present great challenges to immunoassays, especially ELISAs. Furthermore, development of an immunoassay is both costly and time-consuming, usually requiring several months to years for development and validation. The utilization of epitope tags in immunoassays also has the potential disadvantage of having the secondary structure of the polypeptide mask the epitope tag, preventing accurate quantification.
Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has been an indispensible analytical tool to quantify small molecules including pharmaceuticals (Xu et al. (2007) J Pharm Biomed Anal 44:342-355) and pesticide residues (Wong et al. (2010) J Agric Food Chem 58:5897-5903). LC-MS/MS has also been applied to polypeptide quantification in polypeptide biomarker research (Carr and Anderson (2008) Clin Chem 54:1749-1752; Keshishian et al. (2007) Mol Cell Proteomics 6:2212-2229; Seegmiller et al. (2009) Clin Chem 55:1100-1107). Some literature focuses on relative quantification using isotope labeling techniques (Gygi et al. (1999) Nat Biotechnol 17:994-999; Ong et al. (2002) Mol Cell Proteomics 1:376-386; Ross et al. (2004) Mol Cell Proteomics 3:1154-1169), and in other cases absolute polypeptide quantification has been accomplished either by isotope dilution or using a calibration curve generated with peptide standards (Gerber et al. (2003) Proc Natl Acad Sci USA 100:6940-6945; Wienkoop et al. (2006) J Exp Bot 57:1529-1535; Kuhn et al. (2004) Proteomics 4:1175-1186; Lin et al. (2006) Anal Chem 78:5762-5767). In all cases polypeptide quantification using LC-MS/MS has been accomplished by detecting signature peptides, specific amino acid sequences naturally occurring within the polypeptide that are readily detectable by LC-MS/MS and can be used as surrogates to determine polypeptide concentration. However, this technique is limited, as new signature peptides must be identified and validated for each new polypeptide of interest. In most cases a protein of interest is needed for the method development and validation. Therefore method development cannot be done in high-throughput way for large numbers of new proteins.
In consideration of the large number of transgenes being tested in various industries and applications, it remains critically important to develop new high-throughput polypeptide detection and quantification methods that are sensitive, specific and applicable to a wide range of transgenic polypeptides of interest. Furthermore, such methods should have the advantages of shorter development time and reduced cost.