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Endogenous Analyte Quantification by Liquid Chromatography Coupled with Tandem Mass Spectrometry (LC-MS/MS)

IP.com Disclosure Number: IPCOM000241626D
Publication Date: 2015-May-18
Document File: 5 page(s) / 42K

Publishing Venue

The IP.com Prior Art Database

Abstract

Disclosed herein are methods and compositions for the quantification of endogenous analytes in biological samples by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Quantification of endogenous analytes (proteins, peptides, metabolites, small molecules, fatty acids, carbohydrates, etc.) by LC-MS/MS may be challenging because it is often difficult or impossible to obtain target analyte-free matrix which is needed for standard curve preparation. Options to overcome this challenge include preparing the standard curve in a surrogate matrix, using a surrogate analyte as a substitute for the target analyte in the authentic sample matrix, or performing standard addition in the authentic sample matrix. There are drawbacks to each of these options, for example, there may not be a surrogate matrix or surrogate analyte available and standard addition limits throughput and requires larger sample amounts. We propose a solution to the challenge of endogenous analyte quantification. The process of standard addition may be used to accurately determine the concentration of an endogenous analyte in any biological sample. Once the endogenous analyte concentration is determined in one sample, that sample may be used as the matrix for preparation of a standard curve to quantify countless unknown samples. The concentration of the standard curve is adjusted based on the pre-determined concentration in the original sample. In this disclosure, an endogenous protein is quantified in plant extracts as an example of the methodology, but this protocol can be applied to any endogenous analyte in any biological sample. This solution is ideal because there is no need to obtain a surrogate matrix or analyte and it is quick.

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“Endogenous Analyte Quantification by Liquid Chromatography Coupled with Tandem Mass Spectrometry (LC-MS/MS)”

Summary: Disclosed herein are methods and compositions for the quantification of endogenous analytes in biological samples by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Quantification of endogenous analytes (proteins, peptides, metabolites, small molecules, fatty acids, carbohydrates, etc.) by LC-MS/MS may be challenging because it is often difficult or impossible to obtain target analyte-free matrix which is needed for standard curve preparation. Options to overcome this challenge include preparing the standard curve in a surrogate matrix, using a surrogate analyte as a substitute for the target analyte in the authentic sample matrix, or performing standard addition in the authentic sample matrix. There are drawbacks to each of these options, for example, there may not be a surrogate matrix or surrogate analyte available and standard addition limits throughput and requires larger sample amounts.  We propose a solution to the challenge of endogenous analyte quantification. The process of standard addition may be used to accurately determine the concentration of an endogenous analyte in any biological sample. Once the endogenous analyte concentration is determined in one sample, that sample may be used as the matrix for preparation of a standard curve to quantify countless unknown samples. The concentration of the standard curve is adjusted based on the pre-determined concentration in the original sample. In this disclosure, an endogenous protein is quantified in plant extracts as an example of the methodology, but this protocol can be applied to any endogenous analyte in any biological sample. This solution is ideal because there is no need to obtain a surrogate matrix or analyte and it is quick.

Rationale: Protein detection/quantification in genetically engineered crops typically is done by immunoassay such as enzyme-linked immunosorbent assay (ELISA) because of its high sensitivity, specificity, and throughput. However, immunoassays require high quality antibodies that are not always readily available and may take many months to produce. Additionally, proteins that are difficult to extract or solubilize, such as membrane-bound proteins, may require detergents to aid in extraction or to keep the protein in solution and these detergents often interfere with antibody recognition of the target protein.

LC-MS/MS is widely used to quantify small molecules such as pharmaceuticals and pesticides for high throughput sample analysis. LC-MS/MS has become an important tool for the quantification of proteins because there is no need for antibodies and detergents needed for membrane proteins do not interfere with detection/quantification (Dahmani, 2012). Proteins are digested by trypsin and the resulting peptides are detected/quantified by LC-MS/MS as the protein surrogate. One drawback is that LC-MS/MS typically...