Process for ultra-sensitive quantification of target analytes in complex biological systems

Abstract

Antibody-free processes are disclosed that provide accurate quantification of a wide variety of low-abundance target analytes in complex samples. The processes can employ high-pressure, high-resolution chromatographic separations for analyte enrichment. Intelligent selection of target fractions may be performed via on-line Selected Reaction Monitoring (SRM) or off-line rapid screening of internal standards. Quantification may be performed on individual or multiplexed fractions. Applications include analyses of, e.g., very low abundance proteins or candidate biomarkers in plasma, cell, or tissue samples without the need for affinity-specific reagents.

1 . A process for quantifying low-abundance target analytes in an entire sample, comprising the steps of:
 spiking the sample with a known quantity of at least one isotope-labeled target analyte having a pre-identified signal peak;   fractionating the sample with a high-resolution liquid chromatography device to produce a plurality of individual fractions;   identifying individual fractions having a signal peak identical to the pre-identified signal peak and selecting the identified fractions; and   quantifying the low-abundance target analytes in the sample from the ratio of the fractional signal peaks of the low-abundance target analytes to the signal peak of the at least one isotope-labeled target analyte.   
     
     
         2 . The process of  claim 1 , wherein identifying individual fractions further includes combining one or more of the selected individual fractions into individual bins. 
     
     
         3 . The process of  claim 2 , wherein the combining is performed with an N-well plate or a multi-dimensional array. 
     
     
         4 . The process of  claim 2 , wherein the combining includes sorting the selected individual fractions containing the low-abundance target analytes of a similar mass into individual bins as the fractions elute from the high-resolution chromatography device. 
     
     
         5 . The process of  claim 1 , wherein identifying individual fractions includes detecting an SRM signal of the isotope-labeled target analyte in the individual fractions in real time prior to selecting the individual fractions. 
     
     
         6 . The process of  claim 1 , wherein selecting individual fractions includes separating the selected individual fractions from the the high-resolution chromatography device with a high-resolution separation process selected from the group consisting of: reversed-phase liquid chromatography, hydrophilic interaction chromatography, electrostatic repulsion hydrophilic interaction chromatography, capillary electrophoresis, or other chromatography separations, and combinations thereof. 
     
     
         7 . The process of  claim 1 , wherein quantifying the low-abundance target analytes in the sample includes quantifying at a limit of detection, measured in blood plasma or serum, of at least about 100 pg/mL or better. 
     
     
         8 . The process of  claim 1 , wherein identifying individual fractions includes monitoring the plurality of individual fractions released from the high-resolution chromatography device with a process selected from: on-line SRM monitoring, on-line UV detection monitoring, off-line screening, or combinations thereof. 
     
     
         9 . The process of  claim 1 , wherein identifying individual fractions includes monitoring the plurality of individual fractions released from the high-resolution chromatography device with a mass-selective instrument or a UV detection device. 
     
     
         10 . The process of  claim 1 , wherein quantifying the low-abundance target analytes includes multiplexing one or more of the selected individual fractions prior to quantifying the low-abundance target analytes. 
     
     
         11 . The process of  claim 1 , wherein quantifying the low-abundance target analytes includes multiplexing one or more of the selected individual fractions having different elution times to provide sufficient peak separation during a subsequent SRM analysis. 
     
     
         12 . The process of  claim 1 , wherein quantifying the low-abundance target analytes includes multiplexing between four and twelve of the selected individual fractions having different elution times prior to quantifying the low-abundance target analytes. 
     
     
         13 . The process of  claim 1 , wherein quantifying the low-abundance target analytes includes multiplexing between ten and 100 of the selected individual fractions that are at least partially orthogonal prior to quantifying the low-abundance target analytes. 
     
     
         14 . The process of  claim 1 , wherein quantifying the low-abundance target analytes includes multiplexing between 100 and 500 of the selected individual fractions that are at least partially orthogonal prior to quantifying the low-abundance target analytes. 
     
     
         15 . The process of  claim 1 , wherein quantifying the low-abundance target analytes includes multiplexing greater than 500 of the selected individual fractions that are at least partially orthogonal prior to quantifying the low-abundance target analytes. 
     
     
         16 . The process of  claim 1 , wherein the selecting is performed in concert with a metric defined by an elution time of a low-abundance target analyte of interest. 
     
     
         17 . The process of  claim 1 , wherein selecting the individual fractions includes discarding individual fractions released from the high-resolution chromatography device that do not include the isotope-labeled target analyte. 
     
     
         18 . The process of  claim 1 , wherein one or more of the process steps are automated. 
     
     
         19 . The process of  claim 1 , wherein fractionating the sample includes digesting the sample to form peptides of a selected size. 
     
     
         20 . The process of  claim 1 , wherein fractionating the sample includes separating peptides in the sample prior to selecting individual fractions containing the isotope-labeled target analyte. 
     
     
         21 . The process of  claim 1 , further including depleting the sample of high-abundance compounds to concentrate low-abundance target analytes of interest therein. 
     
     
         22 . (canceled) 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . The process of  claim 1 , wherein the sample has a volume up to about 20 uL. 
     
     
         27 . The process of  claim 1 , wherein the sample has a volume greater than 20 uL. 
     
     
         28 . The process of  claim 2 , wherein combining one or more of the selected individual fractions includes combining fractions that are at least partially orthogonal. 
     
     
         29 . The process of  claim 10 , wherein multiplexing one or more of the selected individual fractions includes multiplexing fractions that are at least partially orthogonal.