An antibody-free platform for multiplexed, sensitive quantification of protein biomarkers in complex biomatrices

An, Bo; Sikorsiki, Timothy; Kellie, John F.; Chen, Zhuo; Schneck, Nicole A.; Mehl, John T; Tang, Huaping; Qu, Jun; Shi, Tujin; Gao, Yuqian; Jacobs, Jon; Nandita, Eshani; Soest, Remco van; Jones, Elliott

doi:10.1016/j.chroma.2022.463261

Cited by 4 publications

(4 citation statements)

References 43 publications

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“…Combining biomarker assays into a multiplexed platform has clear advantages for the application of multi-biomarker approaches 5 . Such strategies have already been attempted in a number of areas including; Alzheimer’s 6, 7 , amyotrophic lateral sclerosis 8 , cardiovascular disease 9, 10, 11 , chronic obstructive pulmonary disease 12 , infection 13 and cancers 14, 15, 16, 17, 18, 19 . Many of the methodologies employed rely upon antibody recognition of an epitope and an associated optical readout for each analyte.…”

Section: Mainmentioning

confidence: 99%

Highly multiplexed detection of microRNAs, proteins and small molecules using barcoded molecular probes and nanopore sequencing

Koch

Gutierrez

Lucarelli

et al. 2022

Preprint

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Currently, most blood tests in a clinical setting only investigate a handful of markers. A low-cost, rapid, and highly multiplexed platform for the quantitative detection of blood biomarkers has the potential to advance clinical diagnostics beyond the single biomarker paradigm. In this study, we perform nanopore sequencing of barcoded molecular probes that have been engineered to recognise a panel of biological targets (miRNAs, proteins, and small molecules such as neurotransmitters), allowing for highly multiplexed simultaneous detection. Our workflow is rapid, from sample preparation to results in 1 hour. We also demonstrate that the strategy can be used to detect biomarkers directly from human serum without extraction or amplification. The established method is easily adaptable, as the number and type of targets detected can be greatly expanded depending on the application required.

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Section: Mainmentioning

confidence: 99%

Highly multiplexed detection of microRNAs, proteins and small molecules using barcoded molecular probes and nanopore sequencing

Koch

Gutierrez

Lucarelli

et al. 2022

Preprint

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“…The primary sensitivity limitation comes from significant coeluted interferences in complex samples. 5 Using immuno-enrichment before LC-MS analysis effectively minimizes interference and enhances sensitivity. 6,7 However, the development and optimization of suitable antibodies are often resourceintensive, and the quality of such assays is contingent upon the quality of the immuno-enrichment process.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Although liquid chromatography–mass spectrometry (LC-MS) is a powerful technique, insufficient sensitivity often poses a significant challenge. , For instance, pharmacokinetic studies of monoclonal antibodies (mAbs) and clinical analysis of biomarkers often require quantitative measurements of these proteins at low ng/mL levels, , substantially below the limit of quantification (LOQ) of conventional LC-MS. The primary sensitivity limitation comes from significant coeluted interferences in complex samples . Using immuno-enrichment before LC-MS analysis effectively minimizes interference and enhances sensitivity. , However, the development and optimization of suitable antibodies are often resource-intensive, and the quality of such assays is contingent upon the quality of the immuno-enrichment process.…”

Section: Introductionmentioning

confidence: 99%

A Trapping-Micro-LC-FAIMS/dCV-MS Strategy for Ultrasensitive and Robust Targeted Quantification of Protein Drugs and Biomarkers

Shen,

Pu,

Xue

et al. 2024

Anal. Chem.

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The sensitivity of LC-MS in quantifying target proteins in plasma/tissues is significantly hindered by coeluted matrix interferences. While antibody-based immuno-enrichment effectively reduces interferences, developing and optimizing antibodies are often time-consuming and costly. Here, by leveraging the orthogonal separation capability of Field Asymmetric Ion Mobility Spectrometry (FAIMS), we developed a FAIMS/differential-compensation-voltage (FAIMS/dCV) method for antibody-free, robust, and ultrasensitive quantification of target proteins directly from plasma/tissue digests. By comparing the intensity-CV profiles of the target vs coeluted endogenous interferences, the FAIMS/dCV approach identifies the optimal CV for quantification of each target protein, thus maximizing the signal-to-noise ratio (S/N). Compared to quantification without FAIMS, this technique dramatically reduces endogenous interferences, showing a median improvement of the S/N by 14.8-fold for the quantification of 17 representative protein drugs and biomarkers in plasma or tissues and a 5.2-fold median increase in S/N over conventional FAIMS approach, which uses the peak CV of each target. We also discovered that the established CV parameters remain consistent over months and are matrix-independent, affirming the robustness of the developed FAIMS/dCV method and the transferability of the method across matrices. The developed method was successfully demonstrated in three applications: the quantification of monoclonal antibodies with subng/mL LOQ in plasma, an investigation of the time courses of evolocumab and its target PCSK9 in a preclinical setting, and a clinical investigation of low abundance obesity-related biomarkers. This innovative and easy-to-use method has extensive potential in clinical and pharmaceutical research, particularly where sensitive and high-throughput quantification of protein drugs and biomarkers is required.

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“…To increase the specificity in detecting diseases, multiplexed biomarker assays have been developed. Such strategies have been attempted in several areas, including Alzheimer's disease 6,7 , amyotrophic lateral sclerosis 8 , cardiovascular disease [9][10][11] , chronic obstructive pulmonary disease 12 , infection 13 and cancers [14][15][16][17] . Many of the methodologies employed rely upon antibody recognition of an epitope and an associated optical readout for each analyte, for example, ELISA, which can be sensitive to pM levels of analyte.…”

mentioning

confidence: 99%

Nanopore sequencing of DNA-barcoded probes for highly multiplexed detection of microRNA, proteins and small biomarkers

Koch,

Reilly-O’Donnell,

Gutierrez

et al. 2023

Nat. Nanotechnol.

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There is an unmet need to develop low-cost, rapid and highly multiplexed diagnostic technology platforms for quantitatively detecting blood biomarkers to advance clinical diagnostics beyond the single biomarker model. Here we perform nanopore sequencing of DNA-barcoded molecular probes engineered to recognize a panel of analytes. This allows for highly multiplexed and simultaneous quantitative detection of at least 40 targets, such as microRNAs, proteins and neurotransmitters, on the basis of the translocation dynamics of each probe as it passes through a nanopore. Our workflow is built around a commercially available MinION sequencing device, offering a one-hour turnaround time from sample preparation to results. We also demonstrate that the strategy can directly detect cardiovascular disease-associated microRNA from human serum without extraction or amplification. Due to the modularity of barcoded probes, the number and type of targets detected can be significantly expanded.

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An antibody-free platform for multiplexed, sensitive quantification of protein biomarkers in complex biomatrices

Cited by 4 publications

References 43 publications

Highly multiplexed detection of microRNAs, proteins and small molecules using barcoded molecular probes and nanopore sequencing

Highly multiplexed detection of microRNAs, proteins and small molecules using barcoded molecular probes and nanopore sequencing

A Trapping-Micro-LC-FAIMS/dCV-MS Strategy for Ultrasensitive and Robust Targeted Quantification of Protein Drugs and Biomarkers

Nanopore sequencing of DNA-barcoded probes for highly multiplexed detection of microRNA, proteins and small biomarkers

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