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Fully automated and ultra-high-throughput platform for in-depth single-cell proteomics

Award Number: R01GM138931
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/05/2020
PERIOD OF PERFORMANCE END DATE: 08/31/2024

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Fully automated and ultra-high-throughput platform for in-depth single-cell proteomics - PROJECT SUMMARY/ABSTRACT The development of effective therapies to advance human health requires an in-depth molecular-level understanding of cellular processes and dynamic interactions between individual cells. Conventional population- based biochemical measurements provide limited utility, as contributions from individual cells are averaged and crucial information is lost. Direct measurements of the biochemical makeup of single cells are thus needed to characterize cellular transitions, regulatory mechanisms and the contribution of the microenvironment. Single- cell RNA sequencing is making a tremendous impact on biological research, but proteins mediate the bulk of cellular function and the correlation between RNA and protein abundance is often poor. In addition, RNA measurements are unable to inform on important posttranslational modifications that are readily measured by mass spectrometry. Current efforts to directly quantify targeted proteins in single cells such as CyTOF and immunohistochemistry share common shortcomings in that only a limited number of proteins can be analyzed. There is thus an urgent unmet need for technologies capable of directly generating unbiased and in-depth single- cell protein profiles to provide a more complete picture of cellular processes. We recently developed a proof-of- concept platform termed nanoPOTS (Nanodroplet Processing in One pot for Trace Samples) that effectively downscales sample processing volumes to the nanoliter scale to reduce sample losses. In combination with ultrasensitive liquid chromatography-mass spectrometry (LC-MS), nanoPOTS enables global proteome profiling of ~1000 protein groups in individual dissociated cells isolated by cell sorting or small regions of tissue sections isolated by microdissection. Building upon this proof-of-concept platform, our overall objective is to develop a fully automated prototype that yields far greater proteome coverage and throughput than is currently achievable, providing a capability for direct, in-depth and large-scale protein quantification that is analogous to single-cell RNA-seq. Studies in Aim 1 will focus on fully automating sample preparation and decreasing sample processing volumes at least tenfold to further reduce sample losses and increase proteome coverage. Aim 2 will automate sample transfer to the analytical platform and develop a fully automated and ultrasensitive LC-MS workflow with 100% MS utilization efficiency. Aim 3 will extend these advances in sensitivity, throughput and automation to the multiplexed analysis of single cells based on barcoding with unique isobaric labels. We will combine two distinct multiplexing approaches to enable simultaneous analysis of up to 32 samples in a single run. The completed platform will be fully automated, capable of highly quantitative label-free and multiplexed single cell proteome profiling to a depth of >3000 proteins per cell, and will achieve an unprecedented measurement throughput of >300 single cells per day for multiplexed analyses. This will constitute a unique and broadly enabling technology for the acquisition of basic biomedical knowledge.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = -$11,132 )
2025	2023	BRIGHAM YOUNG UNIVERSITY	A-153 ASB	PROVO	UT	84602	UTAH	USA	Biomedical Research and Research Training	000	4	4/8/2025	NON-COMPETING CONTINUATION	-$6,338
2025	2023	BRIGHAM YOUNG UNIVERSITY	A-153 ASB	PROVO	UT	84602	UTAH	USA	Biomedical Research and Research Training	001	4	4/8/2025	SUPPLEMENT FOR EXPANSION	-$4,794
														Subtotal = -$11,132

Issue Date FY: 2023 ( Subtotal = $580,525 )
2023	2023	BRIGHAM YOUNG UNIVERSITY	A-153 ASB	PROVO	UT	84602	UTAH	USA	Biomedical Research and Research Training	001	4	9/1/2023	SUPPLEMENT FOR EXPANSION	$250,000
2023	2023	BRIGHAM YOUNG UNIVERSITY	A-153 ASB	PROVO	UT	84602	UTAH	USA	Biomedical Research and Research Training	000	4	8/7/2023	NON-COMPETING CONTINUATION	$330,525
														Subtotal = $580,525

Issue Date FY: 2022 ( Subtotal = $330,525 )
2022	2022	BRIGHAM YOUNG UNIVERSITY	A41 ASB	PROVO	UT	84602	UTAH	USA	Biomedical Research and Research Training	000	3	8/15/2022	NON-COMPETING CONTINUATION	$330,525
														Subtotal = $330,525

Issue Date FY: 2021 ( Subtotal = $330,525 )
2021	2021	BRIGHAM YOUNG UNIVERSITY	A41 ASB	PROVO	UT	84602	UTAH	USA	Biomedical Research and Research Training	000	2	8/22/2021	NON-COMPETING CONTINUATION	$330,525
														Subtotal = $330,525

Issue Date FY: 2020 ( Subtotal = $330,525 )
2020	2020	BRIGHAM YOUNG UNIVERSITY	A41 ASB	PROVO	UT	84602	UTAH	USA	Biomedical Research and Research Training	000	1	9/5/2020	NEW	$330,525
														Subtotal = $330,525

Grand Total All Awards = $1,560,968

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Fully automated and ultra-high-throughput platform for in-depth single-cell proteomics

Award Number: R01GM138931

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 09/05/2020

PERIOD OF PERFORMANCE END DATE: 08/31/2024

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