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The Expedit-Isotopomeric CrossLinking Mass Spectrometry (Expedit-ICLMS) technology for mapping global and dynamic protein-protein interaction networks

Award Number: R01GM136974
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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The Expedit-Isotopomeric CrossLinking Mass Spectrometry (Expedit-ICLMS) technology for mapping global and dynamic protein-protein interaction networks - SUMMARY This project seeks to develop new technology to enable global mapping of protein-protein interactions (PPIs) in a condition-specific, timely and affordable manner, by individual researchers interested in specific biological systems. Successful development of this technology is expected to have a transformative effect on all fields of biomedical research, directly addressing the purpose of the Focused Technology Research and Development R01 FOA (PAR-19-253). Global PPI networks are not only the most important resources for understanding the molecular mechanisms underlying normal and aberrant biological processes, but also the bases for understanding genetic interaction networks, constructing gene regulatory networks, and quantitative modeling of biological processes. Current technologies for mapping global PPI networks are labor intensive, time consuming, costly, plagued by false positives/negatives, and do not provide a way for individual researchers to efficiently map global PPI networks for particular biological systems. The proposed technology development is targeted to solve the two most important challenges facing crosslinking-mass spectrometry (CLMS)-based global PPI mapping studies: 1) the complexity of peptide mixtures derived from crosslinking samples with a large number of proteins and a large dynamic range of abundances, and 2) efficient and confident identification of crosslinked peptides by whole proteome database searches. We seek to overcome these challenges by developing a novel crosslinked peptide enrichment strategy, called Expedit, and combining it with the powerful capabilities of ICL crosslinkers, a new class of MS-cleavable, isotopomeric, bi- functional crosslinkers for crosslinked peptide identification. Unique features of ICLs permit 1) efficient determination of individual peptide masses in each crosslink from MS2 spectra, and 2) identification of crosslinked peptides by whole proteome database searching using a single MS2 spectrum per crosslinked peptide. The combination of Expedit with ICLMS is expected to address the major limitations of current CLMS approaches to enable routine large scale PPI studies for the first time. In the Aims, we will first synthesize novel Expedit reagents and evaluate their effectiveness for crosslinked peptide enrichment using increasingly complex mixtures. Once optimized, we will integrate Expedit with ICLMS and evaluate the effectiveness of the technology for building global PPI networks in yeast. We will evaluate the method in terms of the quantity and reproducibility of identified crosslinks/PPIs, the abundances of the identified proteins, their localization, affinities and complex membership (if available). We will compare our PPI networks to previously described yeast PPI networks. The effectiveness of the technology for crosslinked peptide identification will be evaluated by comparing it to state-of-the-art CLMS-based approaches. If successful, this project would provide a general and robust method for studying global PPIs and their dynamics that can be applied to any organism for which a sequenced genome is available.


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 = -$10 )
2025	2023	INSTITUTE FOR SYSTEMS BIOLOGY	401 TERRY AVE N	SEATTLE	WA	98109	KING	USA	Biomedical Research and Research Training	000	4	5/12/2025	NON-COMPETING CONTINUATION	-$10
														Subtotal = -$10

Issue Date FY: 2023 ( Subtotal = $519,905 )
2023	2023	INSTITUTE FOR SYSTEMS BIOLOGY	401 TERRY AVE N	SEATTLE	WA	98109	KING	USA	Biomedical Research and Research Training	001	4	4/4/2023	NON-COMPETING CONTINUATION	$506,436
2023	2022	INSTITUTE FOR SYSTEMS BIOLOGY	401 TERRY AVE N	SEATTLE	WA	98109	KING	USA	Biomedical Research and Research Training	000	3	4/3/2023	NON-COMPETING CONTINUATION	$13,469
														Subtotal = $519,905

Issue Date FY: 2022 ( Subtotal = $492,967 )
2022	2022	INSTITUTE FOR SYSTEMS BIOLOGY	401 TERRY AVE N	SEATTLE	WA	98109	KING	USA	Biomedical Research and Research Training	000	3	3/23/2022	NON-COMPETING CONTINUATION	$443,668
2022	2022	INSTITUTE FOR SYSTEMS BIOLOGY	401 TERRY AVE N	SEATTLE	WA	98109	KING	USA	Biomedical Research and Research Training	001	3	6/29/2022	NON-COMPETING CONTINUATION	$49,299
														Subtotal = $492,967

Issue Date FY: 2021 ( Subtotal = $492,967 )
2021	2021	INSTITUTE FOR SYSTEMS BIOLOGY	401 TERRY AVE N	SEATTLE	WA	98109	KING	USA	Biomedical Research and Research Training	000	2	3/24/2021	NON-COMPETING CONTINUATION	$492,967
														Subtotal = $492,967

Issue Date FY: 2020 ( Subtotal = $542,967 )
2020	2020	INSTITUTE FOR SYSTEMS BIOLOGY	401 TERRY AVE N	SEATTLE	WA	98109	KING	USA	Biomedical Research and Research Training	000	1	3/18/2020	NEW	$542,967
														Subtotal = $542,967

Grand Total All Awards = $2,048,796

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The Expedit-Isotopomeric CrossLinking Mass Spectrometry (Expedit-ICLMS) technology for mapping global and dynamic protein-protein interaction networks

Award Number: R01GM136974

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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