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Protein Sequencing by Tandem Mass Spectrometry

Award Number: R01GM037537
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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Project Summary Proposed here is research to develop a collection of technologies that will make it possible to read the complete amino acid sequence of proteins (forwards and backwards) on a chromatographic time scale at the femtomole level and to also characterize multiple posttranslational modifications that exist on the same protein molecule and together regulate its biological activity. This research is driven by four major innovations from my lab. These include development of (a) electron transfer dissociation (ETD) for fragmentation of peptides and intact proteins, (b) combined ETD and IIPT (ion-ion proton transfer) chemistry to obtain n- and c-terminal sequence information that identifies proteins, (c) front end ETD (FETD) that facilitates a 10-50 fold increase in sensitivity for intact proteins, and (d) a micro column enzyme reactor that is fully active in 8 M urea and can be employed to generate 3-10 KDa protein fragments from large proteins. Going forward, we will implement and optimize ETD and IIPT in combination with parallel ion parking (PIP). This will include the introduction of new ETD reagents and new wave forms to enhance the efficiency of parallel ion parking and new IIPT reagents to extend the usable mass range to m/z 4,000. Also proposed is research to incorporate the powerful micro-column enzyme reactor into a rotation-driven microfluidic disc system where the protease bed length, solvent flow in hundreds of nL/min, and digestion times on the order of hundreds of milliseconds can all be controlled and measured with high accuracy. By using the rotation-driven microfluidic disc system, we expect to increase sample throughput, lower sample quantities, and increase reproducibility of individual sample analyses. All of this will be applied to fully characterize (a) bispecific antibodies, (b) proteins in outer membrane vesicles secreted by the antibiotic resistant, gram negative bacteria, Neisseria gonorrhoeae, (c) plant proteins that are involved in growth or growth inhibition and are targets for O-GlcNAcylation and O-fucosylation, respectively, (d) posttranslational modifications that exist on histone chaperones during the cell cycle, and (e) posttranslational modifications that regulate methylation of Arg residues on histones H3 and H4.


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: 2023 ( Subtotal = $757,113 )
2023	2023	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	36	6/28/2023	NON-COMPETING CONTINUATION	$757,113
														Subtotal = $757,113

Issue Date FY: 2022 ( Subtotal = $757,113 )
2022	2022	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	35	7/12/2022	NON-COMPETING CONTINUATION	$757,113
														Subtotal = $757,113

Issue Date FY: 2021 ( Subtotal = $757,113 )
2021	2021	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	001	34	7/28/2021	NON-COMPETING CONTINUATION	$0
2021	2021	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	34	6/23/2021	NON-COMPETING CONTINUATION	$757,113
														Subtotal = $757,113

Issue Date FY: 2020 ( Subtotal = $757,113 )
2020	2020	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	003	33	8/26/2020	COMPETING CONTINUATION	$118
2020	2020	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	001	33	5/26/2020	COMPETING CONTINUATION	$757,113
2020	2018	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	32	2/23/2020	NON-COMPETING CONTINUATION	$0
2020	2018	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	002	32	8/26/2020	NON-COMPETING CONTINUATION	-$118
														Subtotal = $757,113

Issue Date FY: 2018 ( Subtotal = $718,968 )
2018	2018	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	32	8/31/2018	NON-COMPETING CONTINUATION	$718,968
														Subtotal = $718,968

Issue Date FY: 2017 ( Subtotal = $718,968 )
2017	2017	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	31	8/15/2017	NON-COMPETING CONTINUATION	$718,968
														Subtotal = $718,968

Issue Date FY: 2016 ( Subtotal = $718,968 )
2016	2016	UNIVERSITY OF VIRGINIA	P.O. BOX 400195	CHARLOTTESVILLE	VA	22904	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	30	8/22/2016	NON-COMPETING CONTINUATION	$718,968
														Subtotal = $718,968

Issue Date FY: 2015 ( Subtotal = $718,968 )
2015	2015	UNIVERSITY OF VIRGINIA	P.O. BOX 400195	CHARLOTTESVILLE	VA	22904	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	001	29	9/1/2015	COMPETING CONTINUATION	$718,968
2015	2014	UNIVERSITY OF VIRGINIA	PO BOX 400195	CHARLOTTESVILLE	VA	22904	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	28	8/27/2015	NON-COMPETING CONTINUATION	$0
														Subtotal = $718,968

Issue Date FY: 2014 ( Subtotal = $666,759 )
2014	2014	UNIVERSITY OF VIRGINIA	P.O. BOX 400195	CHARLOTTESVILLE	VA	22904	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	28	8/25/2014	NON-COMPETING CONTINUATION	$666,759
														Subtotal = $666,759

Issue Date FY: 2013 ( Subtotal = $657,679 )
2013	2013	UNIVERSITY OF VIRGINIA	P.O. BOX 400195	CHARLOTTESVILLE	VA	22904	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	27	8/8/2013	NON-COMPETING CONTINUATION	$657,679
														Subtotal = $657,679

Issue Date FY: 2012 ( Subtotal = $692,862 )
2012	2012	UNIVERSITY OF VIRGINIA	P.O. BOX 400195	CHARLOTTESVILLE	VA	22904	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	26	8/31/2012	NON-COMPETING CONTINUATION	$692,862
														Subtotal = $692,862

Issue Date FY: 2011 ( Subtotal = $701,335 )
2011	2011	UNIVERSITY OF VIRGINIA	P.O. BOX 400195	CHARLOTTESVILLE	VA	22904	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	25	8/18/2011	COMPETING CONTINUATION	$701,335
														Subtotal = $701,335

Issue Date FY: 2010 ( Subtotal = $953,009 )
2010	2010	UNIVERSITY OF VIRGINIA	P.O. BOX 400195	CHARLOTTESVILLE	VA	22904	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	24	8/27/2010	NON-COMPETING CONTINUATION	$953,009
														Subtotal = $953,009

Issue Date FY: 2009 ( Subtotal = $939,897 )
2009	2009	UNIVERSITY OF VIRGINIA	P.O. BOX 400195	CHARLOTTESVILLE	VA	22904	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	23	9/1/2009	NON-COMPETING CONTINUATION	$939,897
														Subtotal = $939,897

Issue Date FY: 2008 ( Subtotal = $916,799 )
2008	2008	UNIVERSITY OF VIRGINIA	PO BOX 400195	CHARLOTTESVILLE	VA	22904	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	22	8/22/2008	NON-COMPETING CONTINUATION	$916,799
														Subtotal = $916,799

Issue Date FY: 2007 ( Subtotal = $1,102,771 )
2007	2007	UNIVERSITY OF VIRGINIA	PO BOX 400195	CHARLOTTESVILLE	VA	22904	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	21	9/4/2007	COMPETING CONTINUATION	$1,102,771
														Subtotal = $1,102,771

Issue Date FY: 2006 ( Subtotal = $797,250 )
2006	2006	UNIVERSITY OF VIRGINIA	PO BOX 400195	CHARLOTTESVILLE	VA	22904	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	20	8/25/2006	NON-COMPETING CONTINUATION	$797,250
														Subtotal = $797,250

Issue Date FY: 2005 ( Subtotal = $794,624 )
2005	2005	UNIVERSITY OF VIRGINIA	PO BOX 9003	CHARLOTTESVILLE	VA	22906	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	19	9/1/2005	NON-COMPETING CONTINUATION	$794,624
														Subtotal = $794,624

Issue Date FY: 2004 ( Subtotal = $771,178 )
2004	2004	UNIVERSITY OF VIRGINIA	PO BOX 9003	CHARLOTTESVILLE	VA	22906	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	18	8/27/2004	NON-COMPETING CONTINUATION	$771,178
														Subtotal = $771,178

Issue Date FY: 2003 ( Subtotal = $1,634,479 )
2003	2003	UNIVERSITY OF VIRGINIA	PO BOX 9003	CHARLOTTESVILLE	VA	22906	CHARLOTTESVILLE CITY	USA	Cell Biology and Biophysics Research	000	17	9/8/2003	COMPETING CONTINUATION	$1,634,479
														Subtotal = $1,634,479

Issue Date FY: 2002 ( Subtotal = $436,119 )
2002	2002	UNIVERSITY OF VIRGINIA	PO BOX 9003	CHARLOTTESVILLE	VA	22906	CHARLOTTESVILLE CITY	USA	Cell Biology and Biophysics Research	000	16	8/28/2002	NON-COMPETING CONTINUATION	$436,119
														Subtotal = $436,119

Issue Date FY: 2001 ( Subtotal = $424,035 )
2001	2001	UNIVERSITY OF VIRGINIA	PO BOX 9003	CHARLOTTESVILLE	VA	22906	CHARLOTTESVILLE CITY	USA	Cell Biology and Biophysics Research	000	15	4/23/2001	NON-COMPETING CONTINUATION	$424,035
														Subtotal = $424,035

Issue Date FY: 2000 ( Subtotal = $412,308 ) (Continued on the next page)
Grand Total All Awards = $18,793,535

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Protein Sequencing by Tandem Mass Spectrometry

Award Number: R01GM037537

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

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