BIOANALYTICAL MASS SPECTROMETRY PROTEOMIC APPROACHES TO MAP AND QUANTIFY PROTEIN PHOSPHORYLATION EVENTS AND PROTEIN INTERACTION DYNAMICS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1001973
• Project Start Date: Nov 6, 2013
• Project End Date: Sep 30, 2018
• Program Code: [(N/A)]- (N/A)

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Biochemistry

Non Technical Summary
Signal transduction mechanisms in plants and humans are important to understand since they are involved in a variety of biological processes such as growth and development. The vast amount of research conducted over many decades has revealed that these mechanisms are quite complex, thus requiring the development and application of new methods using cutting-edge technology in order to better understand the details of these pathways that cannot be measured using current methods or protocols. Our research is directed at developing novel approaches in the area of mass spectrometry analysis, so we can characterize the dynamic changes of proteins involved in signal transduction mechanisms in terms of their amount, extent of modification, interactions, and cellular localization and use this information to help increase the production of food crops and provide new avenues for the design of cancer therapeutics.

Animal Health Component

20%

Research Effort Categories

Basic

40%

Applied

20%

Developmental

40%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
203	1460	1000	25%
206	2499	1000	25%
311	7010	1000	25%
404	7410	1000	25%

Knowledge Area
206 - Basic Plant Biology; 311 - Animal Diseases; 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 404 - Instrumentation and Control Systems;

Subject Of Investigation
7010 - Biological Cell Systems; 1460 - Tomato; 7410 - General technology; 2499 - Plant research, general;

Field Of Science
1000 - Biochemistry and biophysics;

Keywords

signal transduction

plant growth

cancer

phosphorylation

protein interactions

mass spectrometry.

Goals / Objectives
Many diseases, including those in plants, can be traced to aberrant protein function. The underlying mechanisms are often related to uncontrolled changes to protein phosphorylation and/or protein interactions. Determining any of these mechanisms is very difficult, requires many scientists, and millions of research dollars. Despite tremendous efforts, there is still no cure for many plant, animal, and human diseases. Thus our goal is to develop new approaches and technologies in order to discover novel protein mechanisms and intervention targets that will provide new insights to understand and combat disease and thus improve agricultural production and human health.

Project Methods
The reversible phosphorylation of proteins and protein interactions play major roles in many vital cellular processes and are used to modulate protein activity and propagate signals within cellular pathways and networks. Because these processes are dynamic and cannot be predicted by an organism's genome, proteomic measurements using mass spectrometry are needed to characterize specific protein phosphorylation states and interactions. Since a high percentage of proteins in higher eukaryotic organisms are predicted to be phosphorylated with an even a larger percentage of possible interactions with other proteins, methods to identify and quantify phosphorylation sites and protein interactions should ideally be high-throughput, enabling proteome-wide changes be identified in a single experiment. Unfortunately, current methodologies have proven inadequate partially due to the difficulties associated with confidently identifying phosphorylated peptides and inter-peptide crosslinks induced by traditional chemical crosslinking reagents. Consequently, two major goals of our research is to develop new and effective techniques to identify (1) protein phosphorylation and (2) protein interactions and measure their relative abundances on a proteome-wide basis at the cellular level using mass spectrometry. (1) Protein Phosphorylation. Due to the low abundance of typical phosphopeptides and the dynamic range of present analytical technologies, the detection of phosphopeptides would be enhanced if more efficient peptide capture and fractionation techniques could be employed. Our efforts focus on developing methods to increase the dynamic range of mass spectrometry detection of low level phosphoproteins by incorporating multidimensional chromatography and affinity enrichment techniques into our approaches. We have successfully developed a variety of stable isotope-coded and label-free approaches to increase the efficiency and sensitivity for isolating and analyzing phosphopeptides and will continue to improve these approaches. We are applying these methods to map out and quantify protein phosphorylation sites involved in cancer, respiratory diseases, virus infection, and plant growth and development. (2) Protein Interactions. Due to the transient nature of protein interactions, characterizing these processes is difficult, especially for very large complexes, and is further impeded by the enormous number of dynamic interactions that are occurring at the cellular level during normal and disease states. To address these challenges, we are developing gas-phase cleavable crosslinking reagents that can react with protein complexes and be utilized to more effectively enhance inter-peptide crosslink identification using multistage tandem mass spectrometry. We have developed several strategies using various mass spectrometry approaches and instruments, and have more advanced designs we will be implementing. In addition we are developing software algorithms to utilize a chemical bar code we have engineered within these reagents that can be used to define protein-protein contacts. Future work will be directed to further developing our technique for high-throughput proteomics studies to better understand protein interaction networks in both plant and human systems.

Progress 11/06/13 to 09/30/18

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Over the course of the final year being reported, 3 undergraduate students, 2 graduate students, and 1 postdoc have been trained in my group in the area of biochemistry regarding kinase function, protein interaction dynamics, quantitative proteomics, and advanced liquid chromatography-tandem mass spectrometry analysis. About 3 other students and postdocs have worked withmy group via collaborations with other researchers at NC State University and neighboring institutions. Many of these folks have gone on to further their education in medicine or other life science areas or are research scientists employed at academic or industrial research institutions. How have the results been disseminated to communities of interest?Publications and presentations as previously described in the other sections of this report. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The project listed involved writing grant proposals, writing research manuscripts, and disseminating our research via presentations at various conferences. The grants that supported research published in this report or active with my role as being PI or CoPI for this HATCH project are listed: NSF EAR-1344198, NSF MCB-1052218, NSF DBI-1126244.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Shen, W., Bobay, B. G., Greeley, L. A., Reyes, M. I., Rajabu, C. A., Blackburn, R. K., Mary Beth Dallas, M. B., Goshe, M. B., Ascencio-Ib��ez, J. T., and Hanley-Bowdoin, L. (2018) Sucrose Nonfermenting 1-Related Protein Kinase 1 Phosphorylates a Geminivirus Rep Protein to Impair Viral Replication and Infection. Plant Physiol. 178(1):372-389. doi: 10.1104/pp.18.00268. Epub 2018 Jul 13. PMID: 30006378.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2018 Citation: Gracieux, M. C., Poynter, D., Struble, J., Baldwin, E. M., Blackburn, K., and Goshe, M. B. (2018) Developing a Quantitative LC/MS/MS Method for High-Throughput Characterization of the Structural Integrity of Protein Therapeutics During Biomanufacturing presented at The 66th ASMS Conference on Mass Spectrometry and Allied Topics held on June 3-7, 2018 at San Diego, CA.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Over the course of the year being reported, 1 undergraduate student, 4 graduate students, and 1 postdoc have been trained in my group in the area of biochemistry regarding kinase function, protein interaction dynamics, quantitative proteomics, and advanced liquid chromatography-tandem mass spectrometry analysis. About 5 other students and postdocs have worked with my group via collaborations with other researchers at NC State University and neighboring institutions. Many of these folks have gone on to further their education in medicine or other life science areas or are professors or staff scientists at academic or industrial research institutions. How have the results been disseminated to communities of interest?Publications and presentations as previously described in the other sections of this report. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The project listed involved writing many grant proposals, writing research manuscripts, and disseminating our research via presentations at various conferences. The grants that supported research published in this report or active with my role as being PI or CoPI are listed: NSF EAR-1344198, NSF MCB-1052218, NSF DBI-1126244. Other accomplishments achieved during this reporting period: 3 research publications in peer-reviewed journals and 1 poster presentation at a conference).

Publications

• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Bender, K.W., Blackburn, R.K,, Monaghan, J., Derbyshire, P., Menke, F.L., Zipfel, C., Goshe, M. B., Zielinski, R.E., and Huber, S.C. (2017) Autophosphorylation-based Calcium (Ca2+) Sensitivity Priming and Ca2+/Calmodulin Inhibition of Arabidopsis thaliana Ca2+-dependent Protein Kinase 28 (CPK28). J. Biol. Chem. 292(10), 3988-4002. doi: 10.1074/jbc.M116.763243. Epub 2017 Jan 30. PMID: 28154194.
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Blackburn, K., Bustamante-Marin, X., Yin, W., Goshe. M.B., and Ostrowski, L.E. (2017) Quantitative Proteomic Analysis of Human Airway Cilia Identifies Previously Uncharacterized Proteins of High Abundance. J. Proteome Res. 16(4), 1579-1592. doi: 10.1021/acs.jproteome.6b00972. Epub 2017 Mar 27. PMID: 28282151.
• Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Imkampe, J., Halter, T., Huang, S., Schulze, S., Mazzotta, S., Schmidt, N., Manstretta, R., Postel, S., Wierzba, M., Yang, Y., van Dongen, WMAM., Stahl, M., Zipfel, C., Goshe, M.B., Clouse, S., de Vries, S.C., Tax, F., Wang, X., and Kemmerling B. (2017) The Arabidopsis Leucine-Rich Repeat Receptor Kinase BIR3 Negatively Regulates BAK1 Receptor Complex Formation and Stabilizes BAK1. Plant Cell 29(9), 2285-2303. doi: 10.1105/tpc.17.00376. Epub 2017 Aug 25. PMID: 28842532.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Over the course of the year being reported, 1 undergraduate student, 5 graduate students, and 1 postdoc have been trained in my group in the area of biochemistry regarding kinase function, protein interaction dynamics, quantitative proteomics, and advanced liquid chromatography-tandem mass spectrometry analysis. About 10 other students and postdocs have worked with my group via collaborations with other researchers at NC State University and neighboring institutions. Many of these folks have gone on to further their education in medicine or other life science areas or are professors or staff scientists at academic or industrial research institutions. How have the results been disseminated to communities of interest?Publications and presentations as previously described in the other sections of this report. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The project listed involved writing many grant proposals, writing research manuscripts, and disseminating our research via presentations at various conferences. The grants that were active at the time of this report and those that were obtained during this project with my role as being PI or CoPI are listed: NSF EAR-1344198, NIH 1R01GM088987-01A2; NSF MCB-1052218. Other accomplishments achieved during this reporting period: 2 research publications in peer-reviewed journals (one published and one submitted), 1 invited research seminar and 2 poster presentations at a conference.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Saha, S., Paul, A., Herring, L., Dutta, A., Bhattacharya, A., Samaddar, S., Goshe, M.B., and DasGupta, M. (2016) Gatekeeper Tyrosine Phosphorylation of SYMRK Is Essential for Synchronizing the Epidermal and Cortical Responses in Root Nodule Symbiosis. Plant Physiol. 171(1), 71-81.
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Ho. T.C., Blackburn, K.B., Goshe, M.B., and Williamson, J.D. (2016) Defining a pathogen-induced interactome associated with unconventional protein secretion in Arabidopsis. J. Cell Bio. (submitted).

Progress 10/01/14 to 09/30/15

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Over the course of the year being reported, 2 undergraduate students, 5 graduate students, and 1 postdoc have been trained in my group in the area of biochemistry regarding kinase function, protein interaction dynamics, quantitative proteomics, and advanced liquid chromatography-tandem mass spectrometry analysis. About 10 other students and postdocs have worked with my group via collaborations with other researchers at NC State University and neighboring institutions. Many of these folks have gone on to further their education in medicine or other life sciences or are professors or staff scientists at academic or industrial research institutions. How have the results been disseminated to communities of interest?Publications and presentations as previously described in the other sections of this report. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The project listed involved writing many grant proposals, writing research manuscripts, and disseminating our research via presentations at various conferences. The grants that were active at the time of this report and those that were obtained during this project with my role as being PI or CoPI are listed: NSF EAR-1344198, NIH 1R01GM088987-01A2; NSF MCB-1052218; NSF MCB-1021363. Other accomplishments achieved during this reporting period: 3 research publications in peer-reviewed journals, 1 invited research seminar, 1 oral presentation at a conference, and 3 poster presentations at a conference.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Argo, A.S., Shi, C., Liu, F., and Goshe, M.B. (2015) Performing Protein Crosslinking using Gas-phase Cleavable Chemical Crosslinkers and Liquid Chromatography-Tandem Mass Spectrometry. Methods. 2015 Jun 16. pii: S1046-2023(15)00253-4. doi:10.1016/j.ymeth. 2015.06.011. [Epub ahead of print]. Mitra S.K., Chen, R., Dhandaydham, M., Wang, X., Blackburn, R.K., Kota, U., Goshe, M.B., Schwartz, D., Huber, S.C., and Clouse, S.D.(2015) An autophosphorylation site database for leucine-rich repeat receptor-like kinases in Arabidopsis thaliana. Plant J. 82(6), 1042-1060. doi: 10.1111/tpj.12863. Epub 2015 May 22. Herring, L.E., Grant, K., Blackburn, K., Haugh, J.M., and Goshe, M.B. (2015) Development of a Tandem Affinity Phosphoproteomic Method with Motif Selectivity and its Application in Analysis of Signal Transduction Networks. J. Chrom. B. 988, 166-174. doi: 10.1016/j.jchromb.2015.02.017. Epub 2015 Feb 19.

Progress 11/06/13 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Over the course of the year being reported, 2 undergraduate students, 3 graduate students, and 3 postdocs have been trained in my group in the area of biochemistry regarding kinase function and advanced liquid chromatography-tandem mass spectrometry analysis. About 10 other students and postdocs have worked with my group via collaborations with other researchers at NC State University and neighboring institutions. Many of these folks have gone on to further their education in medicine or other life sciences or are professors or staff scientists at academic or industrial research institutions. How have the results been disseminated to communities of interest? Publications and presentations as previously described in the other sections of this report. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The project listed involved writing many grant proposals, writing research manuscripts, and disseminating our research via presentations at various conferences. The grants that were active at the time of this report and those that were obtained during this project with my role as being PI or CoPI are listed: NSF EAR-1344198,NIH 1R01GM088987-01A2; NSF MCB-1052218; NSF MCB-1021363; NSF DBI-1126244. Other accomplishments acheived during this reporting period: 2 research publications in peer-reviewed journals, 1 Invited research article, 1 Invited researchseminar, 1 oral presentation at a conference, and 2 poster presentations at a conference.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Shen W., Dallas, M.B., Goshe, M.B., Hanley-Bowdoin, L. (2014) SnRK1 phosphorylation of AL2 delays Cabbage leaf curl virus infection in Arabidopsis. J. Virol. 88(18), 10598-10612. doi: 10.1128/JVI.00761-14. Epub 2014 Jul 2. PMID: 24990996 Schweitzer M.H., Schroeter, E.R., and Goshe, M.B. (2014) Protein molecular data from ancient (>1 million years old) fossil material: pitfalls, possibilities and grand challenges. Anal. Chem. 86(14), 6731-6740. doi: 10.1021/ac500803w. Epub 2014 Jul 1. PMID: 24983800 Ahmed, S., Grant, K.G., Edwards, L.E., Rahman, A., Cirit, M., Goshe, M.B., and Haugh, J.M. (2014) Data-driven modeling reconciles kinetics of ERK phosphorylation, localization, and activity states. Mol. Syst. Biol. 10, 718.