The proposed research is focused on the critical importance of protein conformational dynamics and interactions for the transport and delivery of both cognate ligands and therapeutic agents to cells. Molecular therapeutics places a great emphasis on drug candidates that must precisely deliver the active ingredients to their target sites. One potential method to achieve this aim is to harness the existing cellular machinery, particularly those systems that transport molecules into the cell. This research focuses on the dynamic processes in the transferrin/transferrin receptor system. A powerful and sensitive technique employed in this laboratory for the study of protein higher order structure and dynamics is mass spectrometry. We will develop new experimental strategies combining hydrogen/deuterium exchange in solution (HDX) with electrospray ionization mass spectrometry (ESI MS) detection that use an array of gas phase ion fragmentation techniques to characterize protein conformation and dynamics at an unprecedented level of detail. The new HDX MS methods will enable characterization of protein higher order structure at resolution close to the single amino-acid residue level, in order to pinpoint dynamic regions of proteins critical for function. Application of several recently developed fragmentation techniques will advance current capabilities to include proteins that have otherwise eluded such characterization (such as those rich in disulfide bonds), and also enable selection and characterization of specific conformers. We will use these methods to address pressing biomedical questions related to delivery of therapeutic agents to cells. Specifically we will apply our methodologies to decipher the detailed molecular mechanism of protein-protein interaction in the transferrin-transferrin receptor system and its modulation by metals and conjugated therapeutic agents. The changes in protein dynamics of transferrin in the metal-bound and free form, and comparison of its behavior in the extracellular environment versus the endosome are critical to understanding this transport process. This in vitro model will be verified by correlating the conformational and receptor-binding properties of various transferrin-cytotoxin conjugates with their ability to traverse the blood-brain barrier in vivo and accumulate in malignant cells.

Public Health Relevance

As drug discovery efforts are focusing more on targeting specific cellular processes, a thorough understanding of the machinery involved in transporting biological molecules throughout the body and internalizing within the targeted cell has become critical. Our research seeks to shed light on these processes by developing analytical techniques to investigate the importance of the interactions and dynamic motions of proteins involved in this transport machinery. This knowledge will catalyze the translational research carried out by our collaborators, whose aim is to design new and enhance existing therapeutic strategies using a ubiquitous plasma protein transferrin as a Trojan horse for precise and highly selective cytotoxin delivery to cancer cells. Since transferrin receptor has been identified as a highly promising delivery system for a variety of other therapeutics, particularly those with targets localized in the central nervous system, the proposed work will greatly benefit efforts to design effective therapeutic strategies to treat a variety of other pathological conditions, ranging from Parkinson?s and Alzheimer?s diseases to multiple sclerosis, rare genetic disorders and pain management.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061666-13
Application #
8513342
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Edmonds, Charles G
Project Start
2000-07-01
Project End
2014-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
13
Fiscal Year
2013
Total Cost
$341,536
Indirect Cost
$120,103
Name
University of Massachusetts Amherst
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
153926712
City
Amherst
State
MA
Country
United States
Zip Code
01003
Wang, Guanbo; Kaltashov, Igor A (2014) Approach to characterization of the higher order structure of disulfide-containing proteins using hydrogen/deuterium exchange and top-down mass spectrometry. Anal Chem 86:7293-8
Bobst, Cedric E; Kaltashov, Igor A (2014) Enhancing the quality of H/D exchange measurements with mass spectrometry detection in disulfide-rich proteins using electron capture dissociation. Anal Chem 86:5225-31
Wang, Shunhai; Kaltashov, Igor A (2013) An 18O-labeling assisted LC/MS method for assignment of aspartyl/isoaspartyl products from Asn deamidation and Asp isomerization in proteins. Anal Chem 85:6446-52
Kaltashov, Igor A; Bobst, Cedric E; Nguyen, Son N et al. (2013) Emerging mass spectrometry-based approaches to probe protein-receptor interactions: focus on overcoming physiological barriers. Adv Drug Deliv Rev 65:1020-30
Nguyen, Son N; Bobst, Cedric E; Kaltashov, Igor A (2013) Mass spectrometry-guided optimization and characterization of a biologically active transferrin-lysozyme model drug conjugate. Mol Pharm 10:1998-2007
Kaltashov, Igor A; Bobst, Cedric E; Abzalimov, Rinat R (2013) Mass spectrometry-based methods to study protein architecture and dynamics. Protein Sci 22:530-44
Abzalimov, Rinat R; Bobst, Cedric E; Kaltashov, Igor A (2013) A new approach to measuring protein backbone protection with high spatial resolution using H/D exchange and electron capture dissociation. Anal Chem 85:9173-80
Luck, Ashley N; Bobst, Cedric E; Kaltashov, Igor A et al. (2013) Human serum transferrin: is there a link among autism, high oxalate levels, and iron deficiency anemia? Biochemistry 52:8333-41
Sjoelund, Virginie; Kaltashov, Igor A (2012) Modification of the zonal elution method for detection of transient protein-protein interactions involving ligand exchange. Anal Chem 84:4608-12
Kaltashov, Igor A; Bobst, Cedric E; Abzalimov, Rinat R et al. (2012) Advances and challenges in analytical characterization of biotechnology products: mass spectrometry-based approaches to study properties and behavior of protein therapeutics. Biotechnol Adv 30:210-22

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