PUBLICATIONS: 2009

Investigating Solution-Phase Protein Structure and Dynamics by Hydrogen Exchange Mass Spectrometry.
Morgan CR, Engen JR.
Curr Protoc Protein Sci. 2009. Unit 17.6. In press.

ABSTRACT
Until recently, mass spectrometry (MS) was not often associated with the analysis of protein conformation and dynamics but rather as a method to measure molecular weight and quantify molecules. However, by taking advantage of labeling methods such as hydrogen exchange (HX), many details about protein conformation, dynamics and interactions can be revealed by mass spectrometry. The use of HX MS for investigating protein structure was introduced previously in this series (Wang and Smith, 2002). In the current work we provide an update that covers hydrogen exchange theory as it applies to HX MS protocols, explain in detail the practice of HX MS including data analysis and interpretation, and highlight recent advancements in technology which greatly increase the depth of information gained from the technique.

Pubmed: too new

Conformational Dynamics of Proteins by Mass Spectrometry: Hydrogen / Deuterium Exchange and Covalent Labeling Approaches.
Houde D, Engen JR.
Chimica Oggi / Chemistry Today. 2009. Mar/Apr;27(2):12-15. 

ABSTRACT
As the bioanalytical analysis of proteins becomes evermore important, attention must be paid to the three-dimensional structure of these analytes. Protein function, viability and efficacy in the case of protein-based therapeutics are intricately connected to protein structure. Here we highlight mass spectrometry based approaches for studying protein conformation. By labeling proteins with hydrogen/deuterium exchange or covalent labeling methodology, many challenges associated with biophysical characterisation of protein structure and dynamics can be addressed. There are multiple advantages to using MS, not the least of which is the small amounts of material that are required.

Aspartic Proteinases in Antarctic Fish.
De Luca V, Maria G, De Mauro G, Catara G, Carginale V, Ruggiero G, Capasso A, Parisi E, Brier S, Engen JR, Capasso C.
Marine Genomics. 2009. 2:1-10.

ABSTRACT
The present review surveys several recent studies of the aspartic proteinases from Antarctic Notothenioidei, a dominating fish group that has developed a number of adjustments at the molecular level to maintain metabolic function at low temperatures. Given the unique peculiarities of the Antarctic environment, studying the features of Antarctic aspartic proteinases could provide new insights into the role of these proteins in fish physiology. We describe here: (1) the biochemical properties of a cathepsin D purified from the liver of the hemoglobinless icefish Chionodraco hamatus; (2) the biochemical characterization of Trematomus bernacchii pepsins variants A1 and A2 obtained by heterologous expression in bacteria; and (3) the identification of two closely related, novel aspartic proteinases from the liver of the two Antarctic fish species mentioned above. Overall, the results show that Notothenioidei aspartic proteinases display a number of characteristics that are remarkably different from those of mammalian aspartic proteinases, including high turnover number or high catalytic efficiency. We have named the newly identified aspartic proteinases “Nothepsins” and classified them relative to aspartic proteinases from other species.
 
Pubmed: too new

Isotope Exchange and Covalent Modification Strategies for Studying Protein Structure
and Function.
Chen S, Engen JR.
Curr. Anal. Chem. Special Issue "The exciting ionic life of a protein in the hands of a mass spectrometrist".
2009. Apr 1;5(2):205-212.

ABSTRACT
Mass spectrometry can be used to obtain information about all levels of protein structure. To gain access to the conformation information found in the tertiary and quaternary structure, various labeling methods have been developed. These methods convert structural information into mass differences that can be observed with high-resolution protein/peptide mass spectrometry. Three methods are reviewed here: hydrogen/deuterium exchange, covalent modification (also called chemical modification) and hydroxyl radical footprinting. The general implementation of these methods is described and comparisons are made between the methods.

Characterization of IgG1 Conformation and Conformational Dynamics by
Hydrogen / Deuterium Exchange Mass Spectrometry.
Houde D, Arndt J, Domeier W, Berkowitz SA, Engen JR.
Anal. Chem. 2009. Apr 1;81(7):2644-51.  Errata: Anal. Chem., 2009, 81 (14), p 5966.

ABSTRACT
Protein function is dictated by protein conformation. For the protein biopharmaceutical industry, therefore, it is important to have analytical tools that can detect changes in protein conformation rapidly, accurately and with high sensitivity. In this paper we show that hydrogen/deuterium exchange mass spectrometry (H/DX-MS) can play an important role in fulfilling this need within the industry. H/DX-MS was used to assess both global and local conformational behavior of a recombinant monoclonal IgG1 antibody, a major class of biopharmaceuticals. Analysis of exchange into the intact, glycosylated IgG1 (and the Fab and Fc regions thereof) showed that the molecule was folded, highly stable and highly amenable to analysis by this method using less than a nanomole of material. With improved chromatographic methods, peptide identification algorithms and data-processing steps, the analysis of deuterium levels in peptic peptides produced after labeling was accomplished in 1-2 days. Based on peptic peptide data, exchange was localized to specific regions of the antibody. Changes to IgG1 conformation as a result of deglycosylation were determined by comparing exchange into the glycosylated and deglycosylated forms of the antibody. Two regions of the IgG1 (residues 236-253 and 292-308) were found to have altered exchange properties upon deglycosylation. These results are consistent with previous findings concerning the role of glycosylation in the interaction of IgG1 with Fc receptors. Moreover, the data clearly illustrate how H/DX-MS can provide important characterization information on the higher order structure of antibodies and conformational changes that these molecules may experience upon modification.
 
PDB code: 3FZU

Pubmed: 19265386

Conformational Disturbance in Abl Kinase upon Mutation and Deregulation.
Iacob RE, Dumitrescu TP, Zhang J, Gray NS, Smithgall TE, Engen JR.
Proc Natl Acad Sci U.S.A. 2009. Feb 3;106(5):1386-1391.
 
ABSTRACT
Protein dynamics are inextricably linked to protein function but there are few techniques that allow protein dynamics to be conveniently interrogated. For example, mutations and translocations give rise to aberrant proteins such as Bcr-Abl where changes in protein conformation and dynamics are believed to result in deregulated kinase activity that provides the oncogenic signal in Chronic Myelogeous Leukemia. Although crystal structures of the downregulated c-Abl kinase core have been reported, the conformational impact of mutations that render Abl resistant to small-molecule kinase inhibitors are largely unknown as is the allosteric interplay of the various regulatory elements of the protein. Hydrogen exchange mass spectrometry (HX MS) was employed to compare the conformations of wild-type Abl with a non-myristoylated form and with three clinically relevant imatinib resistance mutants (T315I, Y253H and E255V). A HX-resistant core localized to the interface between the SH2 and kinase domains, a region known to be important for maintaining the downregulated state. Conformational differences upon demyristoylation were consistent with the SH2 domain moving to the top of the small lobe of the kinase domain as a function of activation. There were conformational changes in the T315I mutant but, surprisingly, no major changes in conformation were detected in either the Y253H or the E255V mutants. Taken together, these results provide evidence that allosteric interactions and conformational changes play a major role in Abl kinase regulation in solution. Similar analyses could be performed on any protein to provide mechanistic details about conformational changes and protein function.

Pubmed: 19164531

Protein Analysis with Hydrogen-Deuterium Exchange Mass Spectrometry.
Mitchell JL, Engen JR.
Chapter 4 of “Protein Mass Spectrometry", Comprehensive Analytical Chemistry Series, 2009, Vol. 52, 83-102.
ISBN: 978-0-444-53055-4, Elsevier, Julian Whitelegge, Editor. 

1. Introduction    1.1. Review of Protein Structure    1.2. Obtaining information about conformation and dynamics 2. Experimental protocol    2.1. Deuterium introduction    2.2. Global versus local information    2.3. HPLC and MS    2.4. Data interpretation 3. Illustrative examples    3.1. Protein conformation and the effects of mutation    3.2. Binding interactions    3.3. Investigating proteins lacking structural data 4. Conclusions

ABSTRACT
There are some properties of proteins that remain hidden to a mass spectrometer during simple molecular weight analyses. Some of these hidden protein properties include protein conformation, protein dynamics and protein interactions. How can these properties be revealed when mass spectrometers measure molecular weight not protein conformation? One way to uncover these properties with a mass spectrometer is to use a labeling method that “captures” the structural information before mass analysis occurs. The following chapter will describe one of these labeling methods: hydrogen-deuterium exchange.

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Read a review of this book, published in J Am Soc Mass Spectrom. 2009 Aug;20(8):R29-R30. Direct link. 

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