Semi-automated data processing of hydrogen exchange mass epectra using HX-Express.
Weis DD, Engen JR, Kass IJ.
J Am Soc Mass Spectrom. 2006. 17(12):1700-1703.
 
ABSTRACT
A Microsoft Excel utility, HX-Express, that significantly accelerates the analysis of hydrogen exchange mass spectrometry data is described. HX-Express generates deuterium uptake and peak width plots from peaks in mass spectral data. Data analysis is intentionally semi-automated, requiring that the user find the peaks to be analyzed. The peaks are entered in the form of x, y lists of m/z versus intensity or can be directly imported from Waters MassLynx software. Analysis of data with HX-Express provides the same results as manual data processing but is substantially faster. In addition to speed, HX-Express provides and preserves visual and numeric displays of the analysis process for quality control.
 
Software website:  www.hxms.com/HXExpress
 
Pubmed: 16931036

 
Hydrogen exchange and covalent modification: Focus on biomolecular structure, dynamics, and function. 18th Sanibel Conference on Mass Spectrometry.

Kaltashov IA, Engen JR, Gross ML. J Am Soc Mass Spectrom. 2006 Nov;17(11):I1-I2.

 
Identification and Characterization of EX1 Kinetics in H/D Exchange Mass Spectrometry by Peak Width Analysis.
Weis DD, Wales TE, Engen JR, Hotchko M, Ten Eyck LF.
J Am Soc Mass Spectrom. 2006 Nov;17(11):1498-1509.

ABSTRACT
Proteins that undergo cooperative unfolding events display EX1 kinetic signatures in hydrogen exchange mass spectra. The hallmark bimodal isotope pattern observed for EX1 kinetics is distinct from the binomial isotope pattern for uncorrelated exchange (EX2), the normal exchange regime for folded proteins. Detection and characterization of EX1 kinetics is simple when the cooperative unit is large enough that the isotopic envelopes in the bimodal pattern are resolved in the m/z scale but become complicated in cases where the unit is small or there is a mixture of EX1 and EX2 kinetics. Here we describe a data interpretation method involving peak width analysis that makes characterization of EX1 kinetics simple and rapid. The theoretical basis for EX1 and EX2 isotopic signatures and the effects each have on peak width are described. Modeling of EX2 widening and analysis of empirical data for proteins and peptides containing purely EX2 kinetics showed that the amount of widening attributable to stochastic forward- and back exchange in a typical experiment is small and can be quantified. Proteins and peptides with both obvious and less obvious EX1 kinetics were analyzed with the peak width method. Such analyses provide the half-life for the cooperative unfolding event and the relative number of residues involved. Automated analysis of peak width was performed with custom Excel macros and the DEX software package. Peak width analysis is robust, capable of automation, and provides quick interpretation of the key information contained in EX1 kinetic events.
 
Pubmed: 16875839

 
Altered dynamics in Lck SH3 upon binding to the LBD1 domain of Herpesvirus saimiri Tip.
Weis DD, Kjellen P, Sefton BM, Engen JR.
Protein Sci. 2006 Oct;15(10):2402-10.
 
ABSTRACT
The Tip protein from Herpesvirus saimiri interacts with the SH3 domain from the Src-family kinase Lck via a proline-containing sequence termed LBD1. Src-family kinase SH3 domains related to Lck have been shown to be dynamic in solution and partially unfold under physiological conditions. The rate of such partial unfolding is reduced by viral protein binding. To determine if the Lck SH3 domain displayed similar behavior, the domain was investigated with hydrogen exchange and mass spectrometry. Lck SH3 was found to be highly dynamic in solution. While other SH3 domains require as much as 10,000 sec to become totally deuterated, Lck SH3 became almost completely labeled within 200 sec. A partial unfolding event involving 8-10 residues was observed with a half-life of approximately 10 sec. Tip LBD1 binding did not cause gross structural changes in Lck SH3 but globally stabilized the domain and reduced the rate of partial unfolding by a factor of five. The region of partial unfolding in Lck SH3 was found to be similar to that identified for other SH3 domains that partially unfold. Although the sequence conservation between Lck SH3 and other closely related SH3 domains is high, the dynamics do not appear to be conserved.
 
Pubmed: 17008721

 
Src family kinases phosphorylate the Bcr-Abl SH3-SH2 region and modulate Bcr-Abl transforming activity.
Meyn MA 3rd, Wilson MB, Abdi FA, Fahey N, Schiavone AP, Wu J, Hochrein JM, Engen JR, Smithgall TE.
J Biol Chem. 2006 Oct 13;281(41):30907-16.

ABSTRACT
Bcr-Abl is the oncogenic protein-tyrosine kinase responsible for chronic myelogenous leukemia. Recently, we observed that inhibition of myeloid Src family kinase activity (e.g. Hck, Lyn, and Fyn) induces growth arrest and apoptosis in Bcr-Abl-transformed cells, suggesting that cell transformation by Bcr-Abl involves Src family kinases (Wilson, M. B., Schreiner, S. J., Choi, H. J., Kamens, J., and Smithgall, T. E. (2002) Oncogene 21, 8075-8088). Here, we report the unexpected observation that Hck, Lyn, and Fyn strongly phosphorylate the SH3-SH2 region of Bcr-Abl. Seven phosphorylation sites were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry: Tyr89 and Tyr134 in the Abl-derived SH3 domain; Tyr147 in the SH3-SH2 connector; and Tyr158, Tyr191, Tyr204, and Tyr234 in the SH2 domain. SH3 domain Tyr89, the most prominent phosphorylation site in vitro, was strongly phosphorylated in chronic myelogenous leukemia cells in a Src family kinase-dependent manner. Substitution of the SH3-SH2 tyrosine phosphorylation sites with phenylalanine substantially reduced Bcr-Abl-mediated transformation of TF-1 myeloid cells to cytokine independence. The positions of these tyrosines in the crystal structure of the c-Abl core and the transformation defect of the corresponding Bcr-Abl mutants together suggest that phosphorylation of the SH3-SH2 region by Src family kinases impacts Bcr-Abl protein conformation and signaling.
 
Pubmed: 16912036

 
Conformational features of the full-length HIV and SIV Nef proteins determined by mass spectrometry.
Hochrein JM, Wales TE, Lerner EC, Schiavone AP, Smithgall TE, Engen JR.
Biochemistry. 2006 Jun 27;45(25):7733-9.

ABSTRACT
The Nef protein from human or simian immunodeficiency virus enhances viral replication, downregulates immune cell receptors, and activates multiple host cell signaling pathways. Conformational information about full-length Nef has been difficult to obtain as the full-length protein is not readily amenable to NMR or X-ray crystallography due to aggregation at high concentrations. As an alternative, full-length HIV and SIV Nef were probed with hydrogen exchange mass spectrometry, a method compatible with the low concentration requirements of Nef. The results showed that HIV Nef contains a solvent-protected core, as previously demonstrated with both NMR and X-ray crystallography. SIV Nef, for which there is no structural information, had a similar protected core, although it was more flexible and dynamic than its HIV counterpart. Many of the regions outside the core in both SIV and HIV Nef were highly solvent exposed. However, limited protection from exchange was observed in both N- and C-terminal regions, suggesting the presence of structured elements. Protection from exchange was also observed in a large loop emanating from the core that was deleted for NMR and X-ray analysis. These data show that while the majority of Nef was highly solvent exposed, regions outside the core may have structural attributes which may contribute to Nef functions known to map to these regions.
 
Pubmed: 16784224

Protein interactions probed with mass spectrometry.
Kaveti S, Engen JR.
Methods Mol Biol. 2006;316:179-97.

ABSTRACT
Understanding the interactions of proteins with other proteins and/or with drug molecules is essential for understanding the progression of diseases. In this chapter, we present several methods utilizing mass spectrometry (MS) for the analysis of protein-protein, protein-drug, and protein-metal interactions. We describe the analysis of protein interactions with hydrogen exchange MS methods. Hydrogen exchange methods can be used to analyze conformational changes on binding, to estimate dissociation constants, and to locate the sites of interaction/binding between binding partners. We also discuss more direct MS methods, including the analysis of metal ion complexation with proteins.
 
Pubmed: 16671405

 
Partial unfolding of diverse SH3 domains on a wide timescale.
Wales TE, Engen JR.
J Mol Biol. 2006 Apr 14;357(5):1592-604.

ABSTRACT
SH3 domains are small, modular domains that are found in many proteins, especially signal transduction proteins such as tyrosine kinases. While much is known about the sequences and tertiary structures of SH3 domains, far less is known about their solution dynamics. A slow, partial unfolding event that occurs under physiological conditions was previously identified in the Hck SH3 domain using hydrogen exchange (HX) mass spectrometry (MS). To determine if this unfolding was unique to Hck SH3, HX MS was used to analyze 11 other SH3 domains: seven SH3 domains from Src-family kinases and five SH3 domains from various proteins. A wide variety of unfolding rates were found, with unfolding half-lives ranging from 1s to 1h. The Lyn and alpha-spectrin SH3 domains exhibited slow, partial unfolding in beta strands D and E and part of the RT-loop. Hck SH3 also underwent partial unfolding in the same region, implying that a unique feature in this area of the domains is responsible for the partial unfolding. Partial unfolding was, however, not a function of sequence conservation. Although the Fyn and Yes SH3 domains are very similar to Hck SH3 in sequence, they exhibited no evidence of partial unfolding. Overall, the results suggest that while the tertiary structure of SH3 domains is highly conserved, the dynamics of SH3 domains are variable.
 
Pubmed: 16487539

 
Extensive deuterium back-exchange in certain immobilized pepsin columns used for H/D exchange mass spectrometry.
Wu Y, Kaveti S, Engen JR.
Anal Chem. 2006 Mar 1;78(5):1719-23.

ABSTRACT
Pepsin digestion prior to mass analysis increases the spatial resolution of hydrogen exchange mass spectrometry experiments. Online digestion with immobilized pepsin is advantageous for several reasons including better digestion efficiency. We have found that certain immobilized pepsin columns cause substantial deuterium back-exchange, rendering the data unusable. When pepsin immobilized on a POROS support was used for online digestion, back-exchange was within the expected range and was similar to the back-exchange of deuterated peptides produced by in-solution pepsin digestion. However, when pepsin immobilized onto selected polystyrene-divinylbenzene supports was used for online digestion with the same system, deuterium loss was extremely high. The effect seems linked to the properties of the solid support used to conjugate the pepsin.
 
Pubmed: 16503628

 
Ultra performance liquid chromatography (UPLC) further improves hydrogen/deuterium exchange mass spectrometry.
Wu Y, Engen JR, Hobbins WB.
J Am Soc Mass Spectrom. 2006 Feb;17(2):163-7.

ABSTRACT
Ultra performance liquid chromatography (UPLC) employs particles smaller than 2 microm in diameter to achieve superior resolution, speed, and sensitivity compared with high-performance liquid chromatography (HPLC). We have tested the suitability of UPLC for the analysis of deuterated peptides in hydrogen exchange mass spectrometry experiments. Superior resolution and sample throughput were obtained with UPLC versus HPLC. For highly deuterated model peptides, deuterium loss using UPLC was greater than the deuterium loss observed using a conventional HPLC system, primarily as a result of the injection requirements of the UPLC system. Partially deuterated cytochrome c peptides also lost more deuterium in UPLC versus HPLC, although the effect was not as pronounced as it was for the highly deuterated model peptides. The exceptional chromatographic aspects of UPLC make it a very attractive alternative to HPLC for hydrogen exchange mass spectrometry experiments.
 
Pubmed: 16406808
 

 
Hydrogen exchange mass spectrometry for the analysis of protein dynamics.
Wales TE, Engen JR.
Mass Spectrom Rev. 2006 Jan-Feb;25(1):158-70.

ABSTRACT
Hydrogen exchange coupled to mass spectrometry (MS) has become a valuable analytical tool for the study of protein dynamics. By combining information about protein dynamics with more classical functional data, a more thorough understanding of protein function can be obtained. In many cases, protein dynamics are directly related to specific protein functions such as conformational changes during enzyme activation or protein movements during binding. The method is made possible because labile backbone hydrogens in a protein will exchange with deuterium atoms when the protein is placed in a D2O solution. The subsequent increase in protein mass over time is measured with high-resolution MS. The location of the deuterium incorporation is determined by monitoring deuterium incorporation in peptic fragments that are produced after the labeling reaction. In this review, we will summarize the general principles of the method, discuss the latest variations on the experimental protocol that probe different types of protein movements, and review other recent work and improvements in the field.
 
Pubmed: 16208684

 
An examination of dynamics crosstalk between SH2 and SH3 domains by hydrogen/deuterium exchange and mass spectrometry.
Hochrein JM, Lerner EC, Schiavone AP, Smithgall TE, Engen JR.
Protein Sci. 2006 Jan;15(1):65-73.

ABSTRACT
The ability of proteins to regulate their own enzymatic activity can be facilitated by changes in structure or protein dynamics in response to external regulators. Because many proteins contain SH2 and SH3 domains, transmission of information between the domains is a potential method of allosteric regulation. To determine if ligand binding to one modular domain may alter structural dynamics in an adjacent domain, allowing potential transmission of information through the protein, we used hydrogen exchange and mass spectrometry to measure changes in protein dynamics in the SH3 and SH2 domains of hematopoietic cell kinase (Hck). Ligand binding to either domain had little or no effect on hydrogen exchange in the adjacent domain, suggesting that changes in protein structure or dynamics are not a means of SH2/SH3 crosstalk. Furthermore, ligands of varying affinity covalently attached to SH3/SH2 altered dynamics only in the domain to which they bind. Such results demonstrate that ligand binding may not structurally alter adjacent SH3/SH2 domains and implies that other aspects of protein architecture contribute to the multiple levels of regulation in proteins containing SH3 and SH2 domains.
 
Pubmed: 16322569