Extensive Deuterium Back-Exchange in Certain Immobilized Pepsin Columns Used for H/D Exchange Mass Spectrometry

Wu, Yang-feng; Kaveti, Suma; Engen, John R.

doi:10.1021/ac0518497

Cited by 44 publications

(53 citation statements)

References 16 publications

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“…Thus, only pepsin was used in the following experiments. As was shown previously, the use of immobilized proteases offers several advantages in HDX-MS experiments (16,28,29). Alongside higher protease stability and the absence of autodigestion, more important advantages are the much higher local concentration of protease leading to better digestion efficiency and the possible use of chaotropic reagent such as guanidinium chloride.…”

Section: Resultsmentioning

confidence: 95%

Conformational Dynamics of the Bovine Mitochondrial ADP/ATP Carrier Isoform 1 Revealed by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

Rey

Man

Clémençon

et al. 2010

Journal of Biological Chemistry

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The mitochondrial adenine nucleotide carrier (Ancp) catalyzes the transport of ADP and ATP across the mitochondrial inner membrane, thus playing an essential role in cellular energy metabolism. During the transport mechanism the carrier switches between two different conformations that can be blocked by two toxins: carboxyatractyloside (CATR) and bongkrekic acid. Therefore, our understanding of the nucleotide transport mechanism can be improved by analyzing structural differences of the individual inhibited states. We have solved the three-dimensional structure of bovine carrier isoform 1 (bAnc1p) in a complex with CATR, but the structure of the carrier-bongkrekic acid complex, and thus, the detailed mechanism of transport remains unknown. Improvements in sample processing in the hydrogen/deuterium exchange technique coupled to mass spectrometry (HDX-MS) have allowed us to gain novel insights into the conformational changes undergone by bAnc1p. This paper describes the first study of bAnc1p using HDX-MS. Results obtained with the CATR-bAnc1p complex were fully in agreement with published results, thus, validating our approach. On the other hand, the HDX kinetics of the two complexes displays marked differences. The bongkrekic acidbAnc1p complex exhibits greater accessibility to the solvent on the matrix side, whereas the CATR-bAnc1p complex is more accessible on the intermembrane side. These results are discussed with respect to the structural and biochemical data available on Ancp.

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Section: Resultsmentioning

confidence: 95%

Conformational Dynamics of the Bovine Mitochondrial ADP/ATP Carrier Isoform 1 Revealed by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

Rey

Man

Clémençon

et al. 2010

Journal of Biological Chemistry

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“…Proteins were overexpressed, purified, and labeled as described previously [16,19,20]. Generally speaking, a stock solution of each protein at 100 -200 pmol/L in a physiological buffer (potassium phosphate, Tris, pH 7-8, H 2 O) was prepared.…”

Section: Materials and Deuterium Labelingmentioning

confidence: 99%

“…Before analysis, the quenched exchange sample was incubated with a 1:1 ratio (weight/weight) of protein:pepsin for 5 min on ice or digested with online pepsin columns as described [20,22]. The peptides were fractionated in 5 min by a 5-70% ACN-water gradient at a flow rate of 50 L/min using a 50 ϫ 1.00 mm reversed-phase C18 column (such as Jupiter 4 Proteo 90 Å, Phenomenex, Torrance, CA) maintained at 0°C.…”

Section: Pepsin Digestion and Mass Analysis Of Peptidesmentioning

confidence: 99%

Identification and characterization of EX1 kinetics in H/D exchange mass spectrometry by peak width analysis

Weis

Wales

Engen

et al. 2006

J. Am. Soc. Mass Spectrom.

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213

269

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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

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“…The microreactor approach complicates the frontend fluidic system, and can lead to sample loss and carryover (19). The solution-phase method requires large amounts of enzyme resulting in contamination because of enzyme autolysis (18).…”

mentioning

confidence: 99%

Nepenthesin from Monkey Cups for Hydrogen/Deuterium Exchange Mass Spectrometry

Rey

Yang

Burns

et al. 2013

Molecular & Cellular Proteomics

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Studies of protein dynamics, structure and interactions using hydrogen/deuterium exchange mass spectrometry (HDX-MS) have sharply increased over the past 5-10 years. The predominant technology requires fast digestion at pH 2-3 to retain deuterium label. Pepsin is used almost exclusively, but it provides relatively low efficiency under the constraints of the experiment, and a selectivity profile that renders poor coverage of intrinsically disordered regions. In this study we present nepenthesin-containing secretions of the pitcher plant Nepenthes, commonly called monkey cups, for use in HDX-MS. We show that nepenthesin is at least 1400-fold more efficient than pepsin under HDX-competent conditions, with a selectivity profile that mimics pepsin in part, but also includes efficient cleavage C-terminal to "forbidden" residues K, R, H, and P. High efficiency permits a solution-based analysis with no detectable autolysis, avoiding the complication of immobilized enzyme reactors. Relaxed selectivity promotes high coverage of disordered regions and the ability to "tune" the mass map for regions of interest. Nepenthesin-enriched secretions were applied to an analysis of protein complexes in the nonhomologous endjoining DNA repair pathway. The analysis of XRCC4 binding to the BRCT domains of Ligase IV points to secondary interactions between the disordered C-terminal tail of XRCC4 and remote regions of the BRCT domains, which could only be identified with a nepenthesin-based workflow. HDX data suggest that stalk-binding to XRCC4 primes a BRCT conformation in these remote regions to support tail interaction, an event which may be phosphoregulated. We conclude that nepenthesin is an effective alternative to pepsin for all HDX-MS applications, and especially for the analysis of structural transitions among intrinsically disordered proteins and their binding partners. Molecular & Cellular

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Extensive Deuterium Back-Exchange in Certain Immobilized Pepsin Columns Used for H/D Exchange Mass Spectrometry

Cited by 44 publications

References 16 publications

Conformational Dynamics of the Bovine Mitochondrial ADP/ATP Carrier Isoform 1 Revealed by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

Conformational Dynamics of the Bovine Mitochondrial ADP/ATP Carrier Isoform 1 Revealed by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

Identification and characterization of EX1 kinetics in H/D exchange mass spectrometry by peak width analysis

Nepenthesin from Monkey Cups for Hydrogen/Deuterium Exchange Mass Spectrometry

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