ExMS: Data Analysis for HX-MS Experiments

Kan, Zhong-yuan; Mayne, Leland; Chetty, Palaniappan Sevugan; Englander, S. Walter

doi:10.1007/s13361-011-0236-3

Cited by 119 publications

(136 citation statements)

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“…The fragments then are separated and analyzed for carried deuterium by HPLC and mass spectrometry. Previous papers describe how to maximize the number of peptide fragments obtained (10), how to minimize the back exchange of D label during sample preparation (11), and a program (ExMS) for identifying and characterizing the many peptides in the final MS spectra (12). These methods provided 326 useful peptide fragments; 228 unique fragments for this 155-residue protein are indicated in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…After some folding time, D-to-H exchange at still-exposed sites is induced by a brief highpH pulse (mixer 2) and then is quenched to low pH where HX is very slow (mixer 3). Online proteolysis cuts the protein into many overlapping peptide fragments, and the peptides are separated by LC and MS. (B) The 228 unique peptides used in this work, identified and characterized in the MS data by the ExMS program (12), and plotted as a function of amino acid residue.…”

Section: Resultsmentioning

confidence: 99%

“…Previously described methods (10)(11)(12) were used to obtain the many peptide fragments studied here. Of these, 156 unique peptides (203 with different charge states) were found to pass the ExMS autocheck tests (no operator intervention) and were used in the timed 9-ms to 3-s folding experiments.…”

Section: Methodsmentioning

confidence: 99%

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Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry

Hu¹,

Walters

Kan³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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165

217

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The kinetic folding of ribonuclease H was studied by hydrogen exchange (HX) pulse labeling with analysis by an advanced fragment separation mass spectrometry technology. The results show that folding proceeds through distinct intermediates in a stepwise pathway that sequentially incorporates cooperative native-like structural elements to build the native protein. Each step is seen as a concerted transition of one or more segments from an HX-unprotected to an HX-protected state. Deconvolution of the data to near amino acid resolution shows that each step corresponds to the folding of a secondary structural element of the native protein, termed a "foldon." Each folded segment is retained through subsequent steps of foldon addition, revealing a stepwise buildup of the native structure via a single dominant pathway. Analysis of the pertinent literature suggests that this model is consistent with experimental results for many proteins and some current theoretical results. Two biophysical principles appear to dictate this behavior. The principle of cooperativity determines the central role of native-like foldon units. An interaction principle termed "sequential stabilization" based on nativelike interfoldon interactions orders the pathway.D o proteins fold through varied and multiple tracks, or do they fold through predetermined intermediates according to understandable biophysical principles (1)? This question is fundamental for the interpretation of a large amount of biophysical and biological research. The question could be resolved if it were possible to define the intermediate structures and pathways that unfolded proteins move through on their way to the native state. Unfortunately, transient intermediates cannot be studied by the usual crystallographic and NMR methods. The range of kinetic and spectroscopic methods has been applied to many proteins, but these methods do not yield the necessary structural information.We used a developing technology, hydrogen exchange pulse labeling measured by MS (HX MS), to study the folding of a cysteine-free variant of Escherichia coli ribonuclease H1 (RNase H), a mixed α/β protein that has served as a major proteinfolding model (2-5). Previous studies showed that RNase H folds in a fast, unresolved burst phase (15 ms dead time) to an intermediate termed "I core " and then much more slowly (in seconds) to the native state (3). HX pulse-labeling and equilibrium native-state HX experiments monitored by NMR showed that I core comprises a continuous region of the protein between helix A and strand 5 and that β-strands 1, 2, and 3 and helix E acquire protection much later, consistent with mutational analysis (2-4). Single-molecule and mutational studies indicated that the intermediate is obligatory, on-pathway, and folds first even when I core is not observably populated (6, 7).The HX MS technique used here is able to follow the entire folding trajectory of RNase H in considerable structural and temporal detail. The analysis monitors every amide site, evaluates the folding coopera...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry

Hu¹,

Walters

Kan³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

165

217

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“…Previously described HX MS methods were used (29,30,83). Unfolded MBP, initially fully deuterated at exchangeable hydrogen sites in D 2 O, was allowed to fold for some predetermined time, then probed by a brief pulse of D-to-H labeling to obtain a snapshot of the structure that had been formed to that point.…”

Section: Methodsmentioning

confidence: 99%

“…To study the fast and slow stages of MBP folding at higher structural resolution, we used a quench-flow HX pulse-labeling experiment with analysis by fragment-separation mass spectrometry ( already formed H-bonded structure tend to be protected and remain deuterated, whereas unprotected amides become protonated. The H-labeled protein was immediately quenched into a slow HX condition (pH 2.5, 0°C), injected into an online analysis system, cleaved into small peptide fragments by acid proteolysis (pepsin), the peptide fragments were quickly separated by HPLC and mass spectrometry (LTQ Orbitrap XL) (29), and then identified and analyzed for carried D label (30). The 225 unique peptide fragments obtained, often with multiple charge states, are indicated in Fig.…”

Section: Significancementioning

confidence: 99%

Folding of a large protein at high structural resolution

Walters

Mayne²,

Hinshaw³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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107

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Kinetic folding of the large two-domain maltose binding protein (MBP; 370 residues) was studied at high structural resolution by an advanced hydrogen-exchange pulse-labeling mass-spectrometry method (HX MS). Dilution into folding conditions initiates a fast molecular collapse into a polyglobular conformation (<20 ms), determined by various methods including small angle X-ray scattering. The compaction produces a structurally heterogeneous state with widespread low-level HX protection and spectroscopic signals that match the equilibrium melting posttransition-state baseline. In a much slower step (7-s time constant), all of the MBP molecules, although initially heterogeneously structured, form the same distinct helix plus sheet folding intermediate with the same time constant. The intermediate is composed of segments that are distant in the MBP sequence but adjacent in the native protein where they close the longest residue-to-residue contact. Segments that are most HX protected in the early molecular collapse do not contribute to the initial intermediate, whereas the segments that do participate are among the less protected. The 7-s intermediate persists through the rest of the folding process. It contains the sites of three previously reported destabilizing mutations that greatly slow folding. These results indicate that the intermediate is an obligatory step on the MBP folding pathway. MBP then folds to the native state on a longer time scale (∼100 s), suggestively in more than one step, the first of which forms structure adjacent to the 7-s intermediate. These results add a large protein to the list of proteins known to fold through distinct native-like intermediates in distinct pathways.SAXS | HDX | protein collapse | denatured state ensemble F ifty years after Anfinsen's seminal demonstration that an unfolded protein can refold spontaneously when placed under native conditions, major questions concerning the folding process remain unanswered (1, 2). What is the unfolded state like, its degree of compaction, the reality and character of residual structure before folding begins, and its possible role in guiding the folding process (3-7)? Analogous questions relate to folding intermediates and the folding pathway itself. Do proteins fold through many alternative independent pathways as earlier theoretical investigations have suggested (8-12), or do they fold through necessary intermediates in a distinct pathway (13), as a growing list of experimental observations indicate (14, 15)? To answer these questions, it will be necessary to define experimentally the intermediate forms that proteins move through on their way to the native state. The problem has been that these transient states are beyond the reach of the usual high-resolution crystallographic and NMR structural methods. Most experimental folding studies have therefore relied on low-resolution optical methods that can follow folding in real time but rarely provide the structural information necessary to resolve the basic mechanistic questions.Recent work...

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Data Processing in Bottom‐Up Hydrogen Exchange Mass Spectrometry

Sarpe

Schriemer

2016

Hydrogen Exchange Mass Spectrometry of Proteins

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ExMS: Data Analysis for HX-MS Experiments

Cited by 119 publications

References 14 publications

Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry

Stepwise protein folding at near amino acid resolution by hydrogen exchange and mass spectrometry

Folding of a large protein at high structural resolution

Data Processing in Bottom‐Up Hydrogen Exchange Mass Spectrometry

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