Top-Down HydrogenâDeuterium Exchange Analysis of Protein

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Top-down hydrogen-deuterium exchange analysis of protein structures using ultraviolet photodissociation (UVPD) Nicholas I. Brodie, Romain Huguet, Terry Zhang, Rosa Viner, Vlad Zabrouskov, Jingxi Pan, Evgeniy V. Petrotchenko, and Christoph H. Borchers Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03655 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

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Analytical Chemistry

Top-down hydrogen-deuterium exchange analysis of protein structures using ultraviolet photodissociation (UVPD)

Nicholas I. Brodie1, Romain Huguet2, Terry Zhang2, Rosa Viner2, Vlad Zabrouskov2, Jingxi Pan1, Evgeniy V. Petrotchenko1, Christoph H. Borchers1,3-5*

1. University of Victoria -Genome British Columbia Proteomics Centre, #3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, BC V8Z7X8, Canada 2. Thermo Fisher Scientific, 355 River Oaks Pkwy., San Jose, CA 95134, United States 3. Department of Biochemistry and Microbiology, University of Victoria, Petch Building, Room 270d, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada 4. Gerald Bronfman Department of Oncology, Jewish General Hospital, McGill University, 3755 Côte Ste-Catherine Road, Montreal, Quebec, H3T 1E2, Canada 5. Proteomics Centre, Segal Cancer Centre, Lady Davis Institute, Jewish General Hospital, McGill University, 3755 Côte Ste-Catherine Road, Montreal, Quebec, H3T 1E2, Canada

*Corresponding author: Dr. Christoph H. Borchers University of Victoria - Genome British Columbia Proteomics Centre 3101-4464 Markham St, Victoria, BC Canada V8Z 7X8 Phone (250)483-3221; Fax: (205)483-3238 Email: [email protected]

Running title: Top-down HDX of proteins with UVPD 1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

Top-down hydrogen-deuterium exchange (HDX) analysis using electron capture or transfer dissociation Fourier transform mass spectrometry is a powerful method for the analysis of secondary structure of proteins in solution. The resolution of the method is a function of the extensive fragmentation of backbone bonds in the proteins. While fragmentation is usually extensive near the N- and C-termini, electron capture (ECD) or electron transfer dissociation (ETD) fragmentation methods sometimes lack good coverage of certain regions of the protein – most often in the middle of the sequence. Ultraviolet photodissociation (UVPD) is a recently developed fast-fragmentation technique, which provides extensive backbone fragmentation that can be complementary in sequence coverage to the aforementioned electron-based fragmentation techniques. Here, we explore the application of ESI-UVPD FTMS on an Orbitrap Fusion Lumos Tribrid mass spectrometer to top-down HDX analysis of proteins. We have incorporated UVPDspecific fragment-ion types and fragment-ion mixtures into our isotopic envelope fitting software (HDX Match) for the top-down HDX analysis. We have shown that UVPD data is complementary to ETD, thus improving the overall resolution when used as a combined approach.

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Introduction

Hydrogen-deuterium Exchange (HDX) – a method based on the exchange of a protein’s hydrogen atoms for deuterium atoms when dissolved in a D2O-based solution -- is a valuable method for studying protein structure 1. Amide protons from the main chain of a protein exchange more slowly if they are involved in hydrogen bonding or sequestered from the solvent, allowing the determination of the protein’s secondary structure. The extent of H/D exchange can be conveniently measured by high resolution mass spectrometry due to the 1-Da mass difference between hydrogen and deuterium. To locate sites of exchange or protection along the backbone, the protein can be enzymatically digested and the resulting peptides can then be analyzed by mass spectrometry to determine the deuterium incorporation (i.e., the “bottom-up” approach). Alternatively, the intact protein can be infused into the mass spectrometer, fragmented in the gas phase, and the deuteration level of the fragments can be determined (i.e., the “top-down” approach). By comparing the deuteration levels of consecutive fragments, the degree of exchange or protection can be often determined down to a single amino acid residue (i.e., amino acid-level resolution) 2. In order to get reliable structural information from the top-down method, however, one has to make sure that no H/D scrambling occurs during the protein fragmentation in the gas phase. Collision-induced dissociation (CID) is not generally suitable for top-down HDX-MS measurements because it can induce extensive scrambling 1. By comparing protein amide-deuteration values obtained using top-down ECD/ETD with those obtained using NMR, it was determined that scrambling did not occur, which means that electron based fragmentation methods can be used for structural analysis 2,3. ECD and ETD fragmentation mechanisms usually provide lower sequence coverage of the interior of the protein sequence, thus providing lower HDX resolution in those regions 4. 3 ACS Paragon Plus Environment


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In an attempt to address this issue, we turned our attention to ultraviolet photodissociation (UVPD) fragmentation. UVPD is a recently developed fast-fragmentation technique based on irradiation of the protein ions in the gas phase with far-UV light – at a wavelength where the peptide bond absorbs 5-11. In the study reported here, we applied UVPD fragmentation and topdown HDX analysis, using a commercially-available UVPD-enabled Orbitrap Fusion Lumos Tribrid mass spectrometer.

Experimental section

Myoglobin (Sigma, M1882) was prepared at approximately 0.3 mg/mL in 10 mM ammonium acetate. HDX, followed by top-down ETD-FTMS, was performed as described previously 12. Briefly, a protein solution and D2O from separate syringes were continuously mixed in 1:4 ratio (80% D2O final) at 10 µL/minute via a three-way tee which was connected to a 50 µm x 7 cm capillary, providing a labeling time of 1 s. The outflow from this capillary was mixed with a quenching solution containing 0.4% formic acid in 80% D2O from third syringe via a second three-way tee, and injected into a Thermo Scientific Orbitrap Fusion Lumos TribridTM mass spectrometer, equipped with the Easy-ETD ion source and UVPD 13,14.

ETD spectra were acquired using 5-, 10-, 15-, 20-, and 25-ms reaction times per spectrum, collecting for 1 minute at each reaction time. All of the acquired spectra for the 5-, 10- and 15ms reaction times were averaged, the averaged spectra were used for the data analysis. The UVPD spectra were acquired for 1-, 3-, 5-, 8-, 12-, 17-, 20-, and 25-ms per spectrum, with irradiation from a 2.5-kHz repetition rate (0.4 msec/pulse) 213-nm Nd:YAG (neodymium-doped


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yttrium aluminum garnet) laser (CryLas GmbH) with pulse energy of 1.5 ± 0.2 µJ/pulse and output power of 3.75 ± 0.5 mW, collecting for 1 minute at each reaction time. The beam diameter of 450 ±200 µm including divergence at the center of the ion trap is slightly larger than the simulated ion cloud diameter, and therefore no focusing optics was required. The laser trigger was provided by the instrument as is synched to the scan function. Photoactivation occurs in the low pressure cell of the dual-pressure linear ion trap, while m/z analysis was done in the Orbitrap mass analyzer. All of the acquired spectra for the 8-, 12-, and 17-ms reaction times were averaged, and the averaged spectra were used for the data analysis. Deuteration levels of the amino acid residues were determined using the HDX Match program 15.

Results and discussion

UVPD-FTMS top-down HDX. Top-down HDX analysis was performed by continuous in-line mixing of the protein solution with D2O followed by mixing with an acidic quenching solution and direct infusion into the mass spectrometer. In this experimental setup, exchange and quenching times are determined by the length of the mixing capillaries and the flow rates of the syringes that supply the solutions. One of the advantages of this approach is that total deuteration status of the intact protein ion can be readily determined from the overall mass shift after HDX. It has been shown that, under the conditions used here, total protection of a protein - as determined by HDX -- agrees well with the total number of hydrogen-bonded peptide amide bonds within the secondary structure motifs 4. Thus, for myoglobin, we observed protection of 108 hydrogens, while 112 backbone amides are hydrogen-bonded according to the crystal structure (PDB: 1YMB). 5 ACS Paragon Plus Environment


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A single charge state of myoglobin was isolated and subjected to UVPD fragmentation with a 213 nm laser (2 to 62 laser pulses) in the low pressure cell of the dual-pressure linear ion trap of an Orbitrap Fusion Lumos Tribrid mass spectrometer (Figure S1) 13,14. The fragments generated were detected in the FT mode in the Orbitrap. ETD fragmentation for 1 to 25 ms was performed in parallel. UVPD produced robust fragmentation, spread across the whole protein sequence, with the formation of mainly a-, x- y- and z- ions (Figure S2). The data produced by the 213-nm laser generally reproduced previously reported data obtained with a 193-nm laser (Figure 1-3) 16. Fragment ions which were common between ETD and UVPD showed the same deuteration values, which confirmed the lack of scrambling in UVPD, assuming that ETD fragmentation is scrambling-free 17-19 (Figure S3). Furthermore, the apparent absence of scrambling was confirmed by comparing obtained deuteration data with NMR and X-ray crystallography results (Figure S4). Importantly, the UVPD fragmentation sites were different from – and complementary to -- the ETD fragmentation, resulting in 58%, 58%, and 81% sequence coverage for ETD alone, UVPD alone, and the combination of ETD and UVPD, respectively, according to ProSightPC 20 analysis (Figure S5), more than doubling the number of fragmentation sites suitable for HDX analysis for residues 41-120 region from the interior of the protein, an increase of 123% compared to ETD alone (Figure 2A).

UVPD top-down HDX data analysis. Top-down HDX data analysis was performed by fitting theoretical isotopic envelopes to the experimentally-derived isotopic envelopes for each fragment ion, using an house developed software program called HDX Match 15. Fragment ions are first identified in the non-HDX spectrum by isotopic envelope mass matching, and then corresponding fragments in HDX spectrum are located and are fitted to the theoretical envelopes 6 ACS Paragon Plus Environment

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by varying the number of protected or exchanged protons in the fragment ion, based on its elemental composition. Our original version of the program used ECD/ETD-specific c- and zions; UVPD-specific a-, x-, b- and y-ions have now been included in the program.

We noticed, however, that there was a poor fit to the a-, x-, and y-ion isotopic envelopes in the non-HDX experiments, and that the inclusion of a+1, a+2, x+1, x+2, y-1 or y-2 ions (i.e., fragment ions which had gained or lost 1 or 2 Da) into the analysis only partially alleviated the this problem. It appeared that, in multiple cases, the a-type fragment ions are actually mixtures of a, a+1, a+2, the x-type fragment ions are mixtures of x, x+1, x+2, and the y-type fragment ions are mixtures of y, y-1, and y-2 fragment ions, in varying proportions. This prevented direct determination of the correct fragment-ion deuteration values from the HDX spectrum.

To overcome this problem, we now propose new fragment ion types, which we call “+0/1/2” (i.e., a mixture of the canonical fragment ion and the ions derived from it which gained 1 or 2 Da ) and “-0/1/2” (i.e., a mixture of the canonical fragment ion and the ions derived from it which lost 1 or 2 Da). The contributions (as percent values) from each type of ion in the ternary mixture were determined by iterative two-dimensional coordinate descent minimization, using percent of second and third components as variables. Minima in each dimension were found by the golden section method 21. These percent values were used to produce deuterated superposition envelopes, making the assumption that the added or subtracted hydrogen atoms were exchangeable. This allowed us to improve the envelope-fitting of the non-HDX fragments (Figure S6), and thus to determine the correct deuteration values for the corresponding HDX fragments. This feature was also added to the HDX Match program.



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The multiple fragment ion types obtained from ETD and UVPD experiments can be combined according to the position of the bond being broken relative to the residue amide for the determination of fragment deuteration values. Thus, the deuteration values for the N-terminal ions for residue i amide ai-, bi- and ci-1-ions and the C-terminal ions for a residue i amide xn-i-, yni-

and zn-i+1-ions, where n is the total number of residues, can be combined.

Conclusions

UVPD-FTMS was successfully applied for the top-down HDX analysis of proteins. It is complementary to ETD fragmentation, and the combination of ETD and UVPD datasets improves the overall resolution of the approach. UVPD-specific a-, x- and y- fragment ion types and fragment ion mixtures have now been incorporated into the isotopic envelope-fitting software in our top-down HDX data analysis program, HDX Match, which is available online for no cost. UVPD fragmentation proved to be a valuable addition to the top-down HDX toolbox for the structural analysis of proteins.

Acknowledgements

The University of Victoria-Genome BC Proteomics Centre was supported by the Genomic Innovations Network (GIN) from Genome Canada and Genome British Columbia (project codes 204PRO and 214PRO). CHB would also like to thank the National Sciences and Engineering 8 ACS Paragon Plus Environment

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Research Council of Canada (NSERC) and the Leading Edge Endowment Fund for support. CHB is also grateful for support from the Segal McGill Chair in Molecular Oncology at McGill University (Montreal, Quebec, Canada), and for support from the Warren Y. Soper Charitable Trust and the Alvin Segal Family Foundation to the Jewish General Hospital (Montreal, Quebec, Canada).

Conflict of interest statement

EP and CB are co-founders of Creative Molecules, Inc. RH, TZ, RV, and VZ are employees of Thermo Fisher Scientific. The other authors declare no competing financial interests.



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References

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Figure legends Figure 1. UVPD-HDX analysis of myoglobin. Total deuteration of myoglobin measured in MS1 with no fragmentation of the protein ion. Figure 2: A. Fragments selected for HDX analysis: top, ETD; bottom, UVPD (30, 36, and 57 unique fragmentation sites were observed for ETD, UVPD, and the combination of ETD with UVPD, respectively). B. Deuteration plot for UVPD data. Top: cumulative deuteration values for the fragment ions observed. The solid line represents the number of deuterium atoms which would be incorporated if the protein were fully protected. The marked lines represent the observed number of protected residues. Blue lines are N-terminal fragments, red lines are Cterminal fragments. Bottom: residue-specific deuteration values. The blue line represents Nterminal fragments, the red line indicates C-terminal fragments. Figure 3: Residue deuteration values superimposed on the crystal structure of the myoglobin (1YMB)


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Figure 1.



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Figure 2 A

B


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Figure 3



213 nm UVPD fragments proteins without scrambling in HDX 84x47mm (96 x 96 DPI)

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Top-Down HydrogenâDeuterium Exchange Analysis of Protein

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