Hydrogen/deuterium exchange mass spectrometry with high spatial resolution using gas-phase fragmentation

Daniel T. Weltz Wollenberg

doi:10.21996/m63g-n611

Hydrogen/deuterium exchange mass spectrometry with high spatial resolution using gas-phase fragmentation

Bidragets oversatte titel: Hydrogen/deuterium udveksling målt med massespektrometri med høj rumlig opløsning ved brug af gas-fase fragmentering

Daniel T. Weltz Wollenberg

Publikation: Afhandling › Ph.d.-afhandling

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Abstract

Et proteins funktion er tæt forbundet med dets iboende dynamiske struktur. Det er derfor nødvendigt at undersøge disse flygtige bevægelser, for helt at forstå de allestedsnærværende biologiske maskiner som proteiner er. Hydrogen/deuterium-udveksling (HDX) af amiderne i proteiners rygrad er en sensitiv probe for disse strukturelle dynamikker. Da enhver udveksling fra en isotop til den anden medfører en ændring af proteinets masse, kan massespektrometri (MS) anvendes til at monitorerer denne udveksling. Dette udgør metodologien HDX-MS. HDX-MS er derfor et effektivt værktøj til at studerer proteiners dynamiske
struktur.

Hver aminosyreposition langs proteinets rygrad (bortset fra prolin) udgør en probe for hydrogen/deuterium-udveksling, men den opløselighed som kan opnås med HDX-MS er begrænset af evnen til at generere unikke sekvensfragmenter, som kan bruges til at udlede indholdet af hver isotop (1H dvs. hydrogen eller 2H dvs. deuterium) for et enkelt amid eller en sekvens af amider. Derfor er gas-fase fragmentering blevet undersøgt, som en måde hvorpå isotopindholdet kan bestemmes med stor rummelig
præcision, gennem de seneste to årtier. I denne sammenhæng er det dog en forudsætning, at væskefasens isotopmærkning kan bevares ind i gas-fasen under transport og under selve fragmenteringsprocessen. Dette er imidlertid ikke trivielt på grund af risikoen for opbygning af intern energi under disse processer. Denne energiopbygning kan nemlig resultere i intermolekylær protonvandring, hvorved der sker randomisering af analyttens isotopmærkning, også kaldet hydrogenmigration (scrambling). For at reducere den hydrogenmigration, som sker før gas-fase fragmentering, skal andelen af energi som tilføres under gas-fase transport reduceres. Denne optimering medfører dog en reduktion i transmissionseffektiviteten.

I det første manuskript som er vedlagt denne afhandling, undersøger vi hvordan et optimum kan findes, således at der opnås lav hydrogenmigration med minimale omkostninger for transmissionseffektiviteten. Vi demonstrerer også anvendeligheden af vores metode ved at udføre et bottom-up HDX-MS/MS-forsøg med elektron-overførselsdissociation (ETD) på et velkarakteriseret protein.

Udover ved iontransport, kan hydrogenmigration induceres under selve fragmenteringsprocessen. For nuværende er ETD og elektron-indfangningsdissociation (ECD) veletableret som teknikker, der ikke forsager hydrogenmigration i protonerede peptider. For nyligt blev denne egenskab også påvist for 213 nm
ultraviolet fotodissociation (UVPD), med en unik specialbygget instrumentel implementering. Derfor har vi i det andet manuskript som er vedhæftet denne afhandling, prøvet at gentage disse forsøg på den første kommercielle implementering af UVPD. Til vores overraskelse kunne vi dog vise, at alle UVPD-genererede
fragment-ioner i vores kommercielle implementering af UVPD udviste en høj grad af hydrogenmigration.

Bidragets oversatte titel	Hydrogen/deuterium udveksling målt med massespektrometri med høj rumlig opløsning ved brug af gas-fase fragmentering
Originalsprog	Engelsk
Bevilgende institution	Syddansk Universitet
Vejledere/rådgivere	Jørgensen, Thomas J.D., Hovedvejleder Jørgensen, Christian Isak, Bivejleder, Ekstern person Mouritsen, Jeppe Christian, Bivejleder, Ekstern person Pengelley, Stuart, Bivejleder, Ekstern person
Eksterne samarbejdspartnere	Novozymes A/S Bruker Daltonics
Dato for forsvar	26. aug. 2022
Udgiver	Syddansk Universitet. Det Naturvidenskabelige Fakultet
DOI	https://doi.org/10.21996/m63g-n611
Status	Udgivet - 13. sep. 2022

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10.21996/m63g-n611

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2 Tidsskriftartikel
1 Kommentar/debat

The changing face of SDS denaturation: Complexes of Thermomyces lanuginosus lipase with SDS at pH 4.0, 6.0 and 8.0
Rasmussen, H. Ø., Wollenberg, D. T. W., Wang, H., Andersen, K. K., Oliveira, C. L. P., Jørgensen, C. I., Jørgensen, T. J. D., Otzen, D. E. & Pedersen, J. S., 15. maj 2022, I: Journal of Colloid and Interface Science. 614, s. 214-232 19 s.
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Ultraviolet Photodissociation of Protonated Peptides and Proteins Can Proceed with H/D Scrambling
Modzel, M., Wollenberg, D. T. W., Trelle, M. B., Larsen, M. R. & Jørgensen, T. J. D., 19. jan. 2021, I: Analytical Chemistry. 93, 2, s. 691–696
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Avoiding H/D Scrambling with Minimal Ion Transmission Loss for HDX-MS/MS-ETD Analysis on a High-Resolution Q-TOF Mass Spectrometer
Wollenberg, D. T. W., Pengelley, S., Mouritsen, J. C., Suckau, D., Jørgensen, C. I. & Jørgensen, T. J. D., 2020, I: Analytical Chemistry. 92, 11, s. 7453–7461
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Citationsformater

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abstract = "The function of a protein is intimately linked to the intrinsic dynamics of its structure. Therefore, to fully understand the ubiquitous biological machinery that is proteins, we must probe these elusive motions. Hydrogen/deuterium exchange (HDX) of protein backbone amides provide a sensitive probe for thesestructural dynamics. As each exchange of one isotope for the other incurs a change in the mass of the protein, mass spectrometry (MS) can be used to monitor this exchange in a methodology termed HDXMS. Thus, HDX-MS is a powerful tool for the study of protein structural dynamics.While each amino acid position along the protein backbone (except for proline) provides a probe for hydrogen/deuterium exchange, the resolution offered by HDX-MS is limited to the ability to generate unique backbone fragments which can be used to infer the isotopic occupancy (1H i.e. hydrogen or 2H i.e. deuterium) for a single amide or a range of amides. For this reason, gas-phase fragmentation has been investigated as a means of achieving high resolution amide exchange data for the past two decades. However, a prerequisite for the use of gas-phase fragmentation in HDX-MS is the conservation of the solution phase isotopic labeling into the gas-phase, during gas-phase transport, and during the fragmentation process itself.This is non-trivial due to the risk of internal energy buildup during these processes which can cause intermolecular proton migration, thus essentially randomizing the isotopic labeling of the analyte ion. This process is called hydrogen scrambling. To reduce hydrogen scrambling prior to fragmentation the energyimparted during gas-phase ion transport must be reduced. However, this optimization generally occurs at a cost to ion transmission. In Manuscript I enclosed in this thesis we investigate how an optimum between low hydrogen scrambling and transmission efficiency can be found at minimal cost to transmission efficiency. We also demonstrate the utility of this approach by performing bottom-up HDX-MS/MS using electron transfer dissociation(ETD) on a well-characterized protein.In addition to ion transport, hydrogen scrambling can be induced during the fragmentation process itself. Currently, ETD and electron capture dissociation (ECD) are well established as techniques which do not cause hydrogen scrambling in protonated peptides. More recently, this feature was also demonstrated for 213 nm ultraviolet photodissociation (UVPD) on a unique custom-build instrumental setup. Therefore, in Manuscript II enclosed in the thesis, we set out to replicate these results on the first commercial implementation of UVPD. However, to our surprise, all UVPD generated fragment ions in our commercial setup showed extensive hydrogen scrambling.",

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Hydrogen/deuterium exchange mass spectrometry with high spatial resolution using gas-phase fragmentation

Abstract

Dokumenter og links

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Relaterede publikationer

The changing face of SDS denaturation: Complexes of Thermomyces lanuginosus lipase with SDS at pH 4.0, 6.0 and 8.0

Ultraviolet Photodissociation of Protonated Peptides and Proteins Can Proceed with H/D Scrambling

Avoiding H/D Scrambling with Minimal Ion Transmission Loss for HDX-MS/MS-ETD Analysis on a High-Resolution Q-TOF Mass Spectrometer

Citationsformater