Data-independent acquisition improves quantitative cross-linking mass spectrometry

Quantitative cross-linking mass spectrometry (QCLMS) reveals structural detail on altered protein states in solution. On its way to becoming a routine technology, QCLMS could benefit from data-independent acquisition (DIA), which generally enables greater reproducibility than data-dependent acquisition (DDA) and increased throughput over targeted methods. Therefore, here we introduce DIA to QCLMS by extending a widely used DIA software, Spectronaut, to also accommodate cross-link data. A mixture of seven proteins cross-linked with bis[sulfosuccinimidyl] suberate (BS3) was used to evaluate this workflow. Out of the 414 identified unique residue pairs, 292 (70%) were quantifiable across triplicates with a coefficient of variation (CV) of 10%, with manual correction of peak selection and boundaries for PSMs in the lower quartile of individual CV values. This compares favorably to DDA where we quantified cross-links across triplicates with a CV of 66%, for a single protein. We found DIA-QCLMS to be capable of detecting changing abundances of cross-linked peptides in complex mixtures, despite the ratio compression encountered when increasing sample complexity through the addition of E. coli cell lysate as matrix. In conclusion, the DIA software Spectronaut can now be used in cross-linking and DIA is indeed able to improve QCLMS

Quantitative photo-crosslinking mass spectrometry revealing protein structure response to environmental changes

Protein structures respond to changes in their chemical and physical environment. However, studying such conformational changes is notoriously difficult, as many structural biology techniques are also affected by these parameters. Here, the use of photo-crosslinking, coupled with quantitative crosslinking mass spectrometry (QCLMS), offers an opportunity, since the reactivity of photo-crosslinkers is unaffected by changes in environmental parameters. In this study, we introduce a workflow combining photo-crosslinking using sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA) with our recently developed data-independent acquisition (DIA)-QCLMS. This novel photo-DIA-QCLMS approach is then used to quantify pH-dependent conformational changes in human serum albumin (HSA) and cytochrome C by monitoring crosslink abundances as a function of pH. Both proteins show pH-dependent conformational changes resulting in acidic and alkaline transitions. 93% and 95% of unique residue pairs (URP) were quantifiable across triplicates for HSA and cytochrome C, respectively. Abundance changes of URPs and hence conformational changes of both proteins were visualized using hierarchical clustering. For HSA we distinguished the N–F and the N–B form from the native conformation. In addition, we observed for cytochrome C acidic and basic conformations. In conclusion, our photo-DIA-QCLMS approach distinguished pH-dependent conformers of both proteins

Quantitative Crosslinking Massenspektrometrie : von Entwicklung und Anwendung zu Konformationsänderungen von Proteinen

The dynamics of protein structures are essential for cellular processes but are difficult to monitor by existing technologies. Crosslinking mass spectrometry (CLMS) can provide residue-resolution distance restraints, which may in principle be quantified to obtain unique insights into the structural flexibility of proteins. However, quantitative crosslinking mass spectrometry (QCLMS) needs to be established as a reproducible method before it can develop into a method of choice for studying protein dynamics. This requires the establishment and assessment of experimental workflows. My contributions to the development of QCLMS are presented in this cumulative thesis as four manuscripts: To assess the reproducibility of crosslinking data I first adapted the quantitation software Skyline, which required reformatting crosslinked peptides as linear peptides. Using bis[sulfosuccinimidyl] suberate (BS3)-crosslinked human serum albumin (HSA), I found QCLMS to have a similar reproducibility as general quantitative proteomics. However, in this workflow, quantitation was only possible on precursor level and matching quantitative crosslinked peptide information to residue pair information is error prone. (Müller et al. J. Am. Soc. Mass Spectrom. 2017) To further improve crosslink quantitation and simplify data processing, I switched to data-independent acquisition (DIA) and Spectronaut, as a leading DIA processing software. The DIA-QCLMS workflow improved the reproducibility of QCLMS, as was assessed using a mixture of seven BS3-crosslinked proteins and tolerated even very high sample complexity such as E. coli cell lysate as matrix. (Müller et al. Mol. Cell. Proteomics 2019) In combination with the photoactivatable crosslinker sulfosuccinimidyl 4,4’-azipentanoate (sulfo-SDA), the workflow was then extended to study conformational changes caused by environmental influences, that otherwise affect crosslink reaction activity. The photo-DIA-QCLMS workflow was used to study pH-induced conformation changes in HSA and cytochrome C as model systems and significantly widens the scope of potential scientific applications in quantitative crosslinking. (Müller et al. Anal. Chem. 2019) To make my developments of QCLMS accessible to a broad scientific user base, I prepared a detailed protocol. (Müller et al. submitted) In conclusion, through these developments and applications I have made substantial steps towards the implementation of QCLMS as a routine technology for the analysis of conformational dynamics of proteins and their complexes. Future technical developments in data analysis and detection of crosslinks may allow scaling this towards studying dynamic processes in more complex samples including organelles and whole cells.Die Dynamik von Proteinstrukturen ist für zelluläre Prozesse von wesentlicher Bedeutung, lässt sich jedoch mit vorhanden Technologien nur schwer erfassen. Crosslinking Massenspektrometrie (CLMS) liefert Distanz Informationen innerhalb eines Proteins auf Aminosäuren Ebene. Durch zusätzliche quantitative Experimente erhält man einen einzigartigen Blick in die strukturelle Flexibilität von Proteinen. Die quantitative crosslinking Massenspektrometrie (QCLMS) muss jedoch als reproduzierbare und nützliche Methode erst etabliert werden. Mein Beitrag zu diesem Thema wird in dieser kumulativen Doktorarbeit in vier Manuskripten vorgestellt: Um die Reproduzierbarkeit von crosslinking Daten beurteilen zu können, habe ich zunächst die prozessierung der peptide Daten an die Quantifizierungs-Software Skyline angepasst. Unter Verwendung von Bis[sulfosuccinimidyl] suberat (BS3) vernetztem Human Serum Albumin (HSA) stellte ich fest, dass QCLMS eine ähnliche Reproduzierbarkeit aufweist wie die allgemeine quantitative Proteomik. In diesem Arbeitsablauf war die Quantifizierung jedoch nur auf MS1-Ebene möglich, und die Zuordnung der quantitativen Informationen von vernetzten Peptiden zu nicht redundanten Peptid Paaren war fehleranfällig. (Müller et al. J. Am. Soc. Mass Spectrom. 2017) Um die Quantifizierung der crosslinking Daten weiter zu verbessern und die Datenverarbeitung zu vereinfachen, habe ich sowohl data independent-acquisition (DIA) als auch Spectronaut, eine führende DIA-Verarbeitungssoftware, für den neuen Arbeitsablauf verwendet. Der DIA-QCLMS-Workflow verbesserte die Reproduzierbarkeit von QCLMS, was anhand eines Gemisches aus sieben BS3-vernetzten Proteinen beurteilt wurde, und tolerierte auch komplexe Proben mit E. coli Zelllysat als Matrix. (Müller et al. Mol. Cell. Proteomics 2019) In Kombination mit dem photoaktiven Crosslinker Sulfosuccinimidyl 4,4’-azipentanoate (sulfo-SDA) wurde der Arbeitsablauf erweitert, um Konformationsänderungen von Proteinen durch wechselnde Umweltbedingungen untersuchen zu können. Der (photo)-DIA-QCLMS Arbeitsablauf wurde verwendet, um pH induzierte Konformationsänderungen der Modellsysteme HSA und Cytochrome C zu untersuchen. (Müller et al. Anal. Chem. 2019) Um die Entwicklungen in QCLMS einer breiten wissenschaftlichen Gemeinschaft zugänglich zu machen, habe ich ein detailliertes Protokoll erstellt. (Müller et al. Journal of Proteomics SI: Structural Proteomics 2019 submitted) Zusammenfassend ist festzuhalten, dass ich durch diese Weiterentwicklungen und Anwendungen wesentliche Schritte zur Implementierung von QCLMS als Standard Technologie zur Analyse dynamischer Konformationsänderungen von Proteinen und ihren Komplexen unternommen habe. Zukünftige technische Entwicklungen bei der Datenanalyse und beim Nachweis von Crosslinks könnten die Untersuchungen dynamische Prozesse in komplexeren Proben, einschließlich Organellen und ganzen Zellen, ermöglichen

Chromoplast differentiation in bell pepper (Capsicum annuum\textit {Capsicum annuum}) fruits

We report here a detailed analysis of the proteome adjustments that accompany chromoplast differentiation from chloroplasts during bell pepper ($\textit {Capsicum annuum}$) fruit ripening. While the two photosystems are disassembled and their constituents degraded, the cytochrome b$_6$f complex, the ATPase complex, and Calvin cycle enzymes are maintained at high levels up to fully mature chromoplasts. This is also true for ferredoxin (Fd) and Fd-dependent NADP reductase, suggesting that ferredoxin retains a central role in the chromoplasts’ redox metabolism. There is a significant increase in the amount of enzymes of the typical metabolism of heterotrophic plastids, such as the oxidative pentose phosphate pathway (OPPP) and amino acid and fatty acid biosynthesis. Enzymes of chlorophyll catabolism and carotenoid biosynthesis increase in abundance, supporting the pigment reorganization that goes together with chromoplast differentiation. The majority of plastid encoded proteins decline but constituents of the plastid ribosome and AccD increase in abundance. Furthermore, the amount of plastid terminal oxidase (PTOX) remains unchanged despite a significant increase in phytoene desaturase (PDS) levels, suggesting that the electrons from phytoene desaturation are consumed by another oxidase. This may be a particularity of non-climacteric fruits such as bell pepper that lack a respiratory burst at the onset of fruit ripening