Quantitative crosslinking mass spectrometry

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.

Author dc:creator

• Müller, Fränze

Advisor dc:contributor.advisor

• Rappsilber, Juri

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en

Identifier URI

http://dx.doi.org/10.14279/depositonce-9814

OAI identifier oai:identifier

oai:depositonce.tu-berlin.de:11303/10921