First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study

Iacobucci, Claudio; Piotrowski, Christine; Aebersold, Ruedi; Amaral, B.C. do; Andrews, Philip C.; Bernfur, Katja; Borchers, Christoph H.; Brodie, Nicolas I.; Bruce, James E.; Cao, Yong; Chaignepain, Stéphane; Chavez, Juan D.; Claverol, Stéphane; Cox, Jürgen; Davis, Trisha N.; Degliesposti, Gianluca; Dong, Meng‐Qiu; Edinger, Nufar; Emanuelsson, Cecilia; Gay, Marina; Götze, Michael; Gomes-Neto, Francisco; Gozzo, Fábio C.; Gutierrez, Craig; Haupt, Caroline; Heck, Albert J. R.; Herzog, Franz; Huang, Lan; Hoopmann, Michael R.; Kalisman, Nir; Klykov, Oleg; Kukačka, Zdeněk; Liu, Fan; MacCoss, Michael J.; Mechtler, Karl; Mesika, Ravit; Moritz, Robert L.; Nagaraj, Nagarjuna; Nesati, Victor; Neves-Ferreira, Ana Gisele C.; Ninnis, Robert; Novák, Petr; O’Reilly, Francis J.; Pelzing, Matthias; Petrotchenko, Evgeniy V.; Piersimoni, Lolita; Plasencia, Manolo; Pukala, Tara L.; Rand, Kasper D.; Rappsilber, Juri; Reichmann, Dana; Sailer, Carolin; Sarnowski, Chris P.; Scheltema, Richard A.; Schmidt, Carla; Schriemer, David C.; Shi, Yi; Skehel, J. Mark; Slavin, Moriya; Sobott, Frank; Solis‐Mezarino, Victor; Stephanowitz, Heike; Stengel, Florian; Stieger, Christian E.; Trabjerg, Esben; Trnka, Michael J.; Vilaseca, Marta; Viner, Rosa; Xiang, Yufei; Yılmaz, Şule; Zelter, Alex; Ziemianowicz, Daniel S.; Leitner, Alexander; Sinz, Andrea

doi:10.1021/acs.analchem.9b00658

Cited by 109 publications

(130 citation statements)

References 28 publications

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“…Currently, the most urgent task in the XL-MS community is to harmonize protocols and data reporting formats to ensure obtaining reproducible and reliable results. A recently conducted, community-wide XL-MS harmonization study revealed a great variety in the protocols employed as well as in the outcome, i.e., the number of identified cross-links, between the 32 participating groups [11]. This underlines the need for establishing generally accepted XL-MS protocols as well as common formats for data analysis and reporting results.…”

Section: Discussionmentioning

confidence: 99%

Cross-linking/mass spectrometry at the crossroads

Piersimoni

Sinz

2020

Anal Bioanal Chem

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Cross-linking/mass spectrometry (XL-MS) has come a long way. Originally, XL-MS was used to study relatively small, purified proteins. Meanwhile, it is employed to investigate protein-protein interactions on a proteome-wide level, giving snapshots of cellular processes. Currently, XL-MS is at the intersection of a multitude of workflows and the impact this technique has in addressing specific biological questions is steadily growing. This article is intended to give a bird's-eye view of the current status of XL-MS, the benefits of using MS-cleavable cross-linkers, and the challenges posed in the future development of this powerful technology. We also illustrate how XL-MS can deliver valuable structural insights into protein complexes when used in combination with other structural techniques, such as electron microscopy.

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Section: Discussionmentioning

confidence: 99%

Cross-linking/mass spectrometry at the crossroads

Piersimoni

Sinz

2020

Anal Bioanal Chem

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“…protein sequences and post-translational modifications) are typically not apparent with a technique like cryo-EM but are readily accessible by structural proteomics (3). At the same time, spatial information within and between proteins can be obtained by the use of cross-linking mass spectrometry (XL-MS) (4)(5)(6)(7)(8).…”

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confidence: 99%

Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

Steigenberger

Toorn

Bijl

et al. 2020

Preprint

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Ion mobility separates molecules in the gas-phase on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a time-of-flight mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking mass spectrometry (XL-MS). Cross-linking reagents covalently link amino acids in close proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent -termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500Å 2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50 -70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.

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“…The impact of cross-linker polarity on the final outcome was investigated on a well characterized BSA that was widely used as model protein for several cross-linking studies 20 . In this study, BSA was modified by equimolar mixtures of DSG ("light form") with d6-BS2G ("heavy form") and BS2G ("light form") with d6-DSG ("heavy form").…”

Section: Chemical Cross-linkingmentioning

confidence: 99%

Influence of Cross-Linker Polarity on Selectivity towards Lysine Side Chains

Fiala

Kukačka

Novák

2020

Preprint

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The combination of chemical cross-linking and mass spectrometry is currently a progressive technology for deriving structural information of proteins and protein complexes. In addition, chemical cross-linking is a powerful tool for stabilizing macromolecular complexes for single particle cryo-electron microscopy. Broad pallets of cross-linking chemistry, currently available for the majority of cross-linking experiments, still rely on the amine-reactive N-hydroxysuccinimide esters targeting mainly N-termini and lysine side chains. These cross-linkers are divided into two groups: water soluble and water insoluble; and research teams prefer one or another speculating on the benefits of their choice. However, the effect of cross-linker polarity on the outcome of cross-linking reaction has never been studied. Herein, we use both polar (bis(sulfosuccinimidyl) glutarate) and non-polar (disuccinimidyl glutarate) cross-linkers and systematically investigated the impact of cross-linker hydrophobicity on resulting distance constraints, using bovine serum albumin as a model protein.

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First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study

Cited by 109 publications

References 28 publications

Cross-linking/mass spectrometry at the crossroads

Cross-linking/mass spectrometry at the crossroads

Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

Influence of Cross-Linker Polarity on Selectivity towards Lysine Side Chains

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