Chemical cross‐linking with a diazirine photoactivatable cross‐linker investigated by MALDI‐ and ESI‐MS/MS

Gomes, Alexandre F.; Gozzo, Fábio C.

doi:10.1002/jms.1776

Cited by 68 publications

(51 citation statements)

References 35 publications

(53 reference statements)

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“…On the smaller scale, however, SDA was proved highly effective. For example, Gomes and Gozzo studied cross-linking of model peptides and equine myoglobin using 13.5 Å long, cleavable NHS-diazirine cross-linker, SDAD [93]. As expected, the NHS-diazirine generated higher number of cross-links, compared to DSS.…”

Section: Photo-reactive Groupsmentioning

confidence: 99%

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Large Scale Chemical Cross-linking Mass Spectrometry Perspectives

Zybailov

Glazko

Jaiswal

et al. 2013

J Proteomics Bioinform

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The spectacular heterogeneity of a complex protein mixture from biological samples becomes even more difficult to tackle when one’s attention is shifted towards different protein complex topologies, transient interactions, or localization of PPIs. Meticulous protein-by-protein affinity pull-downs and yeast-two-hybrid screens are the two approaches currently used to decipher proteome-wide interaction networks. Another method is to employ chemical cross-linking, which gives not only identities of interactors, but could also provide information on the sites of interactions and interaction interfaces. Despite significant advances in mass spectrometry instrumentation over the last decade, mapping Protein-Protein Interactions (PPIs) using chemical cross-linking remains time consuming and requires substantial expertise, even in the simplest of systems. While robust methodologies and software exist for the analysis of binary PPIs and also for the single protein structure refinement using cross-linking-derived constraints, undertaking a proteome-wide cross-linking study is highly complex. Difficulties include i) identifying cross-linkers of the right length and selectivity that could capture interactions of interest; ii) enrichment of the cross-linked species; iii) identification and validation of the cross-linked peptides and cross-linked sites. In this review we examine existing literature aimed at the large-scale protein cross-linking and discuss possible paths for improvement. We also discuss short-length cross-linkers of broad specificity such as formaldehyde and diazirine-based photo-cross-linkers. These cross-linkers could potentially capture many types of interactions, without strict requirement for a particular amino-acid to be present at a given protein-protein interface. How these shortlength, broad specificity cross-linkers be applied to proteome-wide studies? We will suggest specific advances in methodology, instrumentation and software that are needed to make such a leap.

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Section: Photo-reactive Groupsmentioning

confidence: 99%

“…Moreover, the existence of predictable fragmentation of the points of carbene insertion as demonstrated [93] can aid in the cross-link identification.…”

Section: Photo-reactive Groupsmentioning

confidence: 99%

Large Scale Chemical Cross-linking Mass Spectrometry Perspectives

Zybailov

Glazko

Jaiswal

et al. 2013

J Proteomics Bioinform

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“…The structure of membranes and associated proteins has also been investigated with diazo-and diazirine-substituted fatty acids [26]. More recently, diazirine precursors have been incorporated into cross-linkers [27,28] and covalent labeling agents [23,29,30]. BCarbene footprinting^has been successfully demonstrated using gaseous diazirine [30], simple aliphatic diazirines [23,31,32], and most recently aromatic diazirine derivatives [29].…”

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confidence: 99%

Amino Acid Insertion Frequencies Arising from Photoproducts Generated Using Aliphatic Diazirines

Ziemianowicz

Bomgarden

Etienne

et al. 2017

J. Am. Soc. Mass Spectrom.

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Abstract. Mapping proteins with chemical reagents and mass spectrometry can generate a measure of accessible surface area, which in turn can be used to support the modeling and refinement of protein structures. Photolytically generated carbenes are a promising class of reagent for this purpose. Substituent effects appear to influence surface mapping properties, allowing for a useful measure of design control. However, to use carbene labeling data in a quantitative manner for modeling activities, we require a better understanding of their inherent amino acid reactivity, so that incorporation data can be normalized. The current study presents an analysis of the amino acid insertion frequency of aliphatic carbenes generated by the photolysis of three different diazirines: 3,3'-azibutyl-1-ammonium, 3,3'-azibutan-1-ol, and 4,4'-azipentan-1-oate. Leveraging an improved photolysis system for single-shot labeling of sub-microliter frozen samples, we used EThCD to localize insertion products in a large population of labeled peptides. Counting statistics were drawn from data-dependent LC-MS 2 experiments and used to estimate the frequencies of insertion as a function of amino acid. We observed labeling of all 20 amino acids over a remarkably narrow range of insertion frequencies. However, the nature of the substituent could influence relative insertion frequencies, within a general preference for larger polar amino acids. We confirm a large (6-fold) increase in labeling yield when carbenes were photogenerated in the solid phase (77 K) relative to the liquid phase (293 K), and we suggest that carbene labeling should always be conducted in the frozen state to avoid information loss in surface mapping experiments.

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“…SDA is a 3.9 Å-short cross-linker, which cross-links free amino-group groups on one protein (via succinimidyl reaction) to any amino-acid on the other protein (via UV-driven decomposition of the diazirine to a reactive carbene). Recently, Gomez et al used the 13.5 Å-long, cleavable NHS-diazirine cross-linker, SDAD to study cross-linking of model peptides and equine myoglobin [22]. As expected, the NHS-diazirine captured more interactions compared to the lysine-to-lysine cross-linker of the similar length.…”

Section: Introductionmentioning

confidence: 98%

Analysis of Protein-protein Interaction Interface between Yeast Mitochondrial Proteins Rim1 and Pif1 Using Chemical Cross-linking Mass Spectrometry

Zybailov¹,

Gokulan²,

Wiese³

2015

J Proteomics Bioinform

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Defining protein-protein contacts is a challenging problem and cross-linking is a promising solution. Here, we present a case of mitochondrial single strand binding protein Rim1 and helicase Pif1, an interaction first observed in immuno-affinity pull-down from yeast cells using Pif1 bait. We found that only the short succinimidyl-diazirine cross-linker or formaldehyde captured the interaction between recombinant Rim1 and Pif1. In addition, Pif1 needed to be stripped of its N-terminal and C-terminal domains, and Rim1's C-terminus needed to be modified for the cross-linked product to become visible. Our report is an example of a non-trivial analysis, where a previously identified stable interaction escapes initial capture with cross-linking agents and requires substantial modification to recombinant proteins and fine-tuning of the mass spectrometry-based methods for the cross-links to become detectable. We used high resolution mass spectrometry to detect the cross-linked peptides. A 1:1 mixture of 15N and 14N-labeled Rim1 was used to validate the cross-links by their mass shift in the LC-MS profiles. Two sites on Rim1 were confirmed: 1) the N-terminus, and 2) the K29 residue. Performing cross-linking with a K29A variant visibly reduced the cross-linked product. Further, K29A-Rim1 showed a five-fold lower affinity to single stranded DNA compared to wild-type Rim1. Both the K29A variant and wild type Rim1 showed similar degrees of stimulation of Pif1 helicase activity. We propose structural models of the Pif1-Rim1 interaction and discuss its functional significance. Our work represents a non-trivial protein-protein interface analysis and demonstrates utility of short and non-specific cross-linkers.

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Chemical cross‐linking with a diazirine photoactivatable cross‐linker investigated by MALDI‐ and ESI‐MS/MS

Cited by 68 publications

References 35 publications

Large Scale Chemical Cross-linking Mass Spectrometry Perspectives

Large Scale Chemical Cross-linking Mass Spectrometry Perspectives

Amino Acid Insertion Frequencies Arising from Photoproducts Generated Using Aliphatic Diazirines

Analysis of Protein-protein Interaction Interface between Yeast Mitochondrial Proteins Rim1 and Pif1 Using Chemical Cross-linking Mass Spectrometry

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