Cross-linking and other structural proteomics techniques: how chemistry is enabling mass spectrometry applications in structural biology

Leitner, Alexander

doi:10.1039/c5sc04196a

Cited by 44 publications

(27 citation statements)

References 123 publications

(125 reference statements)

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“…[5] Hydrogen deuterium exchange is a reversible and nearly universal approach to footprinting [6] but requires fast workup to minimize back exchange. Complementary are chemical reagents that irreversibly react with specific amino-acid side chains, [7] but reactions are slow and not general. Reactive-species footprinting (prototype is •OH) provides general and fast reactions.…”

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confidence: 99%

Laser‐Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass‐Spectrometry‐Based Protein Footprinting

et al. 2017

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We describe a novel, laser-initiated radical trifluoromethylation for protein footprinting and establish its broad residue coverage. •CF3 reacts with 18 of 20 common amino acids including Gly, Ala, Ser, Thr, Asp, Glu that are relatively “silent” with •OH. This new approach to footprinting is a bridge between trifluoromethylation in materials and medicinal chemistry and structural biology and biotechnology. Its application to a membrane protein and to myoglobin show that the approach is sensitive to protein conformational change and solvent accessibility.

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confidence: 99%

Laser‐Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass‐Spectrometry‐Based Protein Footprinting

et al. 2017

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“…The mean separation found for chemical cross-linking is a bit larger than that from X-ray analysis, but observed cross-links were consistent with the published X-ray results. 72–79 The use of cross-linking in structural studies has been the subject of several papers. 80–82 Nuclear magnetic resonance (NMR) is also used to ascertain solution structures for smaller proteins; however, in many cases, loop regions and the C- and N-terminal regions seem to be more flexible as was reported for truncated mouse apoA-I.…”

Section: Discussionmentioning

confidence: 99%

High-Density Lipoprotein Biogenesis: Defining the Domains Involved in Human Apolipoprotein A-I Lipidation

et al. 2016

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The first step in removing cholesterol from a cell is the ATP-binding cassette transporter 1 (ABCA1)-driven transfer of cholesterol to lipid-free or lipid-poor apolipoprotein A-I (apoA-I), which yields cholesterol-rich nascent high-density lipoprotein (nHDL) that then matures in plasma to spherical, cholesteryl ester-rich HDL. However, lipid-free apoA-I has a three-dimensional (3D) conformation that is significantly different from that of lipidated apoA-I on nHDL. By comparing the lipid-free apoA-I 3D conformation of apoA-I to that of 9–14 nm diameter nHDL, we formulated the hypothetical helical domain transitions that might drive particle formation. To test the hypothesis, ten apoA-I mutants were prepared that contained two strategically placed cysteines several of which could form intramolecular disulfide bonds and others that could not form these bonds. Mass spectrometry was used to identify amino acid sequence and intramolecular disulfide bond formation. Recombinant HDL (rHDL) formation was assessed with this group of apoA-I mutants. ABCA1-driven nHDL formation was measured in four mutants and wild-type apoA-I. The mutants contained cysteine substitutions in one of three regions: the N-terminus, amino acids 34 and 55 (E34C to S55C), central domain amino acids 104 and 162 (F104C to H162C), and the C-terminus, amino acids 200 and 233 (L200C to L233C). Mutants were studied in the locked form, with an intramolecular disulfide bond present, or unlocked form, with the cysteine thiol blocked by alkylation. Only small amounts of rHDL or nHDL were formed upon locking the central domain. We conclude that both the N- and C-terminal ends assist in the initial steps in lipid acquisition, but that opening of the central domain was essential for particle formation.

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“…Generally, the choice and length of chemical cross-linkers used will impact the quality and the relevance of data produced. The optimal choice of reactive functional groups and the length of cross-linkers varies based on the objectives of a given study [24,25]. The optimal DSP concentration is cell line- and collagen expression-level dependent and therefore must be experimentally optimized to maximally immunoprecipitate genuine interactors while minimizing non-specific interactions.…”

Section: Notesmentioning

confidence: 99%

Mass Spectrometry-Based Proteomics to Define Intracellular Collagen Interactomes

Doan

DiChiara

Rosario

et al. 2019

Methods in Molecular Biology

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We present the development, optimization, and application of constructs, cell lines, covalent crosslinking methods, and immunoprecipitation strategies that enable robust and accurate determination of collagen interactomes via mass spectrometry-based proteomics. Using collagen type-I as an example, protocols for working with large, repetitive, and GC-rich collagen genes are described, followed by strategies for engineering cells that stably and inducibly express antibody epitope-tagged collagen-I. Detailed steps to optimize collagen interactome crosslinking and perform immunoprecipitations are then presented. We conclude with a discussion of methods to elute collagen interactomes and prepare samples for mass spectrometry-mediated identification of interactors. Throughout, caveats and potential problems researchers may encounter when working with collagen are discussed. We note that the protocols presented herein may be readily adapted to define interactomes of other collagen types, as well as to determine comparative interactomes of normal and disease-causing collagen variants using quantitative isotopic labeling (SILAC)- or isobaric mass tags (iTRAQ or TMT)-based mass spectrometry analysis.

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Cross-linking and other structural proteomics techniques: how chemistry is enabling mass spectrometry applications in structural biology

Abstract: In this perspective, I highlight the contribution of chemical methods to the field of structural proteomics, where mass spectrometry is used to probe the structures of proteins and higher-order protein assemblies.

Cited by 44 publications

References 123 publications

Laser‐Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass‐Spectrometry‐Based Protein Footprinting

Laser‐Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass‐Spectrometry‐Based Protein Footprinting

High-Density Lipoprotein Biogenesis: Defining the Domains Involved in Human Apolipoprotein A-I Lipidation

Mass Spectrometry-Based Proteomics to Define Intracellular Collagen Interactomes

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