Toolkit/top-down mass spectrometry

top-down mass spectrometry

Assay Method·Research·Since 2020

Also known as: top-down MS

Taxonomy: Technique Branch / Method. Workflows sit above the mechanism and technique branches rather than replacing them.

Summary

Top-down mass spectrometry is an assay method for de novo characterization of covalent insulin dimers formed under Fe2+ incubation or UV light stress. In the cited workflow, combined EThcD and 213 nm UVPD enabled identification of cross-link chemical composition and residue-level cross-link sites.

Usefulness & Problems

Why this is useful

This method is useful for structural characterization of covalent protein dimers when both the cross-link chemistry and the residue-level linkage sites must be determined. In the cited insulin application, it resolved cross-link types and chain-to-chain cross-link sites using fragmentation-based top-down analysis.

Source:

The combined utilization of EThcD and 213 nm ultraviolet photodissociation (UVPD) facilitated identification of the chemical composition of the cross-links.

Source:

Identification of cross-link sites between chains at residue level was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.

Source:

we here demonstrate on two case studies of covalent insulin dimers, induced by Fe2+ incubation or ultraviolet (UV) light stress, that de novo characterization in top-down mass spectrometry (MS) workflows can identify cross-link types and sites

Problem solved

It addresses the problem of identifying the chemical nature and exact residue connectivity of covalent insulin dimers generated by Fe2+ incubation or UV light stress. The study specifically showed that MS3 analysis of informative MS2 fragments enabled residue-level site assignment for both dimer types.

Problem links

Need precise spatiotemporal control with light input

Derived

Top-down mass spectrometry is an assay method for de novo characterization of covalent insulin dimers formed under Fe2+ incubation or UV light stress. In the cited workflow, combined EThcD and 213 nm UVPD enabled identification of cross-link chemical composition and residue-level cross-link sites.

Taxonomy & Function

Primary hierarchy

Technique Branch

Method: A concrete measurement method used to characterize an engineered system.

Target processes

No target processes tagged yet.

Input: Light

Implementation Constraints

cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationimplementation constraint: multi component delivery burdenimplementation constraint: spectral hardware requirementoperating role: sensorswitch architecture: cleavageswitch architecture: multi componentswitch architecture: recruitment

The reported workflow used top-down MS with combined EThcD and 213 nm UVPD fragmentation. MS3 analysis of selected MS2 fragments was required for residue-level site assignment in both dimers, although the Fe2+-induced dimer could be localized by UVPD-MS2 without MS3. The analytes were insulin dimers generated by Fe2+ incubation or UV light stress, and interchain disulfide bond cleavage contributed to localization in the MS3 workflow.

MS2 alone was not sufficient for cross-link site identification in the UV light-induced insulin dimer, so deeper fragmentation was required. The evidence is limited to covalent insulin dimers from one study, and broader performance across other proteins, cross-link classes, or sample types was not provided.

Validation

Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication

Supporting Sources

Ranked Claims

Claim 1comparative method performancesupports2020Source 1needs review

UVPD at MS2 provided cross-link site identification for the Fe2+-induced insulin dimer without MS3, but MS2 was not sufficient for cross-link site identification in the UV light-induced dimer.

UVPD provided identification of cross-link sites in the Fe2+-induced dimer without MS3, while cross-link site identification with MS2 was not possible for the UV light-induced dimer.
Claim 2comparative method performancesupports2020Source 1needs review

UVPD at MS2 provided cross-link site identification for the Fe2+-induced insulin dimer without MS3, but MS2 was not sufficient for cross-link site identification in the UV light-induced dimer.

UVPD provided identification of cross-link sites in the Fe2+-induced dimer without MS3, while cross-link site identification with MS2 was not possible for the UV light-induced dimer.
Claim 3comparative method performancesupports2020Source 1needs review

UVPD at MS2 provided cross-link site identification for the Fe2+-induced insulin dimer without MS3, but MS2 was not sufficient for cross-link site identification in the UV light-induced dimer.

UVPD provided identification of cross-link sites in the Fe2+-induced dimer without MS3, while cross-link site identification with MS2 was not possible for the UV light-induced dimer.
Claim 4comparative method performancesupports2020Source 1needs review

UVPD at MS2 provided cross-link site identification for the Fe2+-induced insulin dimer without MS3, but MS2 was not sufficient for cross-link site identification in the UV light-induced dimer.

UVPD provided identification of cross-link sites in the Fe2+-induced dimer without MS3, while cross-link site identification with MS2 was not possible for the UV light-induced dimer.
Claim 5comparative method performancesupports2020Source 1needs review

UVPD at MS2 provided cross-link site identification for the Fe2+-induced insulin dimer without MS3, but MS2 was not sufficient for cross-link site identification in the UV light-induced dimer.

UVPD provided identification of cross-link sites in the Fe2+-induced dimer without MS3, while cross-link site identification with MS2 was not possible for the UV light-induced dimer.
Claim 6comparative method performancesupports2020Source 1needs review

UVPD at MS2 provided cross-link site identification for the Fe2+-induced insulin dimer without MS3, but MS2 was not sufficient for cross-link site identification in the UV light-induced dimer.

UVPD provided identification of cross-link sites in the Fe2+-induced dimer without MS3, while cross-link site identification with MS2 was not possible for the UV light-induced dimer.
Claim 7comparative method performancesupports2020Source 1needs review

UVPD at MS2 provided cross-link site identification for the Fe2+-induced insulin dimer without MS3, but MS2 was not sufficient for cross-link site identification in the UV light-induced dimer.

UVPD provided identification of cross-link sites in the Fe2+-induced dimer without MS3, while cross-link site identification with MS2 was not possible for the UV light-induced dimer.
Claim 8mechanistic explanationsupports2020Source 1needs review

The UV chromophoric side chain of Phe1 was implicated in the Fe2+-induced dimer cross-link, explaining why UVPD-MS2 effectively fragmented the cross-link and nearby backbone bonds.

The UV chromophoric side chain of Phe1 was indicated in the cross-link, explaining why UVPD-MS2 was effective in fragmenting the cross-link and nearby backbone bonds.
Claim 9mechanistic explanationsupports2020Source 1needs review

The UV chromophoric side chain of Phe1 was implicated in the Fe2+-induced dimer cross-link, explaining why UVPD-MS2 effectively fragmented the cross-link and nearby backbone bonds.

The UV chromophoric side chain of Phe1 was indicated in the cross-link, explaining why UVPD-MS2 was effective in fragmenting the cross-link and nearby backbone bonds.
Claim 10mechanistic explanationsupports2020Source 1needs review

The UV chromophoric side chain of Phe1 was implicated in the Fe2+-induced dimer cross-link, explaining why UVPD-MS2 effectively fragmented the cross-link and nearby backbone bonds.

The UV chromophoric side chain of Phe1 was indicated in the cross-link, explaining why UVPD-MS2 was effective in fragmenting the cross-link and nearby backbone bonds.
Claim 11mechanistic explanationsupports2020Source 1needs review

The UV chromophoric side chain of Phe1 was implicated in the Fe2+-induced dimer cross-link, explaining why UVPD-MS2 effectively fragmented the cross-link and nearby backbone bonds.

The UV chromophoric side chain of Phe1 was indicated in the cross-link, explaining why UVPD-MS2 was effective in fragmenting the cross-link and nearby backbone bonds.
Claim 12mechanistic explanationsupports2020Source 1needs review

The UV chromophoric side chain of Phe1 was implicated in the Fe2+-induced dimer cross-link, explaining why UVPD-MS2 effectively fragmented the cross-link and nearby backbone bonds.

The UV chromophoric side chain of Phe1 was indicated in the cross-link, explaining why UVPD-MS2 was effective in fragmenting the cross-link and nearby backbone bonds.
Claim 13mechanistic explanationsupports2020Source 1needs review

The UV chromophoric side chain of Phe1 was implicated in the Fe2+-induced dimer cross-link, explaining why UVPD-MS2 effectively fragmented the cross-link and nearby backbone bonds.

The UV chromophoric side chain of Phe1 was indicated in the cross-link, explaining why UVPD-MS2 was effective in fragmenting the cross-link and nearby backbone bonds.
Claim 14mechanistic explanationsupports2020Source 1needs review

The UV chromophoric side chain of Phe1 was implicated in the Fe2+-induced dimer cross-link, explaining why UVPD-MS2 effectively fragmented the cross-link and nearby backbone bonds.

The UV chromophoric side chain of Phe1 was indicated in the cross-link, explaining why UVPD-MS2 was effective in fragmenting the cross-link and nearby backbone bonds.
Claim 15method capabilitysupports2020Source 1needs review

Combined EThcD and 213 nm UVPD facilitated identification of the chemical composition of cross-links in insulin dimers.

The combined utilization of EThcD and 213 nm ultraviolet photodissociation (UVPD) facilitated identification of the chemical composition of the cross-links.
Claim 16method capabilitysupports2020Source 1needs review

Combined EThcD and 213 nm UVPD facilitated identification of the chemical composition of cross-links in insulin dimers.

The combined utilization of EThcD and 213 nm ultraviolet photodissociation (UVPD) facilitated identification of the chemical composition of the cross-links.
Claim 17method capabilitysupports2020Source 1needs review

Combined EThcD and 213 nm UVPD facilitated identification of the chemical composition of cross-links in insulin dimers.

The combined utilization of EThcD and 213 nm ultraviolet photodissociation (UVPD) facilitated identification of the chemical composition of the cross-links.
Claim 18method capabilitysupports2020Source 1needs review

Combined EThcD and 213 nm UVPD facilitated identification of the chemical composition of cross-links in insulin dimers.

The combined utilization of EThcD and 213 nm ultraviolet photodissociation (UVPD) facilitated identification of the chemical composition of the cross-links.
Claim 19method capabilitysupports2020Source 1needs review

Combined EThcD and 213 nm UVPD facilitated identification of the chemical composition of cross-links in insulin dimers.

The combined utilization of EThcD and 213 nm ultraviolet photodissociation (UVPD) facilitated identification of the chemical composition of the cross-links.
Claim 20method capabilitysupports2020Source 1needs review

Combined EThcD and 213 nm UVPD facilitated identification of the chemical composition of cross-links in insulin dimers.

The combined utilization of EThcD and 213 nm ultraviolet photodissociation (UVPD) facilitated identification of the chemical composition of the cross-links.
Claim 21method capabilitysupports2020Source 1needs review

Combined EThcD and 213 nm UVPD facilitated identification of the chemical composition of cross-links in insulin dimers.

The combined utilization of EThcD and 213 nm ultraviolet photodissociation (UVPD) facilitated identification of the chemical composition of the cross-links.
Claim 22method capabilitysupports2020Source 1needs review

Residue-level identification of cross-link sites between chains was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.

Identification of cross-link sites between chains at residue level was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.
Claim 23method capabilitysupports2020Source 1needs review

Residue-level identification of cross-link sites between chains was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.

Identification of cross-link sites between chains at residue level was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.
Claim 24method capabilitysupports2020Source 1needs review

Residue-level identification of cross-link sites between chains was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.

Identification of cross-link sites between chains at residue level was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.
Claim 25method capabilitysupports2020Source 1needs review

Residue-level identification of cross-link sites between chains was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.

Identification of cross-link sites between chains at residue level was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.
Claim 26method capabilitysupports2020Source 1needs review

Residue-level identification of cross-link sites between chains was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.

Identification of cross-link sites between chains at residue level was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.
Claim 27method capabilitysupports2020Source 1needs review

Residue-level identification of cross-link sites between chains was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.

Identification of cross-link sites between chains at residue level was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.
Claim 28method capabilitysupports2020Source 1needs review

Residue-level identification of cross-link sites between chains was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.

Identification of cross-link sites between chains at residue level was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.
Claim 29method capabilitysupports2020Source 1needs review

Top-down mass spectrometry workflows can identify cross-link types and sites in covalent insulin dimers induced by Fe2+ incubation or UV light stress.

we here demonstrate on two case studies of covalent insulin dimers, induced by Fe2+ incubation or ultraviolet (UV) light stress, that de novo characterization in top-down mass spectrometry (MS) workflows can identify cross-link types and sites
Claim 30method capabilitysupports2020Source 1needs review

Top-down mass spectrometry workflows can identify cross-link types and sites in covalent insulin dimers induced by Fe2+ incubation or UV light stress.

we here demonstrate on two case studies of covalent insulin dimers, induced by Fe2+ incubation or ultraviolet (UV) light stress, that de novo characterization in top-down mass spectrometry (MS) workflows can identify cross-link types and sites
Claim 31method capabilitysupports2020Source 1needs review

Top-down mass spectrometry workflows can identify cross-link types and sites in covalent insulin dimers induced by Fe2+ incubation or UV light stress.

we here demonstrate on two case studies of covalent insulin dimers, induced by Fe2+ incubation or ultraviolet (UV) light stress, that de novo characterization in top-down mass spectrometry (MS) workflows can identify cross-link types and sites
Claim 32method capabilitysupports2020Source 1needs review

Top-down mass spectrometry workflows can identify cross-link types and sites in covalent insulin dimers induced by Fe2+ incubation or UV light stress.

we here demonstrate on two case studies of covalent insulin dimers, induced by Fe2+ incubation or ultraviolet (UV) light stress, that de novo characterization in top-down mass spectrometry (MS) workflows can identify cross-link types and sites
Claim 33method capabilitysupports2020Source 1needs review

Top-down mass spectrometry workflows can identify cross-link types and sites in covalent insulin dimers induced by Fe2+ incubation or UV light stress.

we here demonstrate on two case studies of covalent insulin dimers, induced by Fe2+ incubation or ultraviolet (UV) light stress, that de novo characterization in top-down mass spectrometry (MS) workflows can identify cross-link types and sites
Claim 34method capabilitysupports2020Source 1needs review

Top-down mass spectrometry workflows can identify cross-link types and sites in covalent insulin dimers induced by Fe2+ incubation or UV light stress.

we here demonstrate on two case studies of covalent insulin dimers, induced by Fe2+ incubation or ultraviolet (UV) light stress, that de novo characterization in top-down mass spectrometry (MS) workflows can identify cross-link types and sites
Claim 35method capabilitysupports2020Source 1needs review

Top-down mass spectrometry workflows can identify cross-link types and sites in covalent insulin dimers induced by Fe2+ incubation or UV light stress.

we here demonstrate on two case studies of covalent insulin dimers, induced by Fe2+ incubation or ultraviolet (UV) light stress, that de novo characterization in top-down mass spectrometry (MS) workflows can identify cross-link types and sites
Claim 36method conclusionsupports2020Source 1needs review

HCD, EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.

Our results demonstrated that higher-energy collisional dissociation (HCD), EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.
Claim 37method conclusionsupports2020Source 1needs review

HCD, EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.

Our results demonstrated that higher-energy collisional dissociation (HCD), EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.
Claim 38method conclusionsupports2020Source 1needs review

HCD, EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.

Our results demonstrated that higher-energy collisional dissociation (HCD), EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.
Claim 39method conclusionsupports2020Source 1needs review

HCD, EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.

Our results demonstrated that higher-energy collisional dissociation (HCD), EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.
Claim 40method conclusionsupports2020Source 1needs review

HCD, EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.

Our results demonstrated that higher-energy collisional dissociation (HCD), EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.
Claim 41method conclusionsupports2020Source 1needs review

HCD, EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.

Our results demonstrated that higher-energy collisional dissociation (HCD), EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.
Claim 42method conclusionsupports2020Source 1needs review

HCD, EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.

Our results demonstrated that higher-energy collisional dissociation (HCD), EThcD, and UVPD combined with MS3 were powerful tools for direct de novo characterization of cross-linked insulin dimers.
Claim 43method observationsupports2020Source 1needs review

At the MS2 level, EThcD efficiently cleaved interchain disulfide bonds in insulin dimers to reveal cross-link connectivities between chains.

On the MS2 level, electron-transfer/higher-energy collision dissociation (EThcD) efficiently cleaved the interchain disulfide bonds in the dimers to reveal cross-link connectivities between chains.
Claim 44method observationsupports2020Source 1needs review

At the MS2 level, EThcD efficiently cleaved interchain disulfide bonds in insulin dimers to reveal cross-link connectivities between chains.

On the MS2 level, electron-transfer/higher-energy collision dissociation (EThcD) efficiently cleaved the interchain disulfide bonds in the dimers to reveal cross-link connectivities between chains.
Claim 45method observationsupports2020Source 1needs review

At the MS2 level, EThcD efficiently cleaved interchain disulfide bonds in insulin dimers to reveal cross-link connectivities between chains.

On the MS2 level, electron-transfer/higher-energy collision dissociation (EThcD) efficiently cleaved the interchain disulfide bonds in the dimers to reveal cross-link connectivities between chains.
Claim 46method observationsupports2020Source 1needs review

At the MS2 level, EThcD efficiently cleaved interchain disulfide bonds in insulin dimers to reveal cross-link connectivities between chains.

On the MS2 level, electron-transfer/higher-energy collision dissociation (EThcD) efficiently cleaved the interchain disulfide bonds in the dimers to reveal cross-link connectivities between chains.
Claim 47method observationsupports2020Source 1needs review

At the MS2 level, EThcD efficiently cleaved interchain disulfide bonds in insulin dimers to reveal cross-link connectivities between chains.

On the MS2 level, electron-transfer/higher-energy collision dissociation (EThcD) efficiently cleaved the interchain disulfide bonds in the dimers to reveal cross-link connectivities between chains.
Claim 48method observationsupports2020Source 1needs review

At the MS2 level, EThcD efficiently cleaved interchain disulfide bonds in insulin dimers to reveal cross-link connectivities between chains.

On the MS2 level, electron-transfer/higher-energy collision dissociation (EThcD) efficiently cleaved the interchain disulfide bonds in the dimers to reveal cross-link connectivities between chains.
Claim 49method observationsupports2020Source 1needs review

At the MS2 level, EThcD efficiently cleaved interchain disulfide bonds in insulin dimers to reveal cross-link connectivities between chains.

On the MS2 level, electron-transfer/higher-energy collision dissociation (EThcD) efficiently cleaved the interchain disulfide bonds in the dimers to reveal cross-link connectivities between chains.
Claim 50structural assignmentsupports2020Source 1needs review

In the Fe2+-induced insulin dimer, Phe1 from both B-chains were cross-linked through a -CH2- linkage.

In the Fe2+-induced dimer, Phe1 from both B-chains were cross-linked through a -CH2-.
Claim 51structural assignmentsupports2020Source 1needs review

In the Fe2+-induced insulin dimer, Phe1 from both B-chains were cross-linked through a -CH2- linkage.

In the Fe2+-induced dimer, Phe1 from both B-chains were cross-linked through a -CH2-.
Claim 52structural assignmentsupports2020Source 1needs review

In the Fe2+-induced insulin dimer, Phe1 from both B-chains were cross-linked through a -CH2- linkage.

In the Fe2+-induced dimer, Phe1 from both B-chains were cross-linked through a -CH2-.
Claim 53structural assignmentsupports2020Source 1needs review

In the Fe2+-induced insulin dimer, Phe1 from both B-chains were cross-linked through a -CH2- linkage.

In the Fe2+-induced dimer, Phe1 from both B-chains were cross-linked through a -CH2-.
Claim 54structural assignmentsupports2020Source 1needs review

In the Fe2+-induced insulin dimer, Phe1 from both B-chains were cross-linked through a -CH2- linkage.

In the Fe2+-induced dimer, Phe1 from both B-chains were cross-linked through a -CH2-.
Claim 55structural assignmentsupports2020Source 1needs review

In the Fe2+-induced insulin dimer, Phe1 from both B-chains were cross-linked through a -CH2- linkage.

In the Fe2+-induced dimer, Phe1 from both B-chains were cross-linked through a -CH2-.
Claim 56structural assignmentsupports2020Source 1needs review

In the Fe2+-induced insulin dimer, Phe1 from both B-chains were cross-linked through a -CH2- linkage.

In the Fe2+-induced dimer, Phe1 from both B-chains were cross-linked through a -CH2-.
Claim 57structural assignmentsupports2020Source 1needs review

In the UV light-induced insulin dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain.

in the UV light-induced dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain
Claim 58structural assignmentsupports2020Source 1needs review

In the UV light-induced insulin dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain.

in the UV light-induced dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain
Claim 59structural assignmentsupports2020Source 1needs review

In the UV light-induced insulin dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain.

in the UV light-induced dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain
Claim 60structural assignmentsupports2020Source 1needs review

In the UV light-induced insulin dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain.

in the UV light-induced dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain
Claim 61structural assignmentsupports2020Source 1needs review

In the UV light-induced insulin dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain.

in the UV light-induced dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain
Claim 62structural assignmentsupports2020Source 1needs review

In the UV light-induced insulin dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain.

in the UV light-induced dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain
Claim 63structural assignmentsupports2020Source 1needs review

In the UV light-induced insulin dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain.

in the UV light-induced dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain

Approval Evidence

1 source4 linked approval claimsfirst-pass slug top-down-mass-spectrometry
de novo characterization in top-down mass spectrometry (MS) workflows

Source:

method capabilitysupports

Residue-level identification of cross-link sites between chains was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.

Identification of cross-link sites between chains at residue level was achievable for both dimers with MS3 analysis of MS2 fragments cleaved at the cross-link or additionally the interchain disulfide bonds.

Source:

method capabilitysupports

Top-down mass spectrometry workflows can identify cross-link types and sites in covalent insulin dimers induced by Fe2+ incubation or UV light stress.

we here demonstrate on two case studies of covalent insulin dimers, induced by Fe2+ incubation or ultraviolet (UV) light stress, that de novo characterization in top-down mass spectrometry (MS) workflows can identify cross-link types and sites

Source:

structural assignmentsupports

In the Fe2+-induced insulin dimer, Phe1 from both B-chains were cross-linked through a -CH2- linkage.

In the Fe2+-induced dimer, Phe1 from both B-chains were cross-linked through a -CH2-.

Source:

structural assignmentsupports

In the UV light-induced insulin dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain.

in the UV light-induced dimer, Tyr14 of the A-chain participated in an -O-S- cross-link in which the sulfur was derived either from Cys7 or Cys19 of the B-chain

Source:

Comparisons

Source-backed strengths

Combined EThcD and 213 nm UVPD facilitated identification of cross-link chemical composition in insulin dimers. Residue-level cross-link site identification was achieved for both dimers by MS3 analysis of MS2 fragments cleaved at the cross-link or additionally at interchain disulfide bonds. For the Fe2+-induced dimer, UVPD at MS2 alone was sufficient for cross-link site identification, indicating strong performance in at least one cross-link context.

Source:

UVPD provided identification of cross-link sites in the Fe2+-induced dimer without MS3, while cross-link site identification with MS2 was not possible for the UV light-induced dimer.

top-down mass spectrometry and electron-electron double resonance spectroscopy address a similar problem space.

Shared frame: same top-level item type; shared mechanisms: heterodimerization; same primary input modality: light

top-down mass spectrometry and electron-transfer/higher-energy collision dissociation address a similar problem space.

Shared frame: same top-level item type; shared mechanisms: heterodimerization, photocleavage; same primary input modality: light

Strengths here: looks easier to implement in practice.

top-down mass spectrometry and ultraviolet photodissociation address a similar problem space.

Shared frame: same top-level item type; shared mechanisms: heterodimerization; same primary input modality: light

Ranked Citations

  1. 1.
    StructuralSource 1Journal of the American Society for Mass Spectrometry2020Claim 1Claim 2Claim 3

    Seeded from load plan for claim c6.