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Quantitative Analysis of Samples Labeled with Multiplex iqPIR Cross-Linkers

ABSTRACT: The isobaric quantitative protein interaction reporter (iqPIR) has now been extended to six channels, enabling quantitation of cross-links from six different samples simultaneously. To analyze data for such cross-linkers, the two-channel mode of quantitative analysis was extended to accommodate any number of channels with defined isotope peak offsets. The HeLa 1:1:1:1:1 data was used for the development of the quantitation method of the 6-plex iqPIR.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Hela Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: Helisa Wippel

LAB HEAD: James E. Bruce

PROVIDER: PXD025796 | Pride | 2023-03-11

REPOSITORIES: Pride

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			Action	DRS
	022721_HeLa_6plex_1_1_F10_mango_2rep_1.raw	Raw
	022721_HeLa_6plex_1_1_F10_mango_2rep_2.raw	Raw
	022721_HeLa_6plex_1_1_F11_14_mango_2rep_1.raw	Raw
	022721_HeLa_6plex_1_1_F11_14_mango_2rep_2.raw	Raw
	022721_HeLa_6plex_1_1_F6_7_mango_2rep_1.raw	Raw

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Publications

Multiplexed Cross-Linking with Isobaric Quantitative Protein Interaction Reporter Technology.

Chavez Juan D JD Keller Andrew A Wippel Helisa H HH Mohr Jared P JP Bruce James E JE

Analytical chemistry 20211209 50

Chemical cross-linking with mass spectrometry (XL-MS) has emerged as a useful technique for interrogating protein structures and interactions. When combined with quantitative proteomics strategies, protein conformational and interaction dynamics can be probed. Quantitative XL-MS has been demonstrated with the use of stable isotopes incorporated metabolically or into the cross-linker molecules. Isotope-labeled cross-linkers have primarily utilized deuterium and rely on MS1-based quantitation of p ...[more]

PMID: 34882395

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Multiplexed isobaric quantitative cross-linking reveals drug-induced interactome changes in breast cancer cells

Project description:In vivo application of an extended set of six iqPIR reagents (6-plex iqPIR) allowing multiplexed quantitative interactome analysis of up to six biological samples in a single LC-MS acquisition. Multiplexed iqPIR is demonstrated on MCF-7 breast cancer cells treated with five different Hsp90 inhibitors revealing large scale protein conformational and interaction changes specific to the molecular class of the inhibitors.

2023-03-11 | PXD029350 | Pride

quantitative crosslinking of ADP treated mitochondria isolated from HEK293 cells

Project description:Acute treatment of isolated mitochondria with 1.5 mM ADP. Crosslinking and quantitative comparison to non-treated control using iqPIR technology for quantitative crosslinking.

2022-07-29 | PXD031644 | Pride

Mechanism of pharmacochaperoning in ATP-sensitive potassium channels revealed by cryo-EM

Project description:ATP-sensitive potassium (K-ATP) channels composed of a pore-forming Kir6.2 potassium channel and a regulatory ABC transporter sulfonylurea receptor 1 (SUR1) regulate insulin secretion in pancreatic beta-cells to maintain glucose homeostasis. Mutations that impair channel folding or assembly prevent cell surface expression and cause congenital hyperinsulinism. Structurally diverse K-ATP inhibitors have been shown to act as pharmacochaperones to correct mutant channel expression, but the mechanism is unknown. Here, we compare cryoEM structures of K-ATP channels bound to pharmacochaperones glibenclamide, repaglinide, and carbamazepine. DSP cross-linking mass spectrometry was used to partially confirm cryoEM structures.

2019-11-12 | PXD013873 | Pride

Age induced interactome remodeling in muscle mitochondria

Project description:A novel quantitative crosslinking mass spectrometry technique (qXL-MS) was applied to elucidate interactome changes in aged murine skeletal muscle mitochondria that contribute to age-related mitochondrial functional decline. Reported here are results from initial investigations of murine muscle mitochondrial interactomes that enable identification of statistically significant changes associated with aging. Newly developed isobaric quantitative protein interaction reporter (iqPIR) technologies15 enabled reproducible detection of age-related mitochondrial interactome changes. Muscle mitochondria from young and old mice were isolated, cross-linked with iqPIR molecules, young and old mitochondrial samples were paired, process and analyzed to quantify age-related mitochondrial interactome changes. In parallel, mitochondria were also subjected to additional measurements of mitochondrial protein yield, functional measurements including oxygen consumption rates on Complex I and Complex II substrates, and citrate synthase activity.

2024-05-23 | PXD031643 | Pride

Mechanism of pharmacochaperoning in ATP-sensitive potassium channels revealed by cryo-EM

Project description:ATP-sensitive potassium (K-ATP) channels composed of a pore-forming Kir6.2 potassium channel and a regulatory ABC transporter sulfonylurea receptor 1 (SUR1) regulate insulin secretion in pancreatic beta-cells to maintain glucose homeostasis. Mutations that impair channel folding or assembly prevent cell surface expression and cause congenital hyperinsulinism. Structurally diverse K-ATP inhibitors have been shown to act as pharmacochaperones to correct mutant channel expression, but the mechanism is unknown. Here, we compare cryoEM structures of K-ATP channels bound to pharmacochaperones glibenclamide, repaglinide, and carbamazepine. CyanurBiotinDimercaptoPropionylSuccinimide (CBDPS) cross-linking mass spectrometry was used to partially confirm cryoEM structures.

2019-08-12 | PXD014498 | Pride

Structural basis of human full-length kindlin-3 homotrimer in an auto-inhibited state

Project description:Kindlin-1, -2, and -3, directly bind integrin β cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and link to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin β cytoplasmic tail as the integrin-binding pocket in the F3 sub-domain of one protomer is occluded by the PH domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation. This is also supported by functional assays in which kindlin-3 knockout K562 erythroleukemia cells re-constituted with the mutant kindlin-3 containing trimer-disrupting mutations exhibited an increase in integrin-mediated adhesion and spreading on fibronectin compared to those re-constituted with wild type kindlin-3. Taken together, our findings reveal a novel mechanism of kindlin auto-inhibition that involves its homotrimer formation.

2020-06-08 | PXD019110 | Pride

Nucleotide independant as well as ATP-hydrolysis driven steps of Rea1 Linker remodelling drive the removal of assembly factors from pre-ribosomal particles: PhoX cross-linking of Rea1 in presence of ATPgS, AMP-PNP or Apo state.

Project description:The ribosome maturation factor Rea1 (or Midasin) catalyses the removal of assembly factors from large ribosomal subunit precursors and to promotes their export from the nucleus to the cytosol. Rea1 consists of nearly 5000 amino-acid residues and belongs to the AAA+ protein family. It consists of a ring of six AAA+ domains from which the ≈ 1700 amino-acid residue linker emerges that is subdivided into stem, middle and top domains. A flexible and unstructured D/E rich region connects the linker top to a MIDAS (metal ion dependent adhesion site) domain, which is able to bind the assembly factor substrates. Despite its key importance for ribosome maturation, the Rea1 mechanism driving assembly factor removal by Rea1 is still poorly understood. Here we demonstrate that the Rea1 linker 30 is essential for assembly factor removal. It rotates and swings towards the AAA+ ring following a complex remodelling scheme involving nucleotide independent as well as nucleotide dependent steps. ATP-hydrolysis is required to engage the linker with the AAA+ ring and ultimately with the AAA+ ring docked MIDAS domain. The interaction between the linker top and the MIDAS domain allows direct force transmission for assembly factor removal. To evaluate the conformational changes and spatial proximities in presence or absence of nucleotides we carried out XL-MS experiments using PhoX on purified Rea1 in presence of ATPgS, AMP-PNP or in Apo state (in XL triplicates each).

2024-07-29 | PXD053636 | Pride

Isobaric crosslinking mass spectrometry technology for studying conformational and structural changes in proteins and complexes

Project description:Dynamic conformational and structural changes in proteins and protein complexes play a central and ubiquitous role in the regulation of protein function, yet it is very challenging to study these changes, especially for large protein complexes, under physiological conditions. Here we introduce a novel isobaric crosslinker, Qlinker, for studying conformational and structural changes in proteins and protein complexes using quantitative crosslinking mass spectrometry (qCLMS). Qlinkers are small and simple, amine-reactive molecules with an optimal extended distance of ~10 Å which use MS2 reporter ions for relative quantification of Qlinker-modified peptides derived from different samples. We synthesized the 2-plex Q2linker and showed that the Q2linker can provide quantitative crosslinking data that pinpoints key conformational and structural changes in biosensors, binary and ternary complexes composed of the general transcription factors TBP, TFIIA, and TFIIB, and RNA polymerase II (pol II) complexes.

2024-11-13 | PXD056825 | Pride

Dynamics of different mGBP as revealed by computational and experimental methods

Project description:Guanylate binding proteins (GBP) belong to the superfamily of dynamin proteins. Those might assemble into larger structures upon initial dimerization. We analyzed monomeric and dimerization of mGBP7 via molecular dynamic simulations and compared the developed models with data from crosslinking mass spectrometry experiments.

2024-01-26 | PXD026979 | Pride

Identification of proteins associated with Kv1.2 potassium channels.

Project description:Kv1.2 is a prominent voltage-gated potassium channel that influences action potential generation and propagation in the central nervous system. We explored multi-protein complexes containing Kv1.2 using mass spectrometry of immunoprecipitated samples. Proteins identified as candidate interactors were prioritized based on abundance, and were screened for functional effects on Kv1.2 channels using electrophysiology.

2018-11-04 | PXD011010 | Pride

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