A Tag-Based Affinity Purification Mass Spectrometry Workflow for Systematic Isolation of the Human Mitochondrial Protein Complexes

Wu, Zhuoran; Malty, Ramy H.; Moutaoufik, Mohamed Taha; Zhang, Qingzhou; Jessulat, Matthew; Babu, Mohan

doi:10.1007/978-981-13-8367-0_6

Cited by 3 publications

(4 citation statements)

References 30 publications

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“…All control samples were submitted to the same treatment. Mass spectrometry analysis was performed based on previously described (Wu et al, 2019). Samples were reduced with 5 mM TCEP for 30 min, alkylated with 15 mM iodoacetamide for 30 min in the dark, followed by denaturation with 2M Urea and subsequent digestion by overnight incubation with Trypsin Gold 1:20 (w/w) (Promega) at 37 C. C18 packed TopTip (Glygen) was used to desalt tryptic peptides, that were subsequently dried in a SpeedVac vacuum concentrator (SVC 100H, Savant) and resuspended in 0.1% (v/v) formic acid.…”

Section: Mass Photometrymentioning

confidence: 99%

Assembly principles of the human R2TP chaperone complex reveal the presence of R2T and R2P complexes

et al. 2022

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Section: Mass Photometrymentioning

confidence: 99%

Assembly principles of the human R2TP chaperone complex reveal the presence of R2T and R2P complexes

et al. 2022

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“…In contrast, the second category adopts the affinity purification coupled to the mass spectrometry (AP-MS) technique or its close variants. In such methods, a bait-specific antibody or affinity reagent is used to capture a bait protein from cell lysates, with simultaneous purification of its preys in bulk [4]. Titeca et al named these two respective approaches as binary and co-complex technologies [3].…”

Section: Introductionmentioning

confidence: 99%

Recent progress in mass spectrometry-based strategies for elucidating protein–protein interactions

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Syafruddin

Mohtar

et al. 2021

Cell. Mol. Life Sci.

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Protein-protein interactions are fundamental to various aspects of cell biology with many protein complexes participating in numerous fundamental biological processes such as transcription, translation and cell cycle. MS-based proteomics techniques are routinely applied for characterising the interactome, such as affinity purification coupled to mass spectrometry that has been used to selectively enrich and identify interacting partners of a bait protein. In recent years, many orthogonal MS-based techniques and approaches have surfaced including proximity-dependent labelling of neighbouring proteins, chemical cross-linking of two interacting proteins, as well as inferring PPIs from the co-behaviour of proteins such as the co-fractionating profiles and the thermal solubility profiles of proteins. This review discusses the underlying principles, advantages, limitations and experimental considerations of these emerging techniques. In addition, a brief account on how MS-based techniques are used to investigate the structural and functional properties of protein complexes, including their topology, stoichiometry, copy number and dynamics, are discussed. Keywords Affinity purification coupled to mass spectrometry (AP-MS) • Proximity-dependent biotinylation coupled to MS (PDB-MS) • Cross-linking mass spectrometry (XL-MS) • Co-fractionation mass spectrometry (coFrac-MS) • Thermal proximity coaggregation (TPCA)

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“…Current methods for evaluating mitochondria include fluorescence-activated cell sorting (FACS), 6 confocal laser scanning microscopy (CLSM), 7 positron emission tomography (PET), 8 and mass spectrometry (MS). 9 All these methods rely on the accumulation of mitochondrial-specific fluorescent or radioactive lipophilic cations. Optical imaging reagents, such as inorganic nanoparticles, 10 nanodiamonds, 11 polymer particles embedded with dye, 12 and temperature-sensitive polymer particles, 13 are the most commonly used tools for measuring mitochondrial temperature in vitro.…”

mentioning

confidence: 99%

“…Current methods for evaluating mitochondria include fluorescence-activated cell sorting (FACS), confocal laser scanning microscopy (CLSM), positron emission tomography (PET), and mass spectrometry (MS) . All these methods rely on the accumulation of mitochondrial-specific fluorescent or radioactive lipophilic cations.…”

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confidence: 99%

Small-Molecule Fluorogenic Probe for the Detection of Mitochondrial Temperature In Vivo

et al. 2021

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Mitochondria, as energy factories, participate in many metabolic processes and play vital roles in cell life. Most human diseases are caused by mitochondrial dysfunction, and mitochondrial temperature is an important indicator of mitochondrial function. Despite the biological importance of mitochondria, there are few tools for detecting changes in mitochondrial temperature in living organisms. Here, we report on a thermosensitive rhodamine B (RhB)-derived fluorogenic probe (RhBIV) that enables fluorescent labeling of cell mitochondria at concentrations as low as 1 μM. We demonstrate that this probe exhibits a temperature-dependent response in cell mitochondria. Furthermore, in mice, it has a long half-life (t 1/2 ) and is primarily enriched in the liver. This unique thermosensitive probe offers a simple, nondestructive method for longitudinal monitoring of mitochondrial temperature both in vitro and in vivo to elucidate fundamental physiological and pathological processes related to mitochondrial function.

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A Tag-Based Affinity Purification Mass Spectrometry Workflow for Systematic Isolation of the Human Mitochondrial Protein Complexes

Cited by 3 publications

References 30 publications

Assembly principles of the human R2TP chaperone complex reveal the presence of R2T and R2P complexes

Assembly principles of the human R2TP chaperone complex reveal the presence of R2T and R2P complexes

Recent progress in mass spectrometry-based strategies for elucidating protein–protein interactions

Small-Molecule Fluorogenic Probe for the Detection of Mitochondrial Temperature In Vivo

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