Mass Spectrometry-Based Protein Complex Profiling in Time and Space

Zheng, Jiangnan; Xiong, Chen; Yang, Yun; Tan, Chris Soon Heng; Tian, Ruijun

doi:10.1021/acs.analchem.0c04332

Cited by 20 publications

(15 citation statements)

References 243 publications

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“…2 Thus, efficiently characterizing the protein−protein interactions (PPIs) and dynamic conformations of protein complexes in living cells is essential for understanding the functional mechanism of various biological processes. 3,4 Recently, various technologies have been developed to realize the study of PPIs and protein structures in the cellular content, such as yeast two-hybrid screening, 5 fluorescence resonance energy transfer, 6 enzyme-mediated proximity labeling, 7 and so forth. These methods show superior advantage to the traditional affinity purification mass spectrometry in capturing the weakly/ transiently interacting proteins in living cells.…”

Section: ■ Introductionmentioning

confidence: 99%

In-Depth In Vivo Crosslinking in Minutes by a Compact, Membrane-Permeable, and Alkynyl-Enrichable Crosslinker

et al. 2022

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Chemical crosslinking coupled with mass spectrometry (CXMS) has emerged as a powerful technique to obtain the dynamic conformations and interaction interfaces of protein complexes. Limited by the poor cell membrane permeability, chemical reactivity, and biocompatibility of crosslinkers, in vivo crosslinking to capture the dynamics of protein complexes with finer temporal resolution and higher coverage is attractive but challenging. In this work, a trifunctional crosslinker bis(succinimidyl) with propargyl tag (BSP), involving compact size, proper amphipathy, and enrichment capacity, was developed to enable better cell membrane permeability and efficient crosslinking in 5 min without obvious cellular interference. Followed by a two-step enrichment method based on click chemistry at the peptide level, 13,098 crosslinked peptides (5068 inter-crosslinked peptides and 8030 intra-crosslinked peptides) were identified under the data threshold of peptide-spectrum matches (PSMs) ≥2 on the basic of the FDR control of 1%, which was the most comprehensive dataset for homo species cells by a non-cleavable crosslinker. Besides, the interactome network comprising 1519 proteins connected by 2913 interaction edges in various intracellular compartments, as well as 80S ribosome structural dynamics, were characterized, showing the great potential of our in vivo crosslinking approach in minutes. All these results demonstrated that our developed BSP could provide a valuable toolkit for the in-depth in vivo analysis of protein–protein interactions (PPIs) and protein architectures with finer temporal resolution.

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Section: ■ Introductionmentioning

confidence: 99%

In-Depth In Vivo Crosslinking in Minutes by a Compact, Membrane-Permeable, and Alkynyl-Enrichable Crosslinker

et al. 2022

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“…The use of this protein as a drug is still an extremely common procedure in improving the quality of life for patients with diabetes. The questions of searching for chromatographic methods for specific isolation and determination are still important. − Thin-film materials containing lanthanide atoms, as an example of nanotechnology, are actively used in many fields. − Also, materials containing lanthanide atoms are being actively studied as materials for bioorganic sensors, − including for determining the activity of insulin and analytical applications in the analysis of insulin content in complex systems. ,− Taking into account all of the problems associated with the production and application of such materials for proteomics, the purpose of this work is to demonstrate the possibility of using films containing lanthanide ions for insulin adsorption.…”

Section: Introductionmentioning

confidence: 99%

Thin Films of Lanthanide Stearates as Modifiers of the Q-Sense Device Sensor for Studying Insulin Adsorption

et al. 2022

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This article presents new possibilities of using thin films of lanthanide stearates as sorbent materials. Modification of the Q-sense device resonator with monolayers of lanthanide stearates by the Langmuir–Schaeffer method made it possible to study the process of insulin protein adsorption on the surface of new thin-film sorbents. The resulting films were also characterized by compression isotherms, chemical analysis, scanning electron microscopy, and mass spectrometry. The transition of stearic acid to salt was recorded by IR spectroscopy. Using the LDI MS method, the main component of thin films, lanthanide distearate, was established. The presence of Eu 2+ in thin films was revealed. In the case of europium stearate, the maximum value of insulin adsorption was obtained, −1.67·10 −10 mole/cm 2 . The findings suggest the possibility of using thin films of lanthanide stearates as a sorption material for the proteomics determination of the quantitative protein content in complex fluid systems by specific adsorption on modified surfaces and isolation of such proteins from complex mixtures.

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“…1 Among various analytical methods of choice, affinity purification coupled to mass spectrometry (AP-MS) is a classic but popular method for characterizing protein complex components and association dynamics. 2,3 The AP-MS workflow typically starts with enrichment of interaction partners using synthetic peptide motifs or epitope-tagged proteins as a bait followed by mild washing, on-bead digestion, elution, desalting, liquid chromatography (LC)−MS/MS analysis, and data interpretation. In the last two decades, AP-MS has demonstrated its powerfulness in numerous protein−protein interaction (PPI) profiling works for studying single to thousands of bait proteins.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Deciphering the architecture of protein interaction networks is therefore important for comprehensively understanding the biological processes mediated by protein machinery . Among various analytical methods of choice, affinity purification coupled to mass spectrometry (AP-MS) is a classic but popular method for characterizing protein complex components and association dynamics. , The AP-MS workflow typically starts with enrichment of interaction partners using synthetic peptide motifs or epitope-tagged proteins as a bait followed by mild washing, on-bead digestion, elution, desalting, liquid chromatography (LC)–MS/MS analysis, and data interpretation. In the last two decades, AP-MS has demonstrated its powerfulness in numerous protein–protein interaction (PPI) profiling works for studying single to thousands of bait proteins. , However, the conventional AP-MS protocol is not sensitive enough when starting materials are limited (e.g., clinical samples) or expensive protein affinity ligands are required.…”

Section: Introductionmentioning

confidence: 99%

Fully Integrated and Multiplexed Sample Preparation Technology for Sensitive Interactome Profiling

Mao

Chen

et al. 2021

Anal. Chem.

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Affinity purification coupled to mass spectrometry (AP-MS) is a popular approach for deciphering the architecture of protein interaction networks. Protein lysates (100 μg) are typically required for multistep sample processing in large volumes, which often causes sample loss and reduces the MS analysis sensitivity. Herein, we reported a fully integrated spintip-based AP-MS technology, termed FISAP, for multiplexed and sensitive interactome profiling. The FISAP device can be easily employed for routine use by introducing AP beads into a C18 StageTip. Taking advantage of the switchable functionalization of the C18 matrix by sodium dodecyl sulfate, all the sample preparation steps encompassing peptide or antibody-based AP, reduction, alkylation, tryptic digestion, tandem mass tag (TMT) labeling, and desalting can be performed in a single tip with a benchtop centrifuge in 4 h. Using a biotinylated tyrosine phosphorylated (pTyr) peptide as an affinity ligand, we mapped the pTyr-dependent interactome of the pY191 motif on the immune receptor CD28 cytoplasmic domain. When processing 50 μg of protein lysates, FISAP showed a comparable interactome identification performance but better quantification performance and lower background interference compared to the traditional tube-based method. Furthermore, a costeffective on-column TMT labeling protocol was established and integrated into the FISAP pipeline with increased sensitivity. Compared to the tube-based method, the usage of a synthetic peptide probe and a TMT reagent was both reduced by 20 times. As low as 1 μg of protein lysates could be applied for interactome profiling. Finally, we expanded the applicability of the FISAP technology to epitope tag-based AP-MS for profiling the ILK/PINCH/Parvin complex using 100 times less protein lysate than a previous report. Collectively, FISAP is an easy-to-use and sensitive technology for quantitatively profiling protein complexes when the starting material and affinity reagent are the limitation, especially for applications in biomedical research and chemical biology.

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Mass Spectrometry-Based Protein Complex Profiling in Time and Space

Cited by 20 publications

References 243 publications

In-Depth In Vivo Crosslinking in Minutes by a Compact, Membrane-Permeable, and Alkynyl-Enrichable Crosslinker

In-Depth In Vivo Crosslinking in Minutes by a Compact, Membrane-Permeable, and Alkynyl-Enrichable Crosslinker

Thin Films of Lanthanide Stearates as Modifiers of the Q-Sense Device Sensor for Studying Insulin Adsorption

Fully Integrated and Multiplexed Sample Preparation Technology for Sensitive Interactome Profiling

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