A Membrane‐Permeable and Immobilized Metal Affinity Chromatography (IMAC) Enrichable Cross‐Linking Reagent to Advance In Vivo Cross‐Linking Mass Spectrometry

Jiang, Pin-Lian; Diehl, Anne; Viner, Rosa; Etienne, Chris; Nandhikonda, Premchendar; Foster, Leigh; Bomgarden, Ryan; Liu, Fan

doi:10.1002/ange.202113937

Cited by 6 publications

(9 citation statements)

References 15 publications

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“…We recently described a sample processing workflow, in particular a two-step Immobilized Metal Affinity Chromatography (IMAC) enrichment strategy for interactome profiling of intact cells based on the enrichable cross-linker tert -butyl-PhoX (tBu-PhoX) 17 . In this study, we further augment the tBu-PhoX XL-MS pipeline from the data acquisition perspective by integrating RTLS to advance cross-link detection.…”

Section: Introductionmentioning

confidence: 99%

Real-time library search increases cross-link identification depth across all levels of sample complexity

Ruwolt

Lima

et al. 2022

Preprint

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Cross-linking mass spectrometry (XL-MS) is a universal tool for probing structural dynamics and protein-protein interactions in vitro and in vivo. Although cross-linked peptides are naturally less abundant than their unlinked counterparts, recent experimental advances improved cross-link identification by enriching the cross-linker modified peptides chemically with the use of enrichable cross-linkers. However, mono-links (i.e., peptides modified with a hydrolyzed cross-linker) still hinder efficient cross-link identification since a large proportion of measurement time is spent on their MS2 acquisition. Currently, cross-links and mono-links cannot be separated by sample preparation techniques or chromatography because they are chemically almost identical. Here, we found that based on the intensity ratios of four diagnostic peaks when using PhoX/tBu-PhoX cross-linkers, cross-links and mono-links can be partially distinguished. Harnessing their characteristic intensity ratios for real-time library search (RTLS)-based triggering of high-resolution MS2 scans increased the number of cross-link identifications from both single protein samples and intact E. coli cells. Specifically, RTLS improves cross-link identification from unenriched samples and short gradients, emphasizing its advantages in high-throughput approaches and when instrument time or sample amount is limited.

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Section: Introductionmentioning

confidence: 99%

Real-time library search increases cross-link identification depth across all levels of sample complexity

Ruwolt

Lima

et al. 2022

Preprint

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“…In contrast, subsequent cellular and animal experiments can be conducted to understand whether such an interaction is functionally important in physiological conditions. Although some researchers have developed in vivo cross-linking probes to directly cross-link at the cellular level and identify protein interactions closer to physiological conditions, − they also have the problems at the expense of the number of interprotein cross-links in low-abundance subcellular compartments (Figure S6). APEX-CXMS may be complementary to the in vivo cross-linking.…”

Section: Discussionmentioning

confidence: 99%

Subcellular Interactomes Revealed by Merging APEX with Cross-Linking Mass Spectrometry

et al. 2022

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Subcellular protein−protein interactions (PPIs) are essential to understanding the mechanism of diverse cellular signaling events and the pathogenesis of diseases. Herein, we report an integrated APEX proximity labeling and chemical crosslinking coupled with mass spectrometry (CXMS) platform named APEX-CXMS for spatially resolved subcellular interactome profiling in a high-throughput manner. APEX proximity labeling rapidly captures subcellular proteomes, and the highly reactive chemical cross-linkers can capture weak and dynamic interactions globally without extra genetic manipulation. APEX-CXMS was first applied to mitochondria and identified 653 pairs of interprotein cross-links. Six pairs of new interactions were selected and verified by coimmunoprecipitation, the mammalian two-hybrid system, and surface plasmon resonance method. Besides, our approach was further applied to the nucleus, capturing 336 pairs of interprotein cross-links with approximately 94% nuclear specificity. APEX-CXMS thus provides a simple, fast, and general alternative to map diverse subcellular PPIs.

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“…Therefore, CLASP can readily provide detailed localization predictions for any purifiable organelles but would likely be less powerful when applied to intact cells, since proteome coverage of most in-cell XL-MS workflows is currently still limited. However, with recent technological advancements of XL-MS, identification of tens of thousands of cross-links in intact cells is in reach (Jiang et al ., 2021; Wheat et al ., 2021) and CLASP paves the way to use these data for elucidating protein localizations across the cell.…”

Section: Discussionmentioning

confidence: 99%

“…4 nm and currently limited to cell surface proteins (Geri et al , 2020)). However, even though we and others have developed methods for proteome-wide XL-MS (Liu & Heck, 2015; Mendes et al , 2019; O’Reilly & Rappsilber, 2018) and have shown that these approaches can capture large parts of the proteome in intact cells and organelles (Fasci et al , 2018; Gonzalez-Lozano et al , 2020; Jiang et al , 2021; Liu et al , 2018; Schweppe et al , 2017; Wheat et al , 2021; Wittig et al , 2021), cross-linking has so far only been used to analyze protein structures and interactions.…”

Section: Introductionmentioning

confidence: 99%

Cross-link assisted spatial proteomics to map sub-organelle proteomes and membrane protein topology

Zhu

Akkaya

Lima

et al. 2022

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The specific functions of cellular organelles and sub-compartments depend on their protein content, which can be characterized by spatial proteomics approaches. However, many spatial proteomics methods are limited in their ability to resolve organellar sub-compartments, profile multiple sub-compartments in parallel, and/or characterize membrane-associated proteomes. Here, we develop a cross-link assisted spatial proteomics (CLASP) strategy that addresses these shortcomings. Using human mitochondria as a model system, we show that CLASP can elucidate spatial proteomes of all mitochondrial sub-compartments and provide topological insight into the mitochondrial membrane proteome in a single experiment. Biochemical and imaging-based follow-up studies demonstrate that CLASP allows discovering mitochondria-associated proteins and revising previous protein sub-compartment localization and membrane topology data. This study extends the scope of cross-linking mass spectrometry beyond protein structure and interaction analysis towards spatial proteomics, establishes a method for concomitant profiling of sub-organelle and membrane proteomes, and provides a resource for mitochondrial spatial biology.

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A Membrane‐Permeable and Immobilized Metal Affinity Chromatography (IMAC) Enrichable Cross‐Linking Reagent to Advance In Vivo Cross‐Linking Mass Spectrometry

Cited by 6 publications

References 15 publications

Real-time library search increases cross-link identification depth across all levels of sample complexity

Real-time library search increases cross-link identification depth across all levels of sample complexity

Subcellular Interactomes Revealed by Merging APEX with Cross-Linking Mass Spectrometry

Cross-link assisted spatial proteomics to map sub-organelle proteomes and membrane protein topology

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