Strategies for Membrane Protein Analysis by Mass Spectrometry

Prados-Rosales, Rafael; Aragoneses-Cazorla, Guillermo; Estévez, Héctor; García-Calvo, Estefanía; Machuca, Andres; Luque-García, José L.

doi:10.1007/978-3-030-15950-4_16

Cited by 5 publications

(3 citation statements)

References 76 publications

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“…sodium channels Na v 1.7, Na v 1.8, and Na v 1.9 known to be involved in several pain states. The reasons can be manifold, ranging from relatively low expression levels to insufficient solubilization or localization in detergent-resistant microdomains-all factors known to render membrane protein analysis challenging (Griffin and Schnitzer, 2011;Prados-Rosales et al, 2019). Nonetheless, our results represent the first dataset on proteome dynamics upon paclitaxel treatment providing unprecedented insights into induced molecular alterations during the development and maintenance of CIPN.…”

Section: Discussionmentioning

confidence: 96%

Proteome and Network Analysis Provides Novel Insights Into Developing and Established Chemotherapy-Induced Peripheral Neuropathy

et al. 2022

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Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating side-effect of cancer therapies. So far, the development of CIPN cannot be prevented, neither can established CIPN be reverted, often leading to the cessation of necessary chemotherapy. Thus, there is an urgent need to explore the mechanistic basis of CIPN to facilitate its treatment. Here we used an integrated approach of quantitative proteome profiling and network analysis in a clinically relevant rat model of paclitaxel-induced peripheral neuropathy. We analysed lumbar rat DRG at two critical time points: (1) day 7, right after cessation of paclitaxel treatment, but prior to neuropathy development (pre-CIPN); (2) 4 weeks after paclitaxel initiation, when neuropathy has developed (peak-CIPN). In this way we identified a differential protein signature, which shows how changes in the proteome correlate with the development and maintenance of CIPN, respectively. Extensive biological pathway and network analysis reveals that, at pre-CIPN, regulated proteins are prominently implicated in mitochondrial (dys)function, immune signalling, neuronal damage/regeneration, and neuronal transcription. Orthogonal validation in an independent rat cohort confirmed the increase of β-catenin (CTNNB1) at pre-CIPN. More importantly, detailed analysis of protein networks associated with β-catenin highlights translationally relevant and potentially druggable targets. Overall, this study demonstrates the enormous value of combining animal behaviour with proteome and network analysis to provide unprecedented insights into the molecular basis of CIPN. In line with emerging approaches of network medicine our results highlight new avenues for developing improved therapeutic options aimed at preventing and treating CIPN.

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Section: Discussionmentioning

confidence: 96%

Proteome and Network Analysis Provides Novel Insights Into Developing and Established Chemotherapy-Induced Peripheral Neuropathy

et al. 2022

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“…In contrast, EED and RACK1 proximity to nSMase2 was lost after 2 minutes of stimulation of cells expressing an enzymatically inactive version ofnSMase2: H639A ( Figures 4D, E ). The lack of identification of the TNFR itself may reflect limited solubility and suboptimal retrieval of transmembrane proteins in conventional MS ( 66 ).…”

Section: Discussionmentioning

confidence: 99%

Dynamic changes in the proximitome of neutral sphingomyelinase-2 (nSMase2) in TNFα stimulated Jurkat cells

Schöl,

Schempp,

Hennig

et al. 2024

Front. Immunol.

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Ceramides generated by the activity of the neutral sphingomyelinase 2 (nSMase2) play a pivotal role in stress responses in mammalian cells. Dysregulation of sphingolipid metabolism has been implicated in numerous inflammation-related pathologies. However, its influence on inflammatory cytokine-induced signaling is yet incompletely understood. Here, we used proximity labeling to explore the plasma membrane proximal protein network of nSMase2 and TNFα-induced changes thereof. We established Jurkat cells stably expressing nSMase2 C-terminally fused to the engineered ascorbate peroxidase 2 (APEX2). Removal of excess biotin phenol substantially improved streptavidin-based affinity purification of biotinylated proteins. Using our optimized protocol, we determined nSMase2-proximal biotinylated proteins and their changes within the first 5 min of TNFα stimulation by quantitative mass spectrometry. We observed significant dynamic changes in the nSMase2 microenvironment in response to TNFα stimulation consistent with rapid remodeling of protein networks. Our data confirmed known nSMase2 interactors and revealed that the recruitment of most proteins depended on nSMase2 enzymatic activity. We measured significant enrichment of proteins related to vesicle-mediated transport, including proteins of recycling endosomes, trans-Golgi network, and exocytic vesicles in the proximitome of enzymatically active nSMase2 within the first minutes of TNFα stimulation. Hence, the nSMase2 proximal network and its TNFα-induced changes provide a valuable resource for further investigations into the involvement of nSMase2 in the early signaling pathways triggered by TNFα.

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“…Biological MS is a key and versatile tool to characterize membrane proteins (Kar et al, 2017;Prados-Rosales et al, 2019). Different types of ionization can be used for this purpose: electrospray (ESI) (Pham et al, 2013), laser-induced liquid bead ion desorption (LILBID) (Hellwig et al, 2018), and matrix-assisted laser desorption ionization (MALDI).…”

Section: Introductionmentioning

confidence: 99%

Sequencing intact membrane proteins using MALDI mass spectrometry

Zhamungui Sánchez,

Hijazi,

Haidar

et al. 2023

Front. Anal. Sci.

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Membrane proteins are key players in many cellular events and represent crucial drug targets. Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) is a valuable approach to investigate them. To our knowledge, there are only a few reports of sequencing small membrane proteins using MALDI in-source decay (ISD). We report the successful fragmentation and sequencing of membrane proteins up to 46 kDa by MALDI-ISD. We have 1) investigated key MALDI parameters that influence the sequencing of a soluble protein; 2) used atomic force microscopy to observe our samples and correlate their topological features with MALDI data, which allowed us to optimize fragmentation conditions; 3) sequenced N- and C-termini of three membrane proteins (SpoIIIAF, TIM23, and NOX), solubilized in three different ways. Our results indicate that detergent and buffer type are of key importance for successful MALDI-ISD sequencing. Our findings are significant because sequencing membrane proteins enables the unique characterization of challenging biomolecules. The resulting fragmentation patterns provide key insights into the identity of proteins, their sequences, modifications, and other crucial information, such as the position of unexpected truncation.

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Strategies for Membrane Protein Analysis by Mass Spectrometry

Cited by 5 publications

References 76 publications

Proteome and Network Analysis Provides Novel Insights Into Developing and Established Chemotherapy-Induced Peripheral Neuropathy

Proteome and Network Analysis Provides Novel Insights Into Developing and Established Chemotherapy-Induced Peripheral Neuropathy

Dynamic changes in the proximitome of neutral sphingomyelinase-2 (nSMase2) in TNFα stimulated Jurkat cells

Sequencing intact membrane proteins using MALDI mass spectrometry

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