Investigating Phosphorylation Patterns of the Ion Channel TRPM7 Using Multiple Extraction and Enrichment Techniques Reveals New Phosphosites

Nguyen, Thu T A; Park, Thomas J.; Gong, Liang; Cologna, Stephanie M.

doi:10.1007/s13361-019-02223-5

Cited by 10 publications

(8 citation statements)

References 38 publications

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“…The phosphorylated residues of TRPM7, CNNM3 and CNNM4 identified by MS in the present study are outlined in conjunction with previously published data (62)(63)(64)(65).…”

Section: Excel File Contains One Workheetsupporting

confidence: 75%

The molecular appearance of native TRPM7 channel complexes identified by high-resolution proteomics

Kollewe

Chubanov²,

Tseung³

et al. 2021

Preprint

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The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed membrane protein consisting of ion channel and protein kinase domains. TRPM7 plays a fundamental role in the cellular uptake of divalent cations such as Zn2+, Mg2+ and Ca2+, and thus shapes cellular excitability, plasticity and metabolic activity. The molecular appearance and operation of TRPM7 channel complexes in native tissues have remained unresolved. Here, we investigated the subunit composition of endogenous TRPM7 channels in rodent brain by multi-epitope affinity purification and high-resolution quantitative MS analysis. We found that native TRPM7 channels are high molecular-weight multi-protein complexes that contain the putative metal transporter proteins CNNM1-4 and a small G-protein ARL15. Heterologous reconstitution experiments confirmed the formation of TRPM7/CNNM/ARL15 ternary complexes and indicated that ARL15 effectively and specifically impacts TRPM7 channel activity. These results open up new avenues towards a mechanistic understanding of the cellular regulation and function of TRPM7 channels.Impact StatementHigh-resolution proteomics in conjunction with biochemical and electrophysiological experiments revealed that the channel-kinase TRPM7 in the rodent brain forms macromolecular complexes containing the metal transporters CNNM1-4 and a small G protein ARL15.

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“…The phosphorylated residues of TRPM7, CNNM3 and CNNM4 identified by MS in the present study are outlined in conjunction with previously published data (62)(63)(64)(65).…”

Section: Excel File Contains One Workheetsupporting

confidence: 75%

The molecular appearance of native TRPM7 channel complexes identified by high-resolution proteomics

Kollewe

Chubanov²,

Tseung³

et al. 2021

Preprint

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“…Given the enhanced extraction efficiency of the S-Trap method, this method can be advantageous to various types of biological materials, including formalin-fixed paraffin-embedded (FFPE) samples and transmembrane proteins. , Moreover, the optimized S-Trap methodology may be used as a vital part of a quick workflow to investigate molecular signaling in cancer cells for clinical proteomics . Looking forward, the S-Trap method could facilitate the preparation for other types of PTMs, including glycosylation and methylation.…”

Section: Discussionmentioning

confidence: 99%

Optimized Suspension Trapping Method for Phosphoproteomics Sample Preparation

et al. 2023

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A successful mass spectrometry-based phosphoproteomics analysis relies on effective sample preparation strategies. Suspension trapping (S-Trap) is a novel, rapid, and universal method of sample preparation that is increasingly applied in bottom-up proteomics studies. However, the performance of the S-Trap protocol for phosphoproteomics studies is unclear. In the existing S-Trap protocol, the addition of phosphoric acid (PA) and methanol buffer creates a fine protein suspension to capture proteins on a filter and is a critical step for subsequent protein digestion. Herein, we demonstrate that this addition of PA is detrimental to downstream phosphopeptide enrichment, rendering the standard S-Trap protocol suboptimal for phosphoproteomics. In this study, the performance of the S-Trap digestion for proteomics and phosphoproteomics is systematically evaluated in largescale and small-scale samples. The results of this comparative analysis show that an optimized S-Trap approach, where trifluoroacetic acid is substituted for PA, is a simple and effective method to prepare samples for phosphoproteomics. Our optimized S-Trap protocol is applied to extracellular vesicles to demonstrate superior sample preparation workflow for low-abundance, membrane-rich samples.

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“…In a previous study, S‐Trap and a method known as surfactant and chaotropic agent‐assisted sequential extraction/on‐pellet digestion were combined with titanium dioxide and ferric nitrilotriacetate (Fe‐NTA) phospho‐enrichment to identify the phosphorylation pattern of transient receptor potential cation channel subfamily M member 7 (TRPM7) in transfected HEK293 cells [69]. The combined method detected 22% ± 6% amino acid sequence coverage of TRPM7 (mean ± standard deviation of three runs).…”

Section: Applications Of Suspension Trapping In Recent Proteomics Stu...mentioning

confidence: 99%

A review of suspension trapping digestion method in bottom‐up proteomics

Duong

Park

Lee

2022

J of Separation Science

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The standard bottom‐up proteomic workflow is comprised of sample preparation, data acquisition, and data analysis. While the latter two parts have made considerable advances in the last decade, sample preparation has remained an important challenge within the workflow due to the multi‐step nature of complex biological samples, and still requires much development. Several sample preparation methods have been developed and used in the last two decades, including in‐gel, in‐solution, on‐bead, filter‐aided sample preparation, and suspension trapping, to improve reproducibility, efficiency, scalability, and reduce the handling time of this process. One of the most recent methods developed and applied in proteomics studies in recent years is suspension trapping, which combines rapid detergent removal, reactor‐type protein digestion, and peptide clean‐up in a tip or spin column. Suspension trapping is a simple, rapid, and reproducible digestion method that can effectively handle proteins in low microgram or sub‐microgram amounts. This review discusses the benefits of the suspension trapping digestion method in relation to its development and application in bottom‐up proteomics studies. We also discuss recent applications of suspension trapping digestion to different sample types and the features of the suspension trapping digestion method compared with other sample preparation methods.

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Investigating Phosphorylation Patterns of the Ion Channel TRPM7 Using Multiple Extraction and Enrichment Techniques Reveals New Phosphosites

Cited by 10 publications

References 38 publications

The molecular appearance of native TRPM7 channel complexes identified by high-resolution proteomics

The molecular appearance of native TRPM7 channel complexes identified by high-resolution proteomics

Optimized Suspension Trapping Method for Phosphoproteomics Sample Preparation

A review of suspension trapping digestion method in bottom‐up proteomics

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