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Instant Integrated Ultradeep Quantitative-structural Membrane Proteomics Discovered Post-translational Modification Signatures for Human Cys-loop Receptor Subunit Bias*

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Neurotransmitter ligand-gated ion channels (LGICs) are widespread and pivotal in brain functions. Unveiling their structure-function mechanisms is crucial to drive drug discovery, and demands robust proteomic quantitation of expression, post-translational modifications (PTMs) and dynamic structures. Yet unbiased digestion of these modified transmembrane proteins—at high efficiency and peptide reproducibility—poses the obstacle. Targeting both enzyme-substrate contacts and PTMs for peptide formation and detection, we devised flow-and-detergent-facilitated protease and de-PTM digestions for deep sequencing (FDD) method that combined omni-compatible detergent, tandem immobilized protease/PNGase columns, and Cys-selective reduction/alkylation, to achieve streamlined ultradeep peptide preparation within minutes not days, at high peptide reproducibility and low abundance-bias. FDD transformed enzyme-protein contacts into equal catalytic travel paths through enzyme-excessive columns regardless of protein abundance, removed products instantly preventing inhibition, tackled intricate structures via sequential multiple micro-digestions along the flow, and precisely controlled peptide formation by flow rate. Peptide-stage reactions reduced steric bias; low contamination deepened MS/MS scan; distinguishing disulfide from M oxidation and avoiding gain/loss artifacts unmasked protein-endogenous oxidation states. Using a recent interactome of 285-kDa human GABA type A receptor, this pilot study validated FDD platform's applicability to deep sequencing (up to 99% coverage), H/D-exchange and TMT-based structural mapping. FDD discovered novel subunit-specific PTM signatures, including unusual nontop-surface N-glycosylations, that may drive subunit biases in human Cys-loop LGIC assembly and pharmacology, by redefining subunit/ligand interfaces and connecting function domains.

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Author Contributions: X.Z. conceived and designed study, performed experiments, analyzed data and wrote paper.

*

This work was supported in part by the National Institute of General Medical Sciences (GM 58448, K.W.M.) and the Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This article contains supplemental material.

1

The abbreviations used are:

    TM

    transmembrane

    PTM

    post-translational modification

    FDD

    flow/detergent-facilitated protease and de-PTM digestions for deep sequencing

    DDM

    n-dodecyl-β-D-maltopyranoside

    CHS

    cholesteryl hemisuccinate

    TCEP

    Tris-2-carboxyethylphosphine

    HDX

    H/D exchange

    TMT

    tandem mass tag

    DLT

    DDM-low-TCEP digestion for HDX

    TMD

    TM domain

    ICD

    intracellular domain

    ICL

    intracellular loop

    ECD

    extracellular domain

    C-C

    disulfide-bonded Cys-loop

    h

    human

    LGIC

    ligand-gated ion channel

    GPCR

    G protein-coupled receptor

    GABAAR

    gamma-aminobutyric acid type A receptor

    β2AR

    beta-2 adrenergic receptor

    nAChR

    nicotinic acetylcholine receptor

    AChBP

    acetylcholine-binding protein

    GlyR

    glycine receptor

    5HT3R

    serotonin type 3 receptor

    ER

    endoplasmic reticulum

    HEK293

    human embryonic kidney cell line 293

    MWCO

    molecular weight cutoff

    dial-filtration

    dialysis-filtration

    cmc

    critical micelle concentration

    RP

    reversed-phase

    HCD

    higher energy collisional dissociation

    ETD

    electron-transfer dissociation

    H-L

    high-low, high resolution MS-low resolution MS/MS scans

    H-H

    high-high, high resolution MS and MS/MS scans

    H

    high-abundance

    L

    low-abundance

    PSM

    peptide spectrum match

    FDR

    false discovery rate

    PD

    Proteome Discoverer

    Mox

    Methionine oxidation

    Cb

    carbamidomethylation

    N-glyco

    N-glycosylation

    Me

    methylation

    Ac

    acetylation

    Fm

    formylation

    GlcNAc

    N-acetylglucosamine

    Man

    mannose

    FA

    formic acid

    Cl-DMEA

    (2-chloro)-N,N-dimethylethylamine

    BrE-TMAB

    (2-bromoethyl)-N,N,N-trimethylammonium bromide.