title: Instant Integrated Ultradeep Quantitative-structural Membrane Proteomics Discovered Post-translational Modification Signatures for Human Cys-loop Receptor Subunit Bias.
abstract: 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.
authors.affiliation: From the ‡Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, xi.zhang.edu@gmail.com.|§Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts.
authors.forename: Xi
authors.initials: X
authors.lastname: Zhang
mesh_terms: D001665:Binding Sites; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D010447:Peptide Hydrolases / Q000378:metabolism; D010865:Pilot Projects; D011485:Protein Binding; D060066:Protein Interaction Maps; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D011963:Receptors, GABA-A; D010447:Peptide Hydrolases