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-detergentfacilitated 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/Dexchange 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. Molecular & Cellular

Less is More: Membrane Protein Digestion Beyond Urea–Trypsin Solution for Next-level Proteomics

“…Third, for overall pipelines, amine-free nonionic DDM supports downstream HPLC, MS/MS competition and TMT reactions, can be included throughout the cell lysis-protein extraction-(HDX)-digestion-(TMT)-HPLC-MS/MS process without removal, thus confers the workflows with no interruption, no sample leakage and high sensitivity. DDM at 0.4 -1 mM (0.02-0.05%, 2.3-5.7ϫ cmc) over 1 M proteins (0.04 -0.28 mg/ml) was well tolerated (6,7).…”

“…Thinking outside the box of denaturation and detergent removal, we discovered that nonionic low-cmc amine-free DDM-the most protein nature-promoting and crystallization-successful detergent to date-was completely compatible with structure/protease/TMT/HPLC MS/MS, and devised DDM-based protein extraction, purification and digestion methods that cleared all these challenges of membrane proteins (6,7,66). DDM-low-TCEP (DLT, for HDX) and flow/detergent-facilitated protease and de-PTM digestions (FDD, for deep sequencing and quantitation) both completed peptide preparations within seconds-minutes, at robust coverage and peptide reproducibility supporting full automation, demonstrated by using hGPCR (6) and hLGIC complex (7), respectively.…”

Dodecyl Maltopyranoside Enabled Purification of Active Human GABA Type A Receptors for Deep and Direct Proteomic Sequencing*

“…Direct gel-free digestion of purified GABA A R was tested by using porcine pepsin solution, pepsin-immobilized POROS AL beads slurry, or pepsin-immobilized column. Preparation of pepsin beads and columns was described in another manuscript (57). Purified GABA A R was adjusted to pH 2.5 by mixing with pepsin digestion buffer (0.1 M NaPi-HCl, pH 2.4, 15 mM TCEP, and 0.02% DDM) (52) Proteomic Data Processing-The CID MS/MS spectra were assigned by searching against the GABA A R protein sequences supplied for overexpression or the human proteome containing both forward and reversed sequences (Taplin), using the SEQUEST algorithm (b and y ions), with a fixed modification for cysteine (Cys, C) carbamidomethylation (ϩ57.02146 Da, C-Cb) and dynamic modifications for methionine (Met, M) oxidation (ϩ15.99492 Da, Mox), and N(Q) deamidation (ϩ0.98402 Da, when PNGase F-specific, defined as N(Q)-deglycosylation), at mass variation tolerance of 5 or 10 ppm for MS and 0.8 Da for MS/MS, unless specified otherwise.…”

“…However, only two unique peptides were detected to harbor this site, largely because intertwined C-C bond and bulky glycosylation obstructed effective protease access, a well-established challenge (23) also addressed in another study (57). Strikingly, during the review of this manuscript, crystallography of a truncated construct of (␤3) 5 hGABA A R expressed in HEK293S-GnTI resolved three saccharides for the ␤3 N174CT glycan, confirming it exists and interacts with adjacent ␤-sheet arms of loop C (14).…”

“…We found two sites (N46TT and N146MT) in ␣1 extracellular domain (ECD), consistent with two previous sequencing reports in mouse and rat (28,65), and three new sites (N33MS, N105LT, and N174CT) in ␤3 ECD. We saw no sites on ␥2L for its limited sequence coverage, but solved this problem in another study (57).…”