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Scalable Acid-Aided Lysis of Skin Samples Improves Proteome Coverage

ABSTRACT: In this study, we present a robust and scalable sample preparation protocol based on chemical lysis with trifluoroacetic acid (TFA), adapted from the SPEED method and optimized for human skin samples. The protocol simplifies tissue lysis while selectively leaving ECM scaffolds largely insoluble. As a result, abundant collagens and structural proteins are effectively depleted from the soluble fraction, improving the dynamic range of detectable proteins and increasing sensitivity for less abundant species. Using TFA lysis followed by solid-phase-enhanced sample preparation (SP3), we identified up to 6,200 protein groups from full-thickness human skin punch biopsies. Notably, the protocol enables high-depth proteomic analysis from 2 mm punch biopsies, which are less invasive and more suitable for longitudinal or large-cohort studies. Tape strip samples—commonly used in dermatological research for minimally invasive epidermal sampling—were also compatible with this protocol, yielding up to 2,300 protein groups, representing a two- to threefold increase compared to previously reported studies.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Skin

SUBMITTER: Max Sauerland

LAB HEAD: Mads Gyrd-Hansen

PROVIDER: PXD066029 | Pride | 2025-08-21

REPOSITORIES: Pride

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			Action	DRS
	202400930_DIA_proteome_opt_lysis_15min_S01.raw	Raw
	202400930_DIA_proteome_opt_lysis_15min_S02.raw	Raw
	202400930_DIA_proteome_opt_lysis_15min_S03.raw	Raw
	202400930_DIA_proteome_opt_lysis_RT_S01.raw	Raw
	202400930_DIA_proteome_opt_lysis_RT_S02.raw	Raw

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Project description:the Enhanced Epidermal Antigen Specific Immunotherapy Trial -1 (EEASI) study was a two-centre, open-label, uncontrolled, single-group first-in-human Phase 1A safety study of C19-A3 GNP peptide in individuals with T1D (https://clinicaltrials.gov/ct2/show/NCT02837094). The investigational medicinal product (IMP) was C19-A3 GNP (Midacore™), which comprises Midacore™ GNPs (Midatech Pharma Plc, Cardiff, UK)(1, 20) of a size of less than 5 nm, covalently coupled to an 18-amino acid human peptide, the sequence of which is identical to the residues from position 19 in the C-peptide of proinsulin through to position 3 on the A-chain of the same molecule (GSLQPLALEGSLQKRGIV). The peptide is synthesised with a linker to facilitate binding to the GNPs: 3-mercaptopropionyl-SLQPLALEGSLQKRGIV 2 acetate salt (disulfide bond). The chemical composition of the IMP contained a ratio of 4 C19-A3 peptides: 11 glucose C2: 29 glutathione ligands as determined by 1H-NMR (proton nuclear magnetic resonance). A typical batch contained: [C19-A3 peptide] = 1.33 mg/ml; [gold] = 5.5 mg/ml; [glucose linker] = 0.6 mg/ml; [glutathione linker] = 1.79 mg/ml. As the drug substance was diluted 1:7 to 1:10, depending on the content of C19-A3 peptide per particle, and as 50 μl of the diluted solution was administered to the study participants, this corresponded to C19-A3 peptide: 10 μg; gold: 39 μg; glucose linker: 4.3 μg; glutathione: 12.7 μg. Participants diagnosed with T1D and confirmed to possess the HLA-DRB1∗0401 genotype, were given three doses of C19-A3 GNP at 4-weekly intervals (weeks 0, 4, and 8) in the deltoid region of alternate arms (2 doses in one arm and 1 dose in the other arm) via CE-marked 600 μm length MicronJet600™ hollow microneedles (NanoPass Technologies Ltd. Israel) attached to a standard Luer-lock syringe. The single-dose given in 50 μl volume was equivalent to 10 μg of C19-A3 peptide. Punch skin biopsies of the local area were performed under aseptic conditions and under local anaesthetic (Lidocaine Hydrochloride Injection BP 2%, w/v), using a 6-mm sterile disposable biopsy punch. Following the biopsy, samples were divided with half immediately placed in 10% formalin and transported to the laboratory and the other half placed in RNA laterTM (Fisher Scientific, Loughborough, UK) for bulk RNA sequencing. Control skin samples were obtained from female patients aged 19–82 years, following mastectomy or breast reduction after informed consent. Skin without obvious pathological findings, which was surplus to diagnostic histopathology requirements, was used in the experiments. Control punch biopsy samples were treated in the same manner as described above. After cutting skin samples into small pieces, samples were homogenised and lysed by using Tissue Raptor II and QIAzol® Lysis protocol (QIAzol® Handbook 2021, QIAgen, Crawley, UK). The quality of RNA was routinely assessed by determining the A260:A280 ratio using NanoDrop2000 (Thermo Scientific, UK). RNA libraries were created using TruSeq stranded Total RNA with ribozero GOLD (illumina, UK) and sequenced on an illumina HiSeq 2500 platform with 2 x 75 bp reads

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Scalable Acid-Aided Lysis of Skin Samples Improves Proteome Coverage

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