DOI: 10.25345/c5df6kf39
title: MassIVE MSV000095517 - The proteomic landscape of proteotoxic stress in a fibrogenic liver disease
title: Sample preparation: Patient FFPE samples were stained, imaged, and individual hepatocytes were isolated using laser microdissection into 384-well plates. Please refer to the main paper for details. Peptide preparation: 384-well plates were thawed, and shapes (both combined and individual) were rinsed from the walls into the bottom of the well with 28uL of 100% acetonitrile (ACN). The wells were completely dried in a SpeedVac at 45 deg C, followed by the addition of 6uL of 60mM triethylammonium bicarbonate (TEAB, Supelco 18597) (pH 8.5) supplemented with 0.013% n-Dodecyl-beta-D-maltoside (DDM, Sigma-Aldrich D5172). Plates were sealed and incubated at 95 deg C for one hour. After adjusting to 10% ACN, samples were incubated again at 75 deg C for one hour. Subsequently, 6ng and 4ng of trypsin and Lys-C protease, respectively, in 1 uL of 60 mM TEAB buffer were added to each sample, and proteins were digested for 16 hours at 37 deg C. The reaction was quenched by adding trifluoroacetic acid (TFA) to a final concentration of 1%. Peptide samples were then frozen at -20 deg C. For loading, new Evotips were first soaked in 1-propanol for one minute, then rinsed twice with 50 uL of buffer B (ACN with 0.1% formic acid). After another 1-propanol soaking step for three minutes, the tips were equilibrated with two washes of 50 uL buffer A (0.1% formic acid). Samples were loaded into 70 uL of pre-loaded buffer A. Following one additional buffer A wash, the peptide-containing C18 disk was overlaid with 150uL buffer A and briefly centrifuged through the disk. All centrifugation steps were performed at 700g for one minute. The final tips were stored in buffer A for a maximum of four days prior to LC-MS. LC-MS data acquisition: The peptide samples were analyzed using an Evosep One liquid chromatography (LC) system (Evosep) coupled to an Orbitrap Astral mass spectrometer (Thermo Fisher Scientific). Peptides were eluted from the Evotips with up to 35% acetonitrile (ACN) and separated using an Evosep low-flow 'Whisper' gradient for DVP samples, or an experimental Evosep 'Whisper Zoom' gradient for single shapes and DVP-ML samples, with a throughput of 40 samples per day (SPD) on an Aurora Elite TS column of 15 cm length, 75 um internal diameter (i.d.), packed with 1.7 um C18 beads (IonOpticks). The column temperature was maintained at 50 deg C using a column heater (IonOpticks). The Orbitrap Astral mass spectrometer was equipped with a FAIMS Pro interface and an EASY-Spray source (both Thermo Fisher Scientific). A FAIMS compensation voltage of -40V and a total carrier gas flow of 3.5 L/min were used. An electrospray voltage of 1900V was applied for ionization, and the RF level was set to 40. Orbitrap MS1 spectra were acquired from 380 to 980 m/z at a resolution of 240,000 (at m/z 200) with a normalized automated gain control (AGC) target of 500% and a maximum injection time of 100 ms. For the Astral MS/MS scans in data-independent acquisition (DIA) mode, we experimentally determined the optimal methods across the precursor selection range of 380-980 m/z: (a) For DVP samples, a window width of 5 Th, a maximum injection time of 10 ms, and a normalized AGC target of 800% were used. (b) For DVP-ML samples, a window width of 6 Th, a maximum injection time of 13 ms, and a normalized AGC target of 500% were applied. (c) For single shapes and other DIA scans, the window width was optimized based on precursor density across the selection range of 380-980 m/z. A total of 45 variable-width DIA windows (see supplementary table X) were acquired with a maximum injection time of 28 ms and an AGC target of 800%. The isolated ions were fragmented using higher-energy collisional dissociation (HCD) with 25% normalized collision energy. Detailed method descriptions are provided in a default format with each supplementary data table. Data processing: The raw files were searched together with match-between run in library-free mode within each experimental group with DIA-NN v1.8.1 24. A FASTA file containing only canonical sequences was obtained from Uniprot (20,404 entries, downloaded on 2023-01-02), and the disease-causing amino acid was manually changed (E342K). We allowed a missed cleavage rate of up to 1, and set mass accuracy to 8, MS1 accuracy to 4, and the scan window to 6. Proteins were inferred based on genes, and the neural network classifier was set to 'single-pass mode'. For DVP and DVP-ML samples, precursor intensities in the 'report.tsv' file were then normalized using the directLFQ GUI at standard settings including a minimum number of non-nan ion intensities required to derive a protein intensity of one 25. The single shape data was additionally median normalized to a set of proteins quantified across all samples (621 proteins quantified in 100% of included samples), thereby correcting for the dependence of protein numbers on shape size.