Materials:
Customized peptide standards corresponding to the tryptic peptide fragments (purity >95%) and matched isotopic internal standards for Collagen 1A1 and 3A1 were purchased from Biosynth (supplemental table). LC-MS grade acetonitrile, formic acid (>99%), and xylene were obtained from Fisher Scientific. Ethanol was obtained from Greenfield Inc. Trypsin, ammonium bicarbonate, and acetic acid were purchased from Sigma. Rat tail collagen standard was obtained from Corning Life Sciences.
FFPE tissue specimens:
All experiments involving animals and/or human skin samples received before approval from the Health Sciences Animal Care Committee and the Conjoint Health Ethics Review Board at the University of Calgary, respectively and experiments were performed in accordance with relevant guidelines and regulations. The researchers were blinded to sample type for the duration.
Animals:
Adult reindeer (Rangifer tarandus) were maintained in an outdoor enclosure at the University of Calgary Veterinary Sciences Research Station for the duration of these experiments. Experiments were performed during early antler growth in early spring for males, late spring for females since the timing of antler growth is unique to each sex. Sex was equally distributed across experimental groups, as we have previously noted that there is no significant no sexual dimorphism in the antler velvet or back skin ECM composition. Animals ranged in age from 4-8 years.
All experiments were done in accordance with the University Animal Welfare Committee, the Veterinary Sciences Animal Care Committee and Health Sciences Animal Care Committee at the University of Calgary, the Canadian Council on Animal Care and the Province of Alberta Animal Protection Act and Regulation. Reindeer back skin and antler velvet samples were obtained under University of Calgary animal care protocol AC20-0030 as previously described[3]. In brief, animals were restrained in a hydraulic squeeze and anesthetized (Medetomidine, 0.07-0.15mg/kg I.M. with Azaperone, 0.2mg/kg I.M., if additional duration of anesthesia required). Anesthesia, heart and respiratory rate, and temperature were monitored throughout. For antler tissue collection, local anesthetic was provided (2% Lidocaine – Wyeth) with a ring block and a tourniquet applied; an inverted ‘‘L’’ block was done along the dorsal back. Analgesia was provided (Meloxicam, 0.5mg/kg S.Q.). Back skin and antler velvet were clipped to remove hair, and aseptically prepared using betadine surgical scrub and ethanol. Full-thickness excisional wounds were created using a 12 mm biopsy punch. Sedation was reversed (Atipamezole, IM at 5X the dose of medetomidine used). Tissue samples were fixed in 10% formalin in phosphate-buffered saline overnight at room temperature, then transferred to 70% ethanol, prior to embedding in paraffin.
Human Samples:
Explanted lungs from patients with idiopathic pulmonary fibrosis (IPF) undergoing transplantation were obtained from the University of Alberta under (REB15-1607). Informed consent was obtained from patients prior to any experimentation. Non-transplanted human lungs (controls) from normal donors were obtained from a tissue retrieval service (International Institute for the Advancement of Medicine, Edison, NJ). Ethical approval to receive and use lung tissues was obtained from the Conjoint Health Research Ethics Boards of the University of Calgary and the University of Alberta, and from the Internal Ethics Board of the International Institute for the Advancement of Medicine (REB15-0336). The lungs were sampled, fixed in 10% neutral buffered formalin for 24-48 h, and FFPE, blocks prepared.
Protein extraction and digestion:
Collagen was extracted from FFPE tissues from either scrolls or on side using a method as described previously, with some modifications[14,15]. Tissue was obtained from the FFPE blocks as either 5 µm thick scrolls or sections on glass slides (4 µm sections for lung tissue). Briefly, deparaffinization was performed by three washes with xylene for 10 minutes followed by centrifugation for 2 minutes at 20,000 x g and removal of supernatant (for scrolls) or by tipping the side on its side. Next, tissues were rehydrated by sequential addition of ethanol with increasing amounts of water (100% ethanol, 95% ethanol, 70% ethanol) with a 2-minute incubation followed by centrifugation steps between each addition. The glass mounted sections were scraped off the slides using a razor blade and transferred to a 1.5 mL microcentrifuge tube following the addition of 5-10 µL of water. Tissue lysis and breakage of crosslinking related to formalin fixation was performed through the addition of 50% acetonitrile in 100 mM ammonium bicarbonate followed by sonication for 60 minutes in a sonicating water bath (Branson 1800). Samples were transferred to a heating block and incubated for 60 minutes at 90°C. After each step, samples were subjected to centrifugation to ensure no sample loss. The lysis buffer was then removed by evaporation at 40°C under a flow of air and reconstituted directly into 100 mM ammonium bicarbonate buffer containing trypsin for digestion. At each time point, digestion was stopped by addition of formic acid to a final concentration of 0.1%.
The concentration of peptides in the mixture employed the BCA assay (Peirce) according to manufacturer’s protocols. At this point, COL1A1 and COL3A1 internal standards were added and samples were subject to LC-MS/MS analysis. The workflow is summarized in Figure 1.
Untargeted Post-translational Modification LC-MS/MS analysis:
The untargeted LC-MS/MS analysis was carried out by the Southern Alberta Mass Spectrometry core facility at the University of Calgary, Canada. Analysis was performed on an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific, Mississauga, ON) operated with Xcalibur (version 4.0.21.10) and coupled to a Thermo Scientific Easy-nLC (nanoflow Liquid Chromatography) 1,200 system. Tryptic peptides were loaded onto a C18 trap (75 μm x 2 cm; Acclaim PepMap 100, P/N 164946; Thermo Fisher Scientific) at a flow rate of 2 μL/min of solvent A (0.1% formic acid and 3% acetonitrile in LC-mass spectrometry grade water). Peptides were eluted using a 120 min gradient from 5 to 40% (5% to 28% in 105 min followed by an increase to 40% B in 15min) of solvent B (0.1% formic acid in 80% LC-mass spectrometry grade acetonitrile) at a flow rate of 0.3% μL/min and separated on a C18 analytical column (75 μm x 50 cm; PepMap RSLC C18; P/N ES803; Thermo Fisher Scientific). Peptides were ionised by electrospray using 2.3 kV into the ion transfer tube (300 °C) of the Orbitrap Lumos operating in positive mode. The Orbitrap first performed a full MS scan at a resolution of 120, 000 FWHM to detect the precursor ion having a mass-to-charge ratio (m/z) between 375 and 1,575 and a +2 to +4 charge. The Orbitrap AGC (Auto Gain Control) and the maximum injection time were set at 4 x 105 and 50 ms, respectively. The Orbitrap was operated using the top speed mode with a 3 s cycle time for precursor selection. The most intense precursor ions presenting a peptidic isotopic profile and having an intensity threshold of at least 2 x 104 were isolated using the quadrupole (isolation window of m/z 0.7) and fragmented with HCD (38% collision energy) in the ion routing Multipole. The fragment ions (MS2) were analyzed in the Orbitrap at a resolution of 15,000. The AGC, the maximum injection time and the first mass were set at 1 x 105, 105 ms, and 100 ms, respectively. Dynamic exclusion was enabled for 45 s to avoid of the acquisition of the same precursor ion having a similar m/z (±10 ppm).
Bioinformatic analysis and post translational modification identification
Spectral data were matched to peptide sequences in the bovine UniProt protein database using the MaxQuant software package v.1.6.0.1, peptide-spectrum match false discovery rate (FDR) of <0.01. Search parameters included a mass tolerance of 20 p.p.m. for the parent ion, 0.05 Da for the fragment ion, carbamidomethylation of cysteine residues (+57.021464), variable N-terminal modification by acetylation (+42.010565 Da), variable methionine oxidation (+15.994915 Da) and variable hydroxyproline modification (+15.9949146221).
For the proteomics data, cleavage site specificity was set to Trypsin/P with up to two missed cleavages allowed. Database searches were limited to a maximal length of 40 residues per peptide. Peptide sequences matching reverse or contaminant entries were removed.
Data Availability
The datasets generated and/or analysed during the current study are available in the PRIDE repository[16], http://www.ebi.ac.uk/pride/archive/projects/PXD050877 and PXD050877.
Collagen 1 and 3 Quantification:
For calibration, a surrogate matrix of human serum albumin was chosen as it mimics the protein samples being analyzed without having any collagen in it. Using this method, this method was able to quantify COL1A1 and COL3A1 at concentrations ranging from 1.0 to 1000 ng/mL and 0.5 to 300 ng/mL respectively. Quality control materials were prepared as a bulk digest of a single tissue section which was aliquoted, with a single aliquot thawed prior to analysis of each batch. In addition, a rat tail collagen (Corning, 354236) material was included to monitor processing efficiency however contained only Col1. These materials were used to monitor batch-to-batch consistency and provide precision estimates.
Targeted LC-MS/MS analysis:
Targeted LC-MS/MS analysis was performed using surrogate marker peptides from COL1A1 and COL3A1, with and without hydroxyproline residues. LC-MS/MS parameters for each peptide were obtained from Skyline[17] and tested for uniqueness using a blank matrix of human serum albumin. Optimization of proteolytic digestion conditions, extraction protocols, and chromatographic conditions was performed to minimize time required for processing and analysis.
Tissue immuno-staining and image analysis by SHG:
5 µm thick sections from the FFPE blocks were immuno-stained with rabbit anti-collagen type 3 (600-401-105, Rockland) followed by goat anti-rabbit Alexa488 secondary antibody. Sections were imaged using a multiphoton confocal microscope (Leica SP8 confocal) and second harmonic generation (SHG). First, collagen 3 was imaged using the 488 laser, followed by total collagen imaged with SHG using a Ti:Sa Chameleon multiphoton tunable laser (Coherent, Santa Clara, CA) at 800 nm, both with a 20X water immersion objective lens.
Single Cell RNA Sequencing data analysis:
The scRNAseq data provided in Figure 4 was extracted from a previously published dataset[3]. Briefly, a 12mm biopsy of skin from the back and velvet from the anlters were harvested. The tissue was digested using collagenase IV and single cells isolated using fluorescence associated cell sorting (FACS). Single cells were processed for single cell 3’ gene expression using a 10X Genomics Controller with NextGEM V3.1 chemistry according to the manufacturer’s protocol. cDNA libraries were sequenced using an Illumina NovaSeq. Data was processed using 10X Genomics CellRanger platform, followed by Seurat (version 4) in R for analysis[18].
Data Analysis:
The concentration of each surrogate peptide from COL1A1 and 3A1 were calculated by comparison to a calibration curve. Using these values, the content of Col1 and Col3 were made as follows: