DOI: 10.22002/ee2yc-fg857
title: Age-related matrix stiffening epigenetically regulates α-Klotho expression and compromises chondrocyte integrity

description: Matrix stiffening is a quintessential feature of aged tissues. Authors show that an aged (stiff) matrix epigenetically represses the gene encoding the longevity factor, α-Klotho, resulting in chondrocyte dysfunction, a leading cause of osteoarthritis.
description: LC/MS-MS mass spectrometry-based proteomics Knee cartilage from male and female young, middle-aged, and aged C57/BL6 mice (n=5/age/sex) were microdissected as detailed previously90. Samples were lyophilized overnight and stored at -80°C until shipment to the Proteome Exploratory Laboratory at Caltech.
Cartilage samples from each knee were lysed in 8M urea/100mM TEAB by grinding for 1 min with size tissue grinder pestles (cat. 12141363, Fisher Scientific), tip sonication with a Fisher Scientific 550 Sonic Dismembrator on ice at 20% power using cycles of 20 sec on/20 sec off for 4 minutes total, followed by another grinding step for 1 min. Samples were then clarified by centrifugation at 16,000g for 5 minutes at room temperature. Each lysate was then reduced with 500mM TCEP for 20 min at 37°C and alkylated with 500mM 2-Chloroacetamide for 15 min at 37°C in the dark. Samples were then digested with a 1:200 ratio of LysC to lysate for 4 hr at 37°C, followed by dilution with 100mM TEAB, addition of 100mM CaCl2, and digestion overnight with 1:30 Trypsin at 37°C. Digestions were stopped by acidifying with 20% TFA, desalted on C18 spin columns (cat. 89870, Fisher Scientific) according to manufacturer instructions, and lyophilized to dryness. Peptides were then resuspended in 0.1% formic acid and peptide amounts measured with the Pierce Quantitative Colorimetric Peptide Assay.
15 μg peptides from each sample were lyophilized, resuspended in 100mM TEAB, labeled with TMTpro reagents dissolved in anhydrous acetonitrile for 1 hr at room temperature, and quenched with 5% hydroxylamine for 15 min at room temperature. All 15 male samples were then combined into one sample and all 15 female samples were combined into a second sample 100 ug of each sample was then fractionated with the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo #84868) according to manufacturer instructions and the resulting 8 fractions were lyophilized. Each fraction was resuspended in 20 μl 0.2% formic acid and peptide quantitation performed with the Pierce Quantitative Colorimetric Peptide Assay. Fractions 7 and 8 from both samples had very low peptide amounts and were thus combined with that sample's fraction #6 for a total of 6 fractions per sample.
Liquid chromatography-mass spectrometry (LC-MS) analysis of peptide fractions was carried out on an EASY-nLC 1000 coupled to an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). Each fraction was loaded onto an Aurora 25cm x 75μm ID, 1.6μm C18 reversed phase column (Ion Opticks, Parkville, Victoria, Australia) and separated over 136 min at a flow rate of 350 nL/min with the following gradient: 2–6% Solvent B (7.5 min), 6-25% B (82.5 min), 25-40% B (30 min), 40-98% B (1 min), and 98% B (15 min). MS1 spectra were acquired in the Orbitrap at 120K resolution with a scan range from 350-1800 m/z, an AGC target of 1e6, and a maximum injection time of 50 ms in Profile mode. MS2 scans were then acquired in the Orbitrap at 50K resolution in Centroid mode with the first mass fixed at 110. Cycle time was set at 3 seconds.
Analysis of LCMS proteomic data was performed in Proteome Discoverer 2.5 (Thermo Scientific) utilizing the Sequest HT search algorithm with the mouse proteome (UniProt UP000000589; 55,485 proteins covering 21,989 genes with a BUSCO assessment of 99.8% genetic coverage). Search parameters were as follows: fully tryptic protease rules with 2 allowed missed cleavages, precursor mass tolerance set to 20 ppm, fragment mass tolerance set to 0.05 Da with only b and y ions accounted for. Percolator was used as the validation method, based on q-value, with a maximum FDR set to 0.05. GO Biological Process terms were generated via Proteome Discoverer Sequest HT algorithm and were used for sorting proteins associated with PI3k/Akt signaling.
Quantitative analysis is based on TMT MS2 reporter ions generated from HCD fragmentation, with an average reporter S/N threshold of 10, used a co-isolation threshold of 50 with SPS mass matches set at 65%. Normalization was performed at the peptide level, and protein ratios were calculated from the grouped ion abundances, with protein FDR set to a maximum of 0.05.