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Integrated Dissection of Cysteine Oxidative Modification Proteome During Cardiac Hypertrophy
Cysteine oxidative modification of cellular proteins is crucial for many aspects of cardiac hypertrophy development. However, integrated dissection of multiple types of cysteine oxidation proteome in cardiac hypertrophy is currently missing. Here we developed a novel discovery platform encompassing a customized biotin switch-based quantitative proteomics pipeline and an advanced analytic workflow to comprehensively profile the landscape of cysteine oxidation in ISO-induced cardiac hypertrophy mouse model. Specifically, we identified a total of 3,717 proteins containing 6,837 oxidized cysteine sites by at least one of reversible cysteine oxidation, cysteine sulfinylation (CysSO2H), and cysteine sulfonylation (CysSO3H). Analyzing the hypertrophy signatures that are reproducibly discovered from computational workflow highlighted a group of fatty acid beta-oxidation enzymes with a continual decreased temporal pattern and a significant decreased abundance in reversible oxidation with no temporal or abundance change in total cysteine, revealing the oxidative regulatory map of fatty acid metabolism in cardiac hypertrophy, which is featured by an overall reduced oxidative metabolism. Our cysteine oxidation platform depicts a dynamic and integrated landscape of the cysteine oxidative proteome, extracted molecular signature, and provided mechanistic insights in cardiac hypertrophy.
Sample Processing Protocol
Experimental animal models Male C57BL/6J mice, 9–12 weeks of age (Jackson Laboratories), were housed in a 12-hour light/12-hour dark cycle with controlled temperature, humidity, and access to standard chow and water ad libitum. Mice were surgically implanted with a subcutaneous micro-osmotic pump (ALZET) delivering 15 mg/kg/day isoproterenol (ISO) (Sigma) or saline vehicle. In this treatment protocol, mice develop a gradual cardiac hypertrophy phenotype characterized by significantly increased ejection fraction and HW/BW. Independent groups of 3 mice from each treatment condition were euthanized for sample collection at 1, 3, 5, 7, 10, and 14 days post-implantation. All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals by the National Research Council and approved by the Animal Research Committee at UCLA. Biotin switch-based sample processing Protein extraction: Left ventricles were collected from mouse hearts and placed in 1 mL NP-40 lysis buffer (50 mM Tris–HCl, pH 8, 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mM EDTA) containing 100 mM N-ethylmaleimide (NEM) and Halt™ Protease Inhibitor Cocktail (100X) (ThermoFisher Scientific). Tissue was homogenized using a glass hand homogenizer and mixed by rotation for 2 hr at 4 °C before centrifugation at 13,800 g for 20 min at 4 °C. The protein concentration of the supernatant was measured using the Bio-Rad DC protein assay. Aliquots containing 2 mg protein were prepared, then precipitated by 10% trichloroacetic acid (TCA) and centrifuged at 20,000 g for 15 min at 4 °C. The resulting pellets were stored at -80 °C. Biotin switch-based labeling of reversible cysteine oxidation: After one wash with 1 mL of ice-cold 5% TCA, and two washes with 1 mL of 95% ethanol to get rid of small molecules and reduce acidity, pellets were suspended in 1 mL of urea-containing cysteine modification buffer (CMBU) (0.1 M HEPES-NaOH, pH 7.4, 1% SDS, 10 mM DTPA, 6 M urea) with 0.1 M NEM. After 30 min rotation at room temperature, the samples were reduced by 0.12 M DTT and rotated for another 60 min. To quantify the total abundance of cysteine sites and to preserve cysteine sulfinylation (CysSO¬2H) and sulfonylation (CysSO¬3H), 10% of the lysate was reserved as an unlabeled portion and underwent acetone precipitation. For the remaining 90%, proteins were separated from small molecules by centrifugation with 10% TCA, followed by one wash with 5% TCA and two washes with 95% ethanol. Pellets were suspended in 300 µL CMBU with 0.1 mM maleimide-biotin (Mal-Biotin) (Sigma-Aldrich). After a 30 min rotation at room temperature, unreacted NEM was quenched with 10 mM DTT for an additional 30 min. Small molecules and proteins were separated by TCA and ethanol washes as described above. Digestion and dimethyl labeling: Pellets from both labeled and unlabeled portions were solubilized in 0.1 M triethylammonium bicarbonate buffer (TAEB) with 0.1% Rapigest (Waters) and heated at 60 °C for 45 min. Solubilized proteins were alkylated with 9 mM 2-iodoacetamide (IAM) incubation in the dark at room temperature for 30 min. The alkylated lysate underwent trypsin digestion overnight (16 hr) at 37 °C with a 1:100 ratio of trypsin to protein. A final concentration of 0.16% CH¬2O/C2H2O (Sigma-Aldrich) and 24 mM sodium cyanoborohydride (NaBH3CN) (Sigma-Aldrich) were added to the designated samples with light/medium labeling. Reciprocal labeling was performed on two out of four technical replicates to minimize the technical bias from dimethyl labeling. After a one-hour incubation at room temperature, 0.16% (vol/vol) ammonium solution was added and mixed for 15 min to quench the reaction. Mal-biotin Enrichment: Mal-Biotin and dimethyl labeled peptides were diluted in 1.2 mL PBS with 200 µL pre-washed High-Capacity NeutrAvidin slurry (Thermo Scientific). After overnight incubation, the sample was centrifuged, washed twice with 1 mL PBS, once with 50 mM ammonium bicarbonate w/ 20% methanol, and eluted with 50% Pierce™ acetonitrile (ACN) w/ 0.4% trifluoroacetic acid (TFA). C18 column cleanup: All samples were subjected to 30 min incubation at 37 °C with 1% TFA and centrifugation (13,000 g for 15 min) to remove remaining Rapigest. Samples were cleaned with PierceTM C18 Spin Columns (Thermo Scientific) to remove any interfering substances prior to LC-MS/MS analysis.
Data Processing Protocol
LC-MS/MS analysis LC-MS/MS was performed on our decontaminated protein samples, as previously described. To reduce sample complexity and increase protein coverage, we fractionated peptide samples using high-pH/low–pH two-dimensional reversed-phase chromatography prior to MS/MS. Fifty micrograms of peptides were injected into a Phenomenex C18 column (Jupiter Proteo C12, 4-μm particle, 90-Å pore, 100 mm×1 mm dimension) using a Finnigan Surveyor LC system for the first-dimension (high-pH) separation. We established a gradient between solvent A (20 mM ammonium formate, pH 10) and solvent B (20 mM ammonium formate, 90% acetonitrile, pH 10) at a 50 μL·min−1 flow-rate with the following timing and solvent proportions: 0–5% solvent B in solvent A from 0–2 min; 5–35% solvent B in solvent A from 3–32 min; and, finally, 80% solvent B in solvent A from 32–37 min. Six fractions of peptides were collected from 16–40 min, lyophilized, and re-dissolved in 20 µL 0.5% formic acid with 2% acetonitrile. Each high-pH fraction was injected (10 μL) to an EasySpray C18 column (PepMap, 3-μm particle, 100-Å pore; 75 μm×150 mm dimension; Thermo Scientific) using an auto-sampler on a single Easy-nLC 1000 nano-UPLC system (Thermo Scientific) for second-dimension (low-pH) reversed-phase chromatography analysis. We established a gradient between solvent A (0.1% formic acid, 2% acetonitrile) and solvent B (0.1% formic acid, 80% acetonitrile) at a flow rate of 300 nL·min−1 with the following timing and solvent proportions: 0–40% solvent B from 0–110 min; 40–80% B from 110–117 min; and 80% B from 117–120 min. Column pressure was maintained below 150 bar. High-resolution LC-MS/MS was performed on a single LTQ Orbitrap Elite instrument (Thermo Scientific) through a Thermo EasySpray interface. MS signals were acquired in Fourier-Transform/Ion-Trap (FT/IT) mode: each FT MS1 survey scan was analyzed at 60,000 resolving power in profile mode, followed by rapid IT MS2 scans on the top 15 ions with monoisotopic peak selection. MS1 and MS2 target ion accumulation targets were 104 and 106, respectively. MS1 lock mass (m/z 425.120025) and dynamic exclusion (90 s) were used. Throughout the LC-MS/MS experiment, column temperature was held at a constant 50 °C. Quantification of cysteine modification abundance: The acquired raw mass spectra were processed with MaxQuant software version 126.96.36.199. Peptide identification was performed using the Andromeda search engine, against a reverse-decoyed protein sequence database (UniProt Reference Proteome, reviewed, accessed June-12–2016). First and main searches were performed with precursor mass tolerances of 20 ppm and 4.5 ppm. Specificity for trypsin cleavage was required, allowing up to two missed cleavage sites. Dimethylated peptide labels were identified using the “multiplicity” query, including DimethLys0 and DimethN-term0 as light labels as well as DimethLys4 and DimethN4 as medium labels. Up to four modified sites per peptide were allowed. All forms of cysteine sites were identified by querying variable modification types, including Mal-biotin labeled cysteine (451.1889 Da), NEM-labeled cysteine (125.0477 Da), IAM-labeled cysteine (57.0215 Da), methionine sulfoxidation (15.9949 Da), CysSO2H (31.9898 Da), and CysSO3H (47.9847 Da). Up to five variable modifications were allowed. Tryptic, semi-tryptic, and non-tryptic peptides within a 20 ppm parent mass window surrounding the candidate precursor mass were searched. Peptide ions from up to 3 isotopic peaks with fragment mass tolerance of 600 ppm were allowed. Protein inference required ≤ 5% peptide spectra matching (PSM), posterior error probability (PEP), and protein false discovery rate (FDR), as well as a minimum of 2 ratio counts. Peptides with a cysteine count lower than one were excluded, along with reverse and potential contaminant flagged peptides. Modified peptide identifications with an Andromeda search score greater than 40, a delta score greater than 6, and a localization probability > 0.8 were allowed. All searches for a given data set were based on one set of Andromeda peak list files (apl-files). Each of the cysteine modifications (i.e. Mal-biotin, NEM, IAM, CysSO2H, and CysSO3H) was generated as a separate output file with identified cysteine sites, their extracted ion chromatogram (XIC) values, and normalized ratios of ISO vs. Vehicle conditions calculated from the light and medium dimethyl labeled peptides. The total abundance of one cysteine site is quantified as the sum of XIC values from both modified and unmodified forms of that particular cysteine.
Howard Choi, BD2K Center of Excellence for Big Data Computing at UCLA, UCLA, USA
Peipei Ping, David Geffen School of Medicine, UCLA, USA BD2K Center of Excellence for Big Data Computing at UCLA, UCLA, USA ( lab head )
Wang J, Choi H, Chung NC, Cao Q, Ng DCM, Mirza B, Scruggs SB, Wang D, Garlid AO, Ping P. Integrated Dissection of the Cysteine Oxidative Post-translational Modification Proteome During Cardiac Hypertrophy. J Proteome Res. 2018 PubMed: 30141336
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