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Chemoproteomics reveals unexpected lysine/arginine-specific cleavage of peptide chains as a potential protein degradation machinery Caiping Tian, Keke Liu, Rui Sun, Ling Fu, and Jing Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03237 • Publication Date (Web): 05 Dec 2017 Downloaded from http://pubs.acs.org on December 5, 2017
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Analytical Chemistry
Chemoproteomics reveals unexpected lysine/arginine-specific cleavage of peptide chains as a potential protein degradation machinery Caiping Tian#, Keke Liu#, Rui Sun, Ling Fu, Jing Yang* State Key Laboratory of Proteomics, National Center for Protein Sciences • Beijing, Beijing Proteome Research Center, Beijing Institute of Lifeomics, Beijing 102206, China Supporting Information Placeholder ABSTRACT: Proteins can undergo oxidative cleavage by in vitro metal-catalyzed oxidation (MCO) in either the α-amidation or the diamide pathway. However, whether oxidative cleavage of polypeptide-chain occurs in biological systems remains unexplored. We describe a chemoproteomic approach to globally and site-specifically profile electrophilic protein degradants formed from peptide backbone cleavages in human proteomes, including the known N-terminal α-ketoacyl products and >1000 unexpected N-terminal formyl products. Strikingly, such cleavages predominantly occur at the carboxyl side of lysine (K) and arginine (R) residues across native proteomes in situ, while MCO-induced oxidative cleavages randomly distribute on peptide/protein sequences in vitro. Furthermore, ionizing radiation-induced reactive oxygen species (ROS) also generate random oxidative cleavages in situ. These findings suggest that the endogenous formation of N-formyl and N-α-ketoacyl degradants in biological systems is more likely regulated by a previously unknown mechanism with a trypsin-like specificity, rather than the random oxidative damage as previously thought. More generally, our study highlights the utility of quantitative chemoproteomics in combination with unrestricted search tools as a viable strategy to discover unexpected chemical modifications of proteins labeled with active-based probes.
Reactive oxygen species (ROS) formed from various biological processes or exogenous insults can attack protein backbone or amino acid side chains, yielding electrophilic carbonyl groups, also known as protein carbonylation.1 As first discussed by Garrison in 1987, proteins can undergo ROS-induced cleavage by either the α-amidation or the diamide pathway (Scheme 1).2 Specifically, oxygen radicals attack the protein deposits at the α-carbon sites to form alkoxyl radicals. In the diamide pathway, the C-C bond of the peptide backbone further undergoes homolytic cleavage to produce a diamide and an isocyanate radical, which then undergoes termination to form an isocyanate. Alternatively, in the α-amidation pathway, the C-N bond encompassing the α-carbon site of radicalization can undergo homolytic cleavage to produce an N-α-ketoacyl derivative and an amide radical, which terminates via the addition of peroxyl radical to form an amide. However, these theoretic pathways are proposed mainly based on polypeptide-chain chemistry of proteins in non-physiologically aqueous O2-saturated solutions, involved in both model protein and peptides systems. Whether oxidative cleavage of polypeptidechain also occurs in biological systems has never been investigated, mainly due to lack of analytical methodologies for global profiling of protein carbonylation in native proteomes. Until recently,
Cravatt and his coworkers conducted an elegant study of protein carbonylation in human cells using ‘reverse-polarity’ activitybased protein profiling (RP-ABPP) strategy and identified a few previously unknown endogenous carbonyl products in human cells, including a novel N-terminal glycoxylyl product derived from secernin-3 (SCRN3).3 Nonetheless, the chemical structures of most endogenous protein electrophiles discovered in this study remain to be elucidated. Moreover, global and site-specific mapping of targets of protein carbonylation across native proteomes poses another analytical challenge. Here we describe a quantitative chemoproteomic approach to globally and site-specifically profile electrophilic carbonyl products formed from peptide backbone cleavages in human proteomes. By using an un-restricted database search strategy, we identified >1000 unexpected N-formyl products from cleaved proteins. Our analysis also suggests that these electrophilic protein fragments are generated in an unexpected lysine/arginine-specific manner, a potential protein degradation machinery.
EXPERIMENTAL SECTION Chemicals. HZyne was kindly provided by Dr. Chu Wang at Peking University. 12C and 13C-labeled azido-UV-biotin reagents (Azido-L-biotin and azido-H-biotin) were kindly provided by Dr. Ned Porter at Vanderbilt University. N-formyl NLDIERPTYTNLNR and unmodified NLDIERPTYTNLNR, NLDIEKPTYTNLNR and NLDIEAPTYTNLNR were purchased from Chinese Peptide Company (Hangzhou, China). BSA was purchased from Thermo Fisher. HPLC-grade water, acetonitrile (CAN), and methanol (MeOH) were purchased from J.T.Baker. Other chemicals and reagents were obtained from Sigma-Aldrich unless otherwise indicated. Synthesis of HZyne-derived peptide adducts. 1 mM N-formyl NLDIERPTYTNLNR dissolved in 40 µL (final volume) 1X PBS was incubated in the presence of 1mM HZyne for 1h at room temperature (RT). The resulting peptide adduct mixture was desalted as previously described. Click chemistry then was performed by the addition of 1mM Az-UV-biotin, 10 mM sodium ascorbate, 1 mM TBTA, and 8 mM CuSO4. Click reactions were allowed to proceed at RT for 2 h in the dark with rotation. Reaction was stopped by adding 400 µL 25 mM ammonium bicarbonate (pH 8.0) and transferred to glass tubes and irradiated with 365 nm UV light (Entela, Upland, CA) for 2h at RT with stirring. The resulting peptide mixtures were desalted by 100 µL tips (AXYGEN T-400) containing Durashell C18 (3 µm, 150 Å, Algela, DC930010-L) filled on the C18 membrane (Empore Bioanalytical technologies 3M, 2215-C18).
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Metal-catalyzed oxidation. Metal ion-catalyzed oxidation (MCO) was performed as described previously.4 In brief, 0.2 mg/mL unmodified peptides were incubated overnight (15 h) at 37ºC in 50 mM HEPES-KOH, pH 7.4, containing 100 mM KCl, 10 mM MgCl2, 25 mM ascorbic acid, and 100 µM FeCl3. The reaction mixtures then were desalted and subjected to LC-MS/MS analysis. The MCO of 0.5 mg/mL BSA was performed in the same condition as above. After overnight incubation, an aliquot of 10 µg BSA was resolved by SDS-PAGE and stained with Coomassie blue. The rest BSA sample was diluted with 50 mM ammonium bicarbonate, labeled with HZyne, and then digested with sequencing grade trypsin at a 1:50 (enzyme/substrate) ratio overnight at 37°C. The resulting peptides then were desalted and subjected to LC-MS/MS analysis. Cell culture and treatment. HeLa cells (National Infrastructure of Cell Line Resource, Beijing, China, No. 3111C0001CCC000011) were maintained at 37 °C in a 5% CO2, humidified atmosphere and were cultured in DMEM medium (HyClone, Logan, Utah, USA) containing 10% fetal bovine serum (Life technologies, Gibco). Ionizing radiation (IR). Cells were grown until 70-80% confluent in 15-cm diameter cell culture flasks and irradiated with or without 10 Gy X-rays at 160 kV and a dose rate of 180 cGy/min using the XRAD160 X-ray device (Precision X-Ray, North Branford, CT). Field size for the X-ray irradiation was 20 × 20 cm to fully cover the flasks and the source to surface distance was 50 cm. After irradiation, cells were cultured for 30 min at 37°C, washed with 1X PBS three times, and prepared for the following analysis. Sample preparation for proteomic analysis. Cells were grown until 80-90% confluency, rinsed with 1X PBS quickly. Cells then were lifted with 0.25% trypsin-EDTA (Invitrogen) and harvested by centrifugation at 1,500 g for 3 min. Cell pellets were lysed on ice for 20 min in NETN lysis buffer (50 mM HEPES, 150 mM NaCl, 1% Igepal, pH 7.5) containing inhibitor cocktail and 5 mM HZyne and further incubated at RT for 1h. The lysate was further incubated with 8 mM DTT (Research Products International) at RT for 1h to reduce the reversibly oxidized cysteines. Reduced cysteines then were alkylated with 32 mM IAM for 30 min at RT with light protection. Proteins were then precipitated with methanol-chloroform (aqueous phase/methanol/chloroform, 4:4:1, v/v/v) as previously described.5,6 The precipitated protein pellets were resuspended with 50 mM ammonium bicarbonate. Resuspended protein concentrations were determined with the BCA assay (Pierce Thermo Fisher) and adjusted to a concentration of 2 mg/mL. Resuspended proteins were first digested with sequencing grade trypsin (Promega) at a 1:50 (enzyme/substrate) ratio overnight at 37°C. A secondary digestion was performed by adding additional trypsin to a 1:100 (enzyme/substrate) ratio, followed by incubation at 37°C for additional 4 h. The tryptic digests were desalted with HLB extraction cartridges (Waters). The desalted samples were then evaporated to dryness under vacuum. Click chemistry, capture and enrichment. Desalted tryptic digests were reconstituted in a solution containing 30% ACN at pH 6. Click chemistry was performed by the addition of 0.8 mM either Azido-L-biotin or Azido-H-biotin, 8 mM sodium ascorbate, 1 mM TBTA, and 8 mM CuSO4. Samples were allowed to react at RT for 2 h in the dark with rotation. The Azido-L-biotin and Azido-H-biotin sample were then mixed together immediately following click chemistry. Excess reagents were removed by SCX chromatography as previously described,5,6 and then the extracts
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were allowed to interact with pre-washed streptavidin sepharose for 2 h at RT. Streptavidin sepharose then was washed with 50 mM NaOAc, 50 mM NaOAc containing 2 M NaCl, and water twice each with vortexing and/or rotation to remove non-specific binding peptides, and resuspended in 25 mM ammonium bicarbonate. The suspension of streptavidin sepharose was transferred to several glass tubes (VWR), irradiated with 365 nm UV light (Entela, Upland, CA) for 2 h at room temperature with stirring. The supernatant was collected, evaporated to dryness under vacuum, and stored at −20°C until analysis. LC-MS/MS analysis. LC-MS/MS analyses were performed on Q Exactive plus and Orbitrap Fusion Lumos instruments (Thermo Fisher Scientific) for HCD and EThcD analysis, respectively. Samples were reconstituted in 0.1% formic acid and pressureloaded onto an Easy-nLC1000 system (Thermo Fisher Scientific) equipped with a 360 µm outer diameter × 75 µm inner diameter microcapillary precolumn packed with Jupiter C18 (5 µm, 300 Å, Phenomenex) and then washed with 0.1% formic acid. The precolumn was connected to a 360 µm outer diameter × 50 µm inner diameter microcapillary analytical column packed with the ReproSil-Pur C18-AQ (3 µm, 120 Å, Dr. Maisch) and equipped with an integrated electrospray emitter tip. LC gradient condition consisted of 0 min, 7% B; 14 min, 10% B; 51 min, 20% B; 68 min, 30% B; 69-75 min, 95% B (A = water, 0.1% formic acid; B = ACN, 0.1% formic acid) at a flow rate of 600 nL/min. For HCD analysis, the spray voltage was set to 1.5 kV and the heated capillary temperature to 250°C. HCD MS/MS spectra were recorded in the data-dependent mode using Top 20 method for quantitative analysis, respectively. MS1 spectra were measured with a resolution of 70,000, an AGC target of 3e6, and a mass range from m/z 300 to 1800. HCD MS/MS spectra were acquired with a resolution of 17,500, an AGC target of 2e5, and normalized collision energy of 28. Peptide m/z that triggered MS/MS scans were dynamically excluded from further MS/MS scans for 20 s. For EThcD analysis, the spray voltage was set to 2.0 kV and the heated capillary temperature to 320°C. MS1 spectra were measured with a resolution of 120,000, an AGC target of 5e5, and a mass range from m/z 350 to 1500. EThcD MS/MS spectra were acquired with a resolution of 60,000, an AGC target of 5e4, and normalized collision energy of 30. Peptide identification and quantification. Raw data files were searched against Homo sapiens Uniprot canonical database (Dec 2, 2016, 20,130 entries). Blind search and targeted search were performed with TagRecon (Version 1.4.47)7 and pFind 3.0 software,8-10 respectively. For TagRecon based blind PTM search, the maximum modification mass was 500 Da, precursor ion mass tolerance was 0.01 Da, and fragmentation tolerance was 0.1 Da. For pFind analysis, precursor ion mass and fragmentation tolerance was 10 ppm for the database search. A semi-tryptic search was employed with a maximum of three missed cleavages allowed. The maximum number of modifications allowed per peptide was three. Cysteine iodoacetamide alkylation (+ 57.0214 Da) was searched as fixed modification. Modifications of 15.9949 Da (Methionine oxidation, M), +293.1488 (C13H19N5O3, N-formyl product modified with HZyne-triazohexanoic acid, Any Nterminal), +264.1222 (C12H16N4O3, N-α-ketoacyl product modified with HZyne-triazohexanoic acid, Any N-terminal) were searched as dynamic modifications. A differential modification of 6.0201 Da on HZyne-derived modification was used for all analyses. The FDRs at spectrum, peptide, and protein level were < 1%.
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Analytical Chemistry
Quantification of heavy to light ratios (RH/L) was performed using pQuant as previously described11, which directly uses the RAW files as the input. pQuant calculates RH/L values based on each identified MS scan with a 10 ppm-level m/z tolerance window and assigns an interference score (Int. Score, also known as confidence score σ) to each value from zero to one. In principle, the lower the calculated Int. Score, the less co-elution interference signal was observed in the extracted ion chromatograms. In this regard, the median of peptide ratios with sigma less than or equal 0.5 were considered to calculate site-level ratios. Quantification results were obtained from two biological replicates with single 75-min LC-MS/MS run for each. Quantification results were obtained from at least three biological replicates with singe LCMS/MS run for each. Bioinformatics. Gene ontology (GO) term classification, KEGG pathway and INTERPRO domain (P
