Brownridge, Philip

Jackson, M

McDonagh, Brian

Sakellariou, Yorgos

Smith, Neil

University of Liverpool Repository

Contents lists available at ScienceDirectData in BriefData in Brief 4 (2015) 344–348http://d2352-34(http://cE-mjournal homepage: www.elsevier.com/locate/dibData ArticleRedox proteomic analysis of the gastrocnemiusmuscle from adult and old miceBrian McDonagh a, Giorgos K. Sakellariou a, Neil T. Smith a,Philip Brownridge b, Malcolm J. Jackson aa Skeletal Muscle Pathophysiology Group, MRC-Arthritis Research UK Centre for Integrated Research intoMusculoskeletal Aging (CIMA), Institute of Ageing and Chronic Disease, University of Liverpool, UnitedKingdomb Centre for Proteome Research, Institute of Integrative Biology, University of Liverpool, United Kingdoma r t i c l e i n f oArticle history:Received 21 May 2015Received in revised form15 June 2015Accepted 23 June 2015Available online 2 July 2015Keywords:Redox proteomicsSkeletal muscleAgingOxidative stressx.doi.org/10.1016/j.dib.2015.06.01209/& 2015 The Authors. Published by Elsevreativecommons.org/licenses/by-nc-nd/4.0ail address: b.mcdonagh@liverpool.ac.uk (Ba b s t r a c tThe data provides information in support of the research article,“Differential Cysteine Labeling and Global Label-Free ProteomicsReveals an Altered Metabolic State in Skeletal Muscle Aging”, Journalof Proteome Research, 2014, 13 (11), 2008–21 [1]. Raw data isavailable from ProteomeXchange [2] with identiﬁer PDX001054. Theproteome of gastrocnemius muscle from adult and old mice wasanalyzed by global label-free proteomics and the relative quantiﬁca-tion of speciﬁc reduced and reversibly oxidized Cysteine (Cys)residues was performed using Skyline [3]. Brieﬂy, reduced Cysteine(Cys) containing peptides was alkylated using N-ethylmalemide (d0-NEM). Samples were desalted and reversibly oxidized Cys residueswere reduced using tris(2-carboxyethyl)phosphine (TCEP) and thenewly formed reduced Cys residues were labeled with heavy NEM(d5-NEM). Label-free analysis of the global proteome of adult (n¼5)and old (n¼4) gastrocnemius muscles was performed usingPeaks7™ mass spectrometry data analysis software [4]. Relativequantiﬁcation of Cys containing peptides that were identiﬁed asreduced (d(0) NEM labeled) and reversibly oxidized d(5)–NEMlabeled was performed using the intensity of their precursor ionsin Skyline. Results indicate that muscles from old mice show reducedier Inc. This is an open access article under the CC BY-NC-ND license/).. McDonagh).B. McDonagh et al. / Data in Brief 4 (2015) 344–348 345redox ﬂexibility particularly in proteins involved in the generation ofprecursor metabolites and energy metabolism, indicating a loss in theﬂexibility of the redox energy response.& 2015 The Authors. Published by Elsevier Inc. This is an open accessarticle under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).Speciﬁcations TableSubject area BiologyMore speciﬁc subject area Redox proteomics and agingType of data Excel sheets, ﬁguresHow data was acquired QExactive mass spectrometer coupled with ultimate 3000 RSLC nano system (Thermo Scientiﬁc).Data format Raw and processed data.Experimental factors Initial blocking of free thiols with d(0)NEM, reduction of reversibly oxidized Cys residues andlabeling of newly reduced residues with d(5) NEM.Experimental features Label-free analysis of global proteome and relative quantiﬁcation of oxidation state of redoxsensitive Cysteine residues.Data source location Liverpool, United KingdomData accessibility Data is available through ProteomeXchange with identiﬁer PDX001054 (http://www.ebi.ac.uk/pride/archive/projects/PXD001054).Value of the data A number of metabolic and muscle diseases are associated with aberrant redox regulation. Thecombination of label free analysis and redox proteomic data provides additional information onthe functional proteome. This is the ﬁrst article to our knowledge that combines differential Cys labeling with global label-free analysis of the proteome. Data provides valuable information on the changes that occur in the skeletal muscle proteomewith aging. Skeletal muscle aging is an ideal model for redox proteomics [5] and our ﬁndings suggest thatskeletal muscle aging is associated with a decrease in redox ﬂexibility.1. Data, experimental design, materials and methods1.1. Sample preparationAdult (12 months) and aged (25 months) C57BL/6 male mice were purchased from Charles River andhoused in the Speciﬁc Pathogen-Free (SPF) Facility at the University of Liverpool for at least 2 weeks beforeuse. All experiments were performed in accordancewith United KingdomHome Ofﬁce guidelines under theUnited Kingdom Animals (Scientiﬁc Procedures) Act 1986. Animals were sacriﬁced by cervical dislocationand gastrocnemius muscles were immediately dissected. A gastrocnemius muscle from each mouse wasplaced immediately in a thiol blocking buffer containing (25 mM d(0) NEM, 50 mM ammoniumbicarbonate, pH 8) for redox proteomic analysis. Brieﬂy protein extracts for redox analysis were preparedin the presence of thiol blocking buffer containing d(0) NEM under anaerobic conditions. Homogenizedprotein lysates were cleared by centrifugation at 15,000g for 10 min at 4 1C and protein concentrations werecalculated by Bradford assay (BioRad) using BSA as a standard. Protein extracts for redox analysis weredesalted using Zeba spin desalting columns (Thermo) and protein concentrations were re-calculated asbefore. 200 mg in 160 ml of 25 mM ammonium bicarbonate of the desalted protein extract was denaturedby addition of 10 μL of 1% w/v RapiGest™ (Waters, Manchester, UK) in 25 mM ammonium bicarbonatefollowed by incubation at 80 1C for 10 min. Reversibly oxidized Cys residues were reduced by the addition ofB. McDonagh et al. / Data in Brief 4 (2015) 344–34834610 ml of 100mM TCEP and incubated at 60 1C for 10 min. Newly reduced Cys residues were subsequentlyalkylated with 10 ml of 200mM d(5) NEM and incubated at room temperature for 30 min. Trypsin (Sigma,Poole, UK) was reconstituted in 50 mM acetic acid and 2 μg added to the samples followed by incubationovernight at 37 1C. The digestion was terminated and RapiGest™ removed by acidiﬁcation (3 μL of TFA andincubation at 37 1C for 45 min) and centrifugation (15,000  g for 15 min).1.2. Sample analysisThe data-dependent label-free analysis was performed using an Ultimate 3000 RSLC™ nano systemcoupled to a QExactive™ mass spectrometer (Thermo Scientiﬁc). The sample (5 mL corresponding to200 ng of protein) was loaded onto the trapping column (Thermo Scientiﬁc, PepMap100, C18,75 μm20 mm), using partial loop injection, for 7 min at a ﬂow rate of 4 μL/min with 0.1% (v/v) TFA.The sample was resolved on the analytical column (Easy-Spray C18 75 mm500 mm2 mm column)using a gradient of 97% A (0.1% formic acid) 3% B (99.9% ACN 0.1% formic acid) to 60% A 40% B over120 min at a ﬂow rate of 300 nL/min. Data dependent acquisition consisted of a 70,000 resolution full-scan MS scan (AGC set to 106 ions with a maximum ﬁll time of 250 ms). The 10 most abundant peakswere selected for MS/MS using a 17,000 resolution scan (AGC set to 5104 ions with a maximum ﬁlltime of 250 ms) with an ion selection window of 3 m/z and a normalized collision energy of 30. Toavoid repeated selection of peptides for MS/MS the program used a 30 s dynamic exclusion window.1.3. Data processing and quantiﬁcationRaw spectra were converted to mascot generated ﬁles (mgf) using Proteome Discoverer software(Thermo Scientiﬁc). The resulting mgf ﬁles were searched against Uniprot Mouse database sequencedatabase (12/05/2012, 16376 sequences) using an in-house Mascot server (Matrix Science, London, UK).Search parameters used were: peptide mass tolerances, 10 ppm; fragment mass tolerance, 0.01 Da, 1þ , 2þand 3þ ions; missed cleavages, 1; instrument type, ESI-TRAP. Variable modiﬁcations included were: d(0) NEM, d(5) NEM, mono-, di- and tri-oxidation of Cys residues and oxidation of methionine. Label-freerelative quantiﬁcation software Peaks7™ was used to analyze RAW data ﬁles against the same mouseprotein database used for identiﬁcations with Mascot. Proteins were considered signiﬁcantly changedbetween adult and aged samples using a -10logP score of 20 (equivalent to a p value of 0.01), a fold changeZ1.5, using a quality value of 0.8 and FDR set to 1%. Supplementary ﬁle 1 contains additional informationon the search and result parameters used including a Volcano plot for peptides, the distribution of featurevector ratio by quality, distribution of feature vector ratio by intensity, retention time shift distribution andM/Z Shift distribution. A volcano plot of the expression of proteomic data is provided in Fig. 1 and a list ofproteins identiﬁed and quantiﬁed is included in Supplementary Table 1. An example of a peptide containingFig. 1. Volcano plot showing distribution and log 2 fold change of proteins identiﬁed from Gastrocnemius muscle from adultand old mice.0 20 40 60 80 100Time (min)020406080100020406080100Relative AbundanceR.T. 57.70m/z 957.42R.T. 57.62m/z 959.935Fig. 2. Representative extracted ion chromatogram (XIC) and fragmentation of the double charged Aconitase peptideVGILSCTNNSSYEDMGR (containing Cys385) from a single analysis. The peptide containing the Cys residue labeled with d(0) NEM corresponds to the reduced form of the peptide and the d(5) NEM labeled peptide corresponds to reversibly oxidizedCys residue. The ratio of the intensity of the redox state of the Cys residue can be estimated from the intensity of the precursorions and can be combined with complimentary data on the protein's abundance.B. McDonagh et al. / Data in Brief 4 (2015) 344–348 347a redox sensitive Cys residue (Cys385 from aconitase) is presented in Fig. 2. Analysis of redox peptidesidentiﬁed with both d(0) NEM and d(5) NEMwas performed using Skyline and the relative quantiﬁcation ofthe reversible oxidation state of Cys residues (reduced:reversibly oxidized) was calculated from the intensityof precursor ions and is included in Supplementary Table 2.AcknowledgmentsBMcD is supported by the Wellcome Trust ISSF Fund and the University of Liverpool (097826/Z/11/Z).Appendix A. Supporting informationSupplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.dib.2015.06.012.References[1] B. McDonagh, G.K. Sakellariou, N.T. Smith, P. Brownridge, M.J. Jackson, Differential Cysteine labelling and global lable-freeproteomics reveals an altered metabolic state in skeletal muscle aging, J. Proteome Res. 13 (2014) 2008–2021.B. McDonagh et al. / Data in Brief 4 (2015) 344–348348[2] J.A. Vizcaíno, E.W. Deutsch, R. Wang, A. Csordas, F. Reisinger, D. Ríos, J.A. Dianes, Z. Sun, T. Farrah, N. Bandeira, P.A. Binz,I. Xenarios, M. Eisenacher, G. Mayer, L. Gatto, A. Campos, R.J. Chalkley, H.J. Kraus, J.P. Albar, S. Martinez-Bartolomé,R. Apweiler, G.S. Omenn, L. Martens, A.R. Jones, H. Hermjakob, ProteomeXchange provides globally co-ordinated proteomicsdata submission and dissemination, Nat. Biotechnol. 30 (2014) 223–226.[3] J. Zhang, L. Xin, B. Shan, W. Chen, M. Xie, D. Yuen, W. Zhang, Z. Zhang, G. Lajoie, B. Ma, PEAKS DB: de novo sequencingassisted database search for sensitive and accurate peptide identiﬁcation, Mol. Cell. Proteomics (2011)http://dx.doi.org/10.1074/mcp.M111.010587. M111.010587.[4] B. Schilling, M.J. Rardin, B.X. MacLean, A.M. Zawadzka, B.E. Frewen, M.P. Cusack, D.J. Sorensen, M.S. Bereman, E. Jing, C.C. Wu,E. Verdin, C.R. Kahn, M.J. Maccoss, B.W. Gibson, Platform-independent and label-free quantitation of proteomic data usingMS1 extracted ion chromatograms in skyline: application to protein acetylation and phosphorylation, Mol. Cell. Proteomics11 (2012) 202–214.[5] B. McDonagh, G.K. Sakellariou, M.J. Jackson, Application of redox proteomics to skeletal muscle aging and exercise, Biochem.Soc. Trans. 42 (2014) 965–970.

Redox proteomic analysis of the gastrocnemius muscle from adult and old mice.

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