Novel multifunctional reagents were applied in combination with a lipid probe for affinity enrichment of myristoylated proteins and direct detection of lipid-modified tryptic peptides by mass spectrometry. This method enables high-confidence identification of the myristoylated proteome on an unprecedented scale in cell culture, and allowed the first quantitative analysis of dynamic changes in protein lipidation during vertebrate embryonic development

Boersema

Dieterich

Hannoush

Heal

Speers

Tate

Thinon

Vizcaíno

Wilson

Wright

Zhang

Zheng

Angewandte Chemie International Edition

PubMed

Broncel, Malgorzata

Serwa, Remigiusz A

Ciepla, Paulina

Krause, Eberhard

Dallman, Margaret J

Magee, Anthony I

Tate, Edward W

Hochschulbibliothekszentrum des Landes Nordrhein-Westfalen (hbz)

Multifunctional Reagents for Quantitative Proteome-Wide Analysis of Protein Modification in Human Cells and Dynamic Profiling of Protein Lipidation During Vertebrate Development

Malgorzata Broncel

Remigiusz A. Serwa

Paulina Ciepla

Eberhard Krause

Margaret J. Dallman

Anthony I. Magee

Edward W. Tate

Crossref

Angewandte Chemie

Novel multifunctional reagents were applied in
combination with a lipid probe for affinity enrichment of
myristoylated proteins and direct detection of lipid-modified
tryptic peptides by mass spectrometry. This method enables
high-confidence identification of the myristoylated proteome
on an unprecedented scale in cell culture, and allowed the first
quantitative analysis of dynamic changes in protein lipidation
during vertebrate embryonic development

Broncel, M

Serwa, RA

Ciepla, P

Krause, E

Dallman, MJ

Magee, AI

Tate, EW

Spiral - Imperial College Digital Repository

German Edition: DOI: 10.1002/ange.201500342Proteomics Hot PaperInternational Edition: DOI: 10.1002/anie.201500342Multifunctional Reagents for Quantitative Proteome-Wide Analysis ofProtein Modification in Human Cells and Dynamic Profiling of ProteinLipidation During Vertebrate Development**Malgorzata Broncel, Remigiusz A. Serwa,* Paulina Ciepla, Eberhard Krause,Margaret J. Dallman, Anthony I. Magee, and Edward W. Tate*Abstract: Novel multifunctional reagents were applied incombination with a lipid probe for affinity enrichment ofmyristoylated proteins and direct detection of lipid-modifiedtryptic peptides by mass spectrometry. This method enableshigh-confidence identification of the myristoylated proteomeon an unprecedented scale in cell culture, and allowed the firstquantitative analysis of dynamic changes in protein lipidationduring vertebrate embryonic development.Although advancements in protein mass spectrometry (MS)have enabled global profiling of numerous post-translationalmodifications (PTMs), confident high-throughput identifica-tion of lipidated proteins remains problematic. Lipidationadversely affects solubility, chromatographic properties, andionization of modified peptides, and routine proteome-widedetection by MS and assignment of the modification sitesremains an unsolved challenge.[1] Furthermore, lipidatedproteins are often present at relatively low abundance incells, and lipid-specific enrichment is required to reducesample complexity and improve discovery. Enrichment strat-egies can exploit natural features of the lipid, for example, theuse of anti-farnesylpeptide antibodies;[2] indirect analysisthrough acyl–biotin exchange for S-acylation;[3] or moregeneralizable introduction of a functional handle throughmetabolic incorporation of a bioorthogonal chemical tag.[4]Co-translational myristoylation is a specific class ofprotein lipidation whereby N-myristoyl transferases (NMTs)catalyze the covalent and irreversible addition of a tetradeca-noyl chain at an N-terminal glycine (Gly), revealed by theaction of methionine aminopeptidases during protein syn-thesis at ribosomes.[5] Metabolic tagging has become thetechnique of choice for whole-proteome profiling of thecellular myristoylated proteome. As previously demon-strated,[6] metabolic tagging of a cellular proteome withtetradec-13-ynoic acid (YnMyr) and subsequent elaborationthrough ligation with secondary reporters (capture reagents)enables visualization, enrichment, and MS-based identifica-tion of myristoylated proteins. This chemoproteomic method-ology delivers greatly improved processing time and sensi-tivity over traditional radioisotope or direct MS approaches,but there are also limitations. Firstly, as with any affinityenrichment technique, unspecific protein background is oftenobserved when coupled to high-sensitivity MS detec-tion. Secondly, protein fatty acyl transferases that utilizelonger acyl-CoAs as substrates may also use YnMyr eithernatively or following chain extension, which may lead tofalse positive identifications.[7] Finally, robust and generalmethods for confirmation of the lipidation site have yet to beestablished.To overcome these limitations, we report a portfolio ofreagents for the enrichment and visualization of myristoy-lated proteomes and direct MS detection of lipid modificationof protein N termini. The design of these reagents is based oncompound 1 (Figure 1a), a robust tool for enrichment and in-gel fluorescence (igFl) analysis of metabolically taggedproteomes.[8] To make 1 amenable to the detection of lipid-modified peptides, we envisioned the introduction of a linker(Figure S1 in the Supporting Information) to facilitatecleavage as an integral part of a proteomic workflow. Ina standard tagging experiment (Figure 1b), cells or organismsare treated with YnMyr or a myristic acid (Myr) control tometabolically tag proteins via activation by cellular acyl-CoAsynthase followed by transfer by NMTs. The cells/organismsare harvested and the metabolically tagged proteins ligated toa capture reagent through copper-catalyzed alkyne azide[*] Dr. M. Broncel,[+] Dr. R. A. Serwa,[+] P. Ciepla, Prof. E. W. TateDepartment of Chemistry, Imperial College LondonExhibition Road, London SW7 2AZ (UK)E-mail: r.serwa@imperial.ac.uke.tate@imperial.ac.ukDr. E. KrauseLeibniz-Institut fír Molekulare Pharmakologie (FMP),Robert-Roessle-Str. 10, 13125 Berlin (Germany)Prof. M. J. DallmanDepartment of Life Sciences, Imperial College LondonExhibition Road, London SW7 2AZ (UK)Prof. A. I. MageeNational Heart and Lung Institute, Imperial College LondonExhibition Road, London SW7 2AZ (UK)[+] These authors contributed equally to this work.[**] This work was supported by Deutsche Forschungsgemeinschaft (Br4387/1-1) and European Union (PIEF-GA-2011-299740) fellowshipsto M.B.; European Union fellowship (PIEF-GA-2010-273868) toR.A.S.; Imperial College London Institute of Chemical BiologyEPSRC Centre for Doctoral Training studentship (EP/F500416/1) toP.C. MS proteomics data was deposited to the ProteomeXchangeConsortium[14] via the PRIDE partner repository with the datasetidentifiers PXD001863 and PXD001876.Supporting information for this article is available on the WWWunder http://dx.doi.org/10.1002/anie.201500342.Ó 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co.KGaA. This is an open access article under the terms of the CreativeCommons Attribution License, which permits use, distribution andreproduction in any medium, provided the original work is properlycited..AngewandteCommunications5948 Ó 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2015, 54, 5948 –5951cycloaddition (CuAAC) and affinity-enriched. The enrichedproteomes are visualized by igFl to assess metabolic taggingand enrichment efficiency, or analyzed through on-beadproteolytic digestion, MS analysis, and software-aided proteinidentification.We synthesized a library of reagents bearing enzymati-cally cleavable linkers (Figure 1a, Table S1 in the SupportingInformation). Reagents 2–5contain arginine (Arg) resi-dues and reagent 6 containsa lysine (Lys) residue toenable proteolysis by tryp-sin and simultaneous en-hancement ofthe ionization properties oflipidated peptides. Reagent7 in turn incorporates phe-nylalanine (Phe) andreagents 8–9 incorporateaspartic acid (Asp) to facil-itate cleavage by chymo-trypsin and AspN, respec-tively. These proteases areless commonly used in pro-teomic workflows, howeverthey may offer complemen-tary protein sequence cov-erage, thus improving over-all modified peptide discov-ery. Similarly to 1, com-pounds 2 and 6 wereequipped with theTAMRA fluorophore (R1)for fast visualization by igFl.All other reagents lack thefluorophore to reduce bulkand possible steric hin-drance, since biotin (con-tained in R2) is sufficientfor visualization (e.g., byusing streptavidin conju-gated to horseradish perox-idase (streptavidin–HRP)).In addition, 5 was equippedwith a-azido-arginine to fur-ther minimizethe size of the cleaved prod-uct. Finally, we aimed toenhance the ionizationproperties of the lipidatedpeptides still further byincorporating trimethyl (4,7, 8) or dimethyl (9) lysineresidues.Taking advantage ofboth the TAMRA andbiotin moieties, we firstinvestigated the efficiencyof 2–9 for the capture andenrichment of myristoylated proteins. HEK293 cells weretreated with YnMyr or Myr for 24 h followed by lysis, CuAACwith 1–9, and affinity enrichment on streptavidin-conjugatedbeads. Captured proteins were eluted by thermal denatura-tion, resolved by SDS-PAGE, and visualized by igFl orWestern Blotting with streptavidin–HRP (Figure S2a and b).We were pleased to observe that the majority of the reagentsFigure 1. a) Structures of capture reagents 1–9. Protease (trypsin, chymotrypsin, AspN) cleavage sites areindicated by arrows. b) General proteomic workflow; the capture reagent is shown schematically as a cartoon:star= TAMRA (R1), fused pentagons = biotin (R2).AngewandteChemie5949Angew. Chem. Int. Ed. 2015, 54, 5948 –5951 Ó 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.orgshowed comparable enrichment efficiency to 1 and that therewas negligible background signal, as shown by the Myrcontrol. However, the performance of 8 and 9 was clearlydiminished, which may indicate lower efficiency of CuAAC,and these reagents were excluded from further analysis. Wealso observed that despite their bulkier structures, 2 and 6performed as robustly as 1, which was demonstrated by twoalternative readouts.We then evaluated the capacity of thesereagents to identify proteins with a consen-sus myristoylation sequence (MG-pro-teins) and, most importantly, N-terminallymyristoylated peptides. Samples were pre-pared as described above, subjected toCuAAC with 2–7, affinity-enriched, anddigested with either trypsin (2–6) or chy-motrypsin (7), followed by MS analysis.Five reagents (2–4 and 6–7) identified38 lipid-modified peptides with high(> 99%) confidence (Figure S2c andTable S2 in the Supporting Information).Nearly 90 % of these peptides were discov-ered with 2, thus demonstrating its higheffectiveness despite the presence of theTAMRA dye and lower predicted netcharge compared to 3 and 4, respectively.Reagent 2 was also superior to 6, thussuggesting that the proteolysis step and/orthe ionization properties of the Arg-con-taining conjugates were improved com-pared to Lys since CuAAC and enrichmentwere similar (Figure S2). These steps alsoseemed efficient for 5, however, no lipi-dated peptides were detected; we speculatethat proximity of the triazole to the cleav-age site reduces the affinity for trypsin.Regardless of the reagent type, approxi-mately 30% of all of the proteins identifiedcarried an N-terminal MG motif(Table S3), which is more than four-foldhigher than in analysis of the nativeproteome, where the abundance of MGproteins is approximately 7%.[9] Theremaining 70 % of the detected proteinsrepresent tagging at sites other than the N terminus as a resultof the promiscuity of lipid transferases and proteins binding tobeads and/or tagged proteins, thus highlighting the utility ofmethodologies that allow either quantification of NMTinhibition (if reliable inhibitors are available)[6b] or directdetection of lipidated peptides to improve modified proteinID confidence.Having selected the best performing capture reagent (2),we next aimed to maximize the discovery of myristoylatedpeptides and the corresponding endogenous protein sub-strates of human NMT. Larger-scale chemical proteomicsexperiments were undertaken with three commonly used celllines: HeLa, MCF7, and HEK293. Two complementarysoftware packages were employed for MS data analysis,with primary identification of lipid-modified peptides per-formed with PEAKS7, and MaxQuant1.5 used as a secondaryplatform.[10] The latter was also used to quantify protein levelsbetween YnMyr- and Myr-treated samples, with Myr servingas a control for both nonspecific protein identification andreliability of the modified peptide identifications. 81 lipid-modified peptides were detected in PEAKS7 analysis, ofwhich approximately 40 % were common to all three cell lines(Figure 2a). MaxQuant analysis delivered 62 lipidated pep-tides, four of which were not observed in PEAKS7.[11]Importantly, each search engine returned only one lipidatedpeptide common to both YnMyr and Myr control samples,which was assigned as a false positive and excluded from thelist of myristoylated peptides (Table S4).Based on statistically significant enrichment of MGproteins pulled down from YnMyr-treated samples comparedto Myr controls, we identified 170, 163, and 145 candidatemyristoylated proteins for HEK293, HeLa, and MCF7 cells,respectively (Table S5). However, a previous comprehensivestudy of quantitative dose response to NMT inhibitionshowed that enrichment is only a simple and indirect measureof N-terminal myristoylation since only 70 out of 169 enrichedMG HeLa proteins robustly responded to NMT inhibition.[6b]In agreement with this alternative approach, our datasetFigure 2. a) Modified peptide discovery in human cells. b) Time-dependent metabolictagging with YnMyr visualized by igFl. c) Quantitative MS-based analysis of myristoylation indeveloping zebrafish embryos. Color coding represents normalized levels (log2 scale) ofmyristoylation. Protein targets are reported by gene names with peptide indicated as (+)..AngewandteCommunications5950 www.angewandte.org Ó 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2015, 54, 5948 –5951shows direct evidence for 87 of the candidate proteins in thethree cell lines. Herein, we provide direct proof for myris-toylation in Hela cells for 47 previously reported proteins, aswell as significantly increased coverage of since direct MS/MSevidence for myristoylated peptides was detected for 69enriched MG targets in HeLa, 65 in HEK293, and 50 in MCF7cells (Table S5). This represents the largest directly validateddatabase reported to date for the N-myristoylated humanproteome.We next applied 2 to profile the myristoylated proteomefor the first time in a developing organism. Followingevaluation of YnMyr tagging specificity (Figure S3), Daniorerio (zebrafish) embryos were metabolically tagged inspecific time windows (0–24, 48–72, and 96–120 hours post-fertilization (hpf)) and processed according to the standardworkflow. Chemical proteomics experiments revealed thescope of myristoylation in developing zebrafish; 72 potentialmyristoylation targets were identified based on statisticalanalyses of YnMyr/Myr enrichment and 56 lipid-modifiedpeptides were discovered (Tables S6 and S7). Notably, in theabsence of validated NMT inhibitors for zebrafish, themethodology presented herein is currently the only approachfor confident assignment of myristoylated proteins in thisanimal model.We also investigated the dynamic nature of myristoylationthrough pulsed YnMyr tagging as described above, coupledwith visualization by igFl and quantification through triplexdimethyl labeling[12] (Figure 2b and c, respectively). IgFlanalysis indicates development-stage-specific D. rerio proteinmyristoylation profiles that reflect differential protein expres-sion during embryonic development. Further MS-basedanalysis allowed the quantification of myristoylation dynam-ics for 54 zebrafish proteins (Figure 2c), which revealed thatmyristoylation is most prominent in early development.Several proteins myristoylated within 24 hpf (e.g., prkacaand gnai families) participate in progesterone-mediatedmaturation, the hedgehog and wnt signaling pathways,melanogenesis, and meiosis, all of which are critical in earlydevelopment (Table S8).In summary, the multifunctional capture reagentsreported herein enable robust identification of metabol-ically-tagged myristoylated proteomes, with unprecedentedconfidence resulting from the combination of chemical-probe-based enrichment and release and direct detection oflipid-modified peptides by MS. Previously reportedreagents[13] typically required an extra proteolytic step andtheir capacity to enable the detection of lipidated peptides hasnot been demonstrated. Herein, we report the largest data-base (87 counts) of experimentally validated human proteinsthat are myristoylated at an endogenous level in living cells.We also present the first profile of myristoylation in a livingmulticellular organism and the confident identification ofover 50 novel targets. Importantly, this work provides the firstexample of the analysis of any protein lipidation event duringvertebrate development. We envision that our reagents willfind application in the quantitative and dynamic analysis ofother PTMs, and in related workflows such as activity-basedprotein profiling, both in cells and in multicellular organisms.Keywords: capture reagents · lipidation · mass spectrometry ·post-translational modification · proteomicsHow to cite: Angew. Chem. Int. Ed. 2015, 54, 5948–5951Angew. Chem. 2015, 127, 6046–6049[1] H. Lin, X. Su, B. He, ACS Chem. Biol. 2012, 7, 947 – 960.[2] H. P. Lin, S. C. Hsu, J. C. Wu, I. J. Sheen, B. S. Yan, W. J. Syu, J.Gen. Virol. 1999, 80, 91 – 96.[3] R. N. Hannoush, J. Sun, Nat. Chem. Biol. 2010, 6, 498 – 506.[4] E. W. Tate, K. A. Kalesh, T. Lanyon-Hogg, E. M. Storck, E.Thinon, Curr. Opin. Chem. Biol. 2015, 24, 48 – 57.[5] M. H. Wright, W. P. Heal, D. J. Mann, E. W. Tate, J. Chem. Biol.2010, 3, 19 – 35.[6] a) M. H. Wright, B. Clough, M. D. Rackham, K. Rangachari,J. A. Brannigan, M. Grainger, D. K. Moss, A. R. Bottrill, W. P.Heal, M. Broncel, et al., Nat. Chem. 2014, 6, 112 – 121; b) E.Thinon, R. A. Serwa, M. Broncel, J. A. Brannigan, U. Brassat,M. H. Wright, W. P. Heal, A. J. Wilkinson, D. J. Mann, E. W.Tate, Nat. Commun. 2014, 5, 4919.[7] J. P. Wilson, A. S. Raghavan, Y. Y. Yang, G. Charron, H. C.Hang, Mol. Cell. Proteomics 2011, 10, M110 001198.[8] W. P. Heal, M. H. Wright, E. Thinon, E. W. Tate, Nat. Protoc.2012, 7, 105 – 117.[9] Uniprot.org.[10] a) J. Cox, M. Mann, Nat. Biotechnol. 2008, 26, 1367 – 1372; b) J.Zhang, L. Xin, B. Shan, W. Chen, M. Xie, D. Yuen, W. Zhang, Z.Zhang, G. A. Lajoie, B. Ma, Mol. Cell. Proteomics 2012, 11,M111 010587.[11] Distinct user-defined sets of parameters were chosen (typicallydefault) to perform database searches with MaxQuant1.5 andPEAKS7. The outcome is therefore not directly comparable. Seethe Supporting Information for details.[12] P. J. Boersema, R. Raijmakers, S. Lemeer, S. Mohammed, A. J.Heck, Nat. Protoc. 2009, 4, 484 – 494.[13] a) A. E. Speers, B. F. Cravatt, J. Am. Chem. Soc. 2005, 127,10018 – 10019; b) D. C. Dieterich, A. J. Link, J. Graumann, D. A.Tirrell, E. M. Schuman, Proc. Natl. Acad. Sci. USA 2006, 103,9482 – 9487; c) T. Zheng, H. Jiang, P. Wu, Bioconjugate Chem.2013, 24, 859 – 864.[14] J. A. Vizcano, E. W. Deutsch, R. Wang, A. Csordas, F. Reisinger,D. Ros, J. A. Dianes, Z. Sun, T. Farrah, N. Bandeira, et al., Nat.Biotechnol. 2014, 30, 223 – 226.Received: January 14, 2015Revised: February 14, 2015Published online: March 25, 2015AngewandteChemie5951Angew. Chem. Int. Ed. 2015, 54, 5948 –5951 Ó 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org

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Multifunctional Reagents for Quantitative Proteome-Wide Analysis of Protein Modification in Human Cells and Dynamic Profiling of Protein Lipidation During Vertebrate Development

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