Abstract
Xenopus oocytes and embryos are model systems optimally suited for quantitative proteomics. This is due to the availability of large amount of protein material and the ease of physical manipulation. Furthermore, facile in vitro fertilization provides superbly synchronized embryos for cell cycle and developmental stages. Here, we detail protocols developed over the last few years for sample preparation of multiplexed proteomics with TMT-tags followed by quantitative mass spectrometry analysis using the MultiNotch MS3 approach. In this approach, each condition is barcoded with an isobaric tag at the peptide level. After barcoding, samples are combined and the relative abundance of ~100,000 peptides is quantified on a mass spectrometer. High reproducibility of the sample preparation process prior to peptides being tagged and combined is of upmost importance for obtaining unbiased data. Otherwise, differences in sample handling can inadvertently appear as biological changes. We detail and exemplify the application of our sample workflow on an embryonic time-series of ten developmental stages of Xenopus laevis embryos ranging from the egg to stage 35 (just before hatching). Our accompanying paper (Chapter 14) details a bioinformatics pipeline to analyze the quality of the given sample preparation and strategies to convert spectra of X. laevis peptides into biologically interpretable data.
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References
Swammerdam J (1737) Bibilia Naturae; Sive historia insectorum, in classes certas redact 2
Prevost JL, Dumas J-B (1824) Nouvelle théorie de la génération. Ann Sci Nat 2
Gurdon JB, Elsdale TR, Fischberg M (1958) Sexually mature individuals of Xenopus laevis from the transplantation of single somatic nuclei. Nature 182(4627):64–65
Paine PL, Moore LC, Horowitz SB (1975) Nuclear envelope permeability. Nature 254(5496):109–114
Session AM, Uno Y, Kwon T, Chapman JA, Toyoda A, Takahashi S, Fukui A, Hikosaka A, Suzuki A, Kondo M, van Heeringen SJ, Quigley I, Heinz S, Ogino H, Ochi H, Hellsten U, Lyons JB, Simakov O, Putnam N, Stites J, Kuroki Y, Tanaka T, Michiue T, Watanabe M, Bogdanovic O, Lister R, Georgiou G, Paranjpe SS, van Kruijsbergen I, Shu S, Carlson J, Kinoshita T, Ohta Y, Mawaribuchi S, Jenkins J, Grimwood J, Schmutz J, Mitros T, Mozaffari SV, Suzuki Y, Haramoto Y, Yamamoto TS, Takagi C, Heald R, Miller K, Haudenschild C, Kitzman J, Nakayama T, Izutsu Y, Robert J, Fortriede J, Burns K, Lotay V, Karimi K, Yasuoka Y, Dichmann DS, Flajnik MF, Houston DW, Shendure J, DuPasquier L, Vize PD, Zorn AM, Ito M, Marcotte EM, Wallingford JB, Ito Y, Asashima M, Ueno N, Matsuda Y, Veenstra GJ, Fujiyama A, Harland RM, Taira M, Rokhsar DS (2016) Genome evolution in the allotetraploid frog Xenopus laevis. Nature 538(7625):336–343. https://doi.org/10.1038/nature19840
Gurdon JB, Wakefield, L (1986) Microinjection of amphibian oocytes and eggs for the analysis of transcription. Microinjection and Organelle Transplantation Techniques 269–299.
Markow TA, Beall S, Matzkin LM (2009) Egg size, embryonic development time and ovoviviparity in Drosophila species. J Evol Biol 22(2):430–434. https://doi.org/10.1111/j.1420-9101.2008.01649.x
Tartia AP, Rudraraju N, Richards T, Hammer MA, Talbot P, Baltz JM (2009) Cell volume regulation is initiated in mouse oocytes after ovulation. Development 136(13):2247–2254. https://doi.org/10.1242/dev.036756
Smits AH, Lindeboom RG, Perino M, van Heeringen SJ, Veenstra GJ, Vermeulen M (2014) Global absolute quantification reveals tight regulation of protein expression in single Xenopus eggs. Nucleic Acids Res 42(15):9880–9891. https://doi.org/10.1093/nar/gku661
Lombard-Banek C, Moody SA, Nemes P (2016) High-sensitivity mass spectrometry for probing gene translation in single embryonic cells in the early frog (Xenopus) embryo. Front Cell Dev Biol 4:100. https://doi.org/10.3389/fcell.2016.00100
Wühr M, Güttler T, Peshkin L, McAlister GC, Sonnett M, Ishihara K, Groen AC, Presler M, Erickson BK, Mitchison TJ, Kirschner MW, Gygi SP (2015) The nuclear proteome of a vertebrate. Curr Biol 25(20):2663–2671. https://doi.org/10.1016/j.cub.2015.08.047
Boke E, Ruer M, Wühr M, Coughlin M, Lemaitre R, Gygi SP, Alberti S, Drechsel D, Hyman AA, Mitchison TJ (2016) Amyloid-like self-assembly of a cellular compartment. Cell 166(3):637–650. https://doi.org/10.1016/j.cell.2016.06.051
Peshkin L, Wühr M, Pearl E, Haas W, Freeman RM Jr, Gerhart JC, Klein AM, Horb M, Gygi SP, Kirschner MW (2015) On the relationship of protein and mRNA dynamics in vertebrate embryonic development. Dev Cell 35(3):383–394. https://doi.org/10.1016/j.devcel.2015.10.010
Lombard-Banek C, Moody SA, Nemes P (2016) Single-cell mass spectrometry for discovery proteomics: quantifying translational cell heterogeneity in the 16-cell frog (Xenopus) embryo. Angew Chem Int Ed Engl 55(7):2454–2458. https://doi.org/10.1002/anie.201510411
Sun L, Bertke MM, Champion MM, Zhu G, Huber PW, Dovichi NJ (2014) Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development. Sci Rep 4:4365. https://doi.org/10.1038/srep04365
Presler MS, Van Itallie E, Klein AM, Kunz R, Coughlin P, Peshkin L, Gygi S, Wühr M, Kirschner M (2017) Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in the Xenopus egg. Proc Natl Acad Sci U S A. 2017 Dec 12;114(50):E10838-E10847. doi: 10.1073/pnas.1709207114
Sawin KE, Mitchison TJ (1991) Mitotic spindle assembly by two different pathways in vitro. J Cell Biol 112(5):925–940
Reinsch S, Karsenti E (1997) Movement of nuclei along microtubules in Xenopus egg extracts. Curr Biol 7(3):211–214
Wühr M, Chen Y, Dumont S, Groen AC, Needleman DJ, Salic A, Mitchison TJ (2008) Evidence for an upper limit to mitotic spindle length. Curr Biol 18(16):1256–1261. https://doi.org/10.1016/j.cub.2008.07.092
Gache V, Waridel P, Winter C, Juhem A, Schroeder M, Shevchenko A, Popov AV (2010) Xenopus meiotic microtubule-associated interactome. PLoS One 5(2):e9248. https://doi.org/10.1371/journal.pone.0009248
Liska AJ, Popov AV, Sunyaev S, Coughlin P, Habermann B, Shevchenko A, Bork P, Karsenti E, Shevchenko A (2004) Homology-based functional proteomics by mass spectrometry: application to the Xenopus microtubule-associated proteome. Proteomics 4(9):2707–2721. https://doi.org/10.1002/pmic.200300813
Wühr M, Freeman RM Jr, Presler M, Horb ME, Peshkin L, Gygi S, Kirschner MW (2014) Deep proteomics of the Xenopus laevis egg using an mRNA-derived reference database. Curr Biol 24(13):1467–1475. https://doi.org/10.1016/j.cub.2014.05.044
Deshmukh AS, Murgia M, Nagaraj N, Treebak JT, Cox J, Mann M (2015) Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors. Mol Cell Proteomics 14(4):841–853. https://doi.org/10.1074/mcp.M114.044222
Eng JK, McCormack AL, Yates JR (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5(11):976–989. https://doi.org/10.1016/1044-0305(94)80016-2
Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1(5):376–386
Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M (2014) Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics 13(9):2513–2526. https://doi.org/10.1074/mcp.M113.031591
Rosenberger G, Bludau I, Schmitt U, Heusel M, Hunter CL, Liu Y, MacCoss MJ, MacLean BX, Nesvizhskii AI, Pedrioli PGA, Reiter L, Rost HL, Tate S, Ting YS, Collins BC, Aebersold R (2017) Statistical control of peptide and protein error rates in large-scale targeted data-independent acquisition analyses. Nat Methods 14(9):921–927. https://doi.org/10.1038/nmeth.4398
McAlister GC, Nusinow DP, Jedrychowski MP, Wühr M, Huttlin EL, Erickson BK, Rad R, Haas W, Gygi SP (2014) Multinotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal Chem 86(14):7150–7158. https://doi.org/10.1021/ac502040v
Wenger CD, Lee MV, Hebert AS, McAlister GC, Phanstiel DH, Westphall MS, Coon JJ (2011) Gas-phase purification enables accurate, multiplexed proteome quantification with isobaric tagging. Nat Methods 8(11):933–935. https://doi.org/10.1038/nmeth.1716
Ting L, Rad R, Gygi SP, Haas W (2011) MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat Methods 8(11):937–940. https://doi.org/10.1038/nmeth.1714
Wühr M, Haas W, McAlister GC, Peshkin L, Rad R, Kirschner MW, Gygi SP (2012) Accurate multiplexed proteomics at the MS2 level using the complement reporter ion cluster. Anal Chem 6;84(21):9214–9221
Wessel D, Flugge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138 (1):141-143. doi:0003-2697(84)90782-6 [pii]
Chevallet M, Diemer H, Van Dorssealer A, Villiers C, Rabilloud T (2007) Toward a better analysis of secreted proteins: the example of the myeloid cells secretome. Proteomics 7(11):1757–1770. https://doi.org/10.1002/pmic.200601024
Mitulovic G, Stingl C, Steinmacher I, Hudecz O, Hutchins JR, Peters JM, Mechtler K (2009) Preventing carryover of peptides and proteins in nano LC-MS separations. Anal Chem 81(14):5955–5960. https://doi.org/10.1021/ac900696m
Edwards A, Haas W (2016) Multiplexed quantitative proteomics for high-throughput comprehensive proteome comparisons of human cell lines. Methods Mol Biol 1394:1–13. https://doi.org/10.1007/978-1-4939-3341-9_1
Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2(8):1896–1906. https://doi.org/10.1038/nprot.2007.261
Lyon RP, Setter JR, Bovee TD, Doronina SO, Hunter JH, Anderson ME, Balasubramanian CL, Duniho SM, Leiske CI, Li F, Senter PD (2014) Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates. Nat Biotechnol 32(10):1059–1062. https://doi.org/10.1038/nbt.2968
Dworkin MB, Dworkin-Rastl E (1989) Metabolic regulation during early frog development: glycogenic flux in Xenopus oocytes, eggs, and embryos. Dev Biol 132(2):512–523
Thompson A, Schafer J, Kuhn K, Kienle S, Schwarz J, Schmidt G, Neumann T, Johnstone R, Mohammed AK, Hamon C (2003) Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem 75(8):1895–1904
Sonnett M, Yeung E, Wühr M (2018) Accurate, Sensitive, and Precise Multiplexed Proteomics Using the Complement Reporter Ion Cluster. Analytical Chemistry 90(8):5032–5039
Acknowledgments
We thank Thao Nguyen for help collecting the Xenopus embryonic time series and Felix Keber for comments and suggestions on the manuscript. M.P. was supported by NIH grant R01GM103785. M.S. was supported by NIH F31 predoctoral fellowship 5F31GM116451. This work was supported by NIH grant 1R35GM128813 and Princeton University startup funding.
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Gupta, M., Sonnett, M., Ryazanova, L., Presler, M., Wühr, M. (2018). Quantitative Proteomics of Xenopus Embryos I, Sample Preparation. In: Vleminckx, K. (eds) Xenopus. Methods in Molecular Biology, vol 1865. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8784-9_13
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DOI: https://doi.org/10.1007/978-1-4939-8784-9_13
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