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Quantitative Proteomics of Xenopus Embryos I, Sample Preparation

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1865))

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

  1. Swammerdam J (1737) Bibilia Naturae; Sive historia insectorum, in classes certas redact 2

    Google Scholar 

  2. Prevost JL, Dumas J-B (1824) Nouvelle théorie de la génération. Ann Sci Nat 2

    Google Scholar 

  3. 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

    Article  CAS  PubMed  Google Scholar 

  4. Paine PL, Moore LC, Horowitz SB (1975) Nuclear envelope permeability. Nature 254(5496):109–114

    Article  CAS  PubMed  Google Scholar 

  5. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Gurdon JB, Wakefield, L (1986) Microinjection of amphibian oocytes and eggs for the analysis of transcription. Microinjection and Organelle Transplantation Techniques 269–299.

    Google Scholar 

  7. 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

    Article  CAS  PubMed  Google Scholar 

  8. 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

    Article  CAS  PubMed  Google Scholar 

  9. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. 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

    Article  PubMed Central  PubMed  Google Scholar 

  11. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. 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

    Article  CAS  Google Scholar 

  17. Sawin KE, Mitchison TJ (1991) Mitotic spindle assembly by two different pathways in vitro. J Cell Biol 112(5):925–940

    Article  CAS  PubMed  Google Scholar 

  18. Reinsch S, Karsenti E (1997) Movement of nuclei along microtubules in Xenopus egg extracts. Curr Biol 7(3):211–214

    Article  CAS  PubMed  Google Scholar 

  19. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  21. 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

    Article  CAS  PubMed  Google Scholar 

  22. 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

    Article  PubMed  Google Scholar 

  23. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. 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

    Article  CAS  PubMed  Google Scholar 

  25. 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

    Article  CAS  PubMed  Google Scholar 

  26. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. 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

    Article  PubMed  PubMed Central  Google Scholar 

  32. 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]

    Google Scholar 

  33. 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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. 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

    Article  CAS  PubMed  Google Scholar 

  35. 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

    Article  CAS  PubMed  Google Scholar 

  36. 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

    Article  CAS  PubMed  Google Scholar 

  37. 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

    Article  CAS  PubMed  Google Scholar 

  38. 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

    Article  CAS  PubMed  Google Scholar 

  39. 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

    Article  CAS  PubMed  Google Scholar 

  40. 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

    Google Scholar 

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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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Authors and Affiliations

  1. Department of Molecular Biology and Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA

    Meera Gupta, Matthew Sonnett, Lillia Ryazanova & Martin Wühr

  2. Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, USA

    Meera Gupta

  3. Department of Systems Biology, Harvard Medical School, Boston, MA, USA

    Marc Presler

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  1. Meera Gupta
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  2. Matthew Sonnett
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  3. Lillia Ryazanova
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  4. Marc Presler
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Corresponding author

Correspondence to Martin Wühr .

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Editors and Affiliations

  1. Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium

    Kris Vleminckx

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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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