Skip to main content

Advertisement

SpringerLink
Log in

Coupling suspension trapping–based sample preparation and data-independent acquisition mass spectrometry for sensitive exosomal proteomic analysis

  • Paper in Forefront
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

It has been a challenge to analyze minute amounts of proteomic samples in a facile and robust manner. Herein, we developed a quantitative proteomics workflow by integrating suspension trapping (S-Trap)–based sample preparation and label-free data-independent acquisition (DIA) mass spectrometry and then applied it for the analysis of microgram and even nanogram amounts of exosome samples. S-Trap–based sample preparation outperformed the traditional in-solution digestion-based approach and the commonly used filter-aided sample preparation (FASP)–based approach with regard to the number of proteins and peptides identified. Moreover, S-Trap–based sample preparation coupled with DIA mass spectrometry also showed the highest reproducibility for protein quantification. In addition, this approach allowed for identification and quantification of exosome proteins with low starting amounts (down to 50 ~ 200 ng). Finally, the proposed method was successfully applied to label-free quantification of exosomal proteins extracted from MDA-MB-231 breast cancer cells and MCF-10A non-tumorigenic epithelial breast cells. Prospectively, we envision the integrated S-Trap sample preparation coupled with DIA quantification strategy as a promising alternative for highly efficient and sensitive analysis of trace amounts of proteomic samples (e.g., exosomal samples).

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Aebersold R, Mann M. Mass-spectrometric exploration of proteome structure and function. Nature. 2016;537(7620):347–55.

    Article  CAS  Google Scholar 

  2. Zhang Y, Fonslow BR, Shan B, Baek M-C, Yates JR. Protein analysis by shotgun/bottom-up proteomics. Chem Rev. 2013;113(4):2343–94.

    Article  CAS  Google Scholar 

  3. Ye X, Tang J, Mao Y, Lu X, Yang Y, Chen W, et al. Integrated proteomics sample preparation and fractionation: method development and applications. TrAC Trends Anal Chem. 2019;120:115667.

    Article  CAS  Google Scholar 

  4. Ma J, Zhang L, Liang Z, Shan Y, Zhang Y. Immobilized enzyme reactors in proteomics. TrAC Trends Anal Chem. 2011;30(5):691–702.

    Article  CAS  Google Scholar 

  5. Wiśniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis. Nat Methods. 2009;6(5):359–62.

    Article  Google Scholar 

  6. Liebler DC, Ham A-JL. Spin filter–based sample preparation for shotgun proteomics. Nat Methods. 2009;6(11):785–785.

    Article  CAS  Google Scholar 

  7. Kulak NA, Pichler G, Paron I, Nagaraj N, Mann M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat Methods. 2014;11(3):319–24.

    Article  CAS  Google Scholar 

  8. Zhu Y, Piehowski PD, Zhao R, Chen J, Shen Y, Moore RJ, et al. Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells. Nat Commun. 2018;9(1):882.

    Article  Google Scholar 

  9. Yi L, Piehowski PD, Shi T, Smith RD, Qian W-J. Advances in microscale separations towards nanoproteomics applications. J Chromatogr A. 2017;1523:40–8.

    Article  CAS  Google Scholar 

  10. Zougman A, Selby PJ, Banks RE. Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis. Proteomics. 2014;14(9):1006–1000.

    Article  CAS  Google Scholar 

  11. HaileMariam M, Eguez RV, Singh H, Bekele S, Ameni G, Pieper R, et al. S-Trap, an ultrafast sample-preparation approach for shotgun proteomics. J Proteome Res. 2018;17(9):2917–24.

    Article  CAS  Google Scholar 

  12. Hughes CS, Moggridge S, Müller T, Sorensen PH, Morin GB, Krijgsveld J. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat Protoc. 2019 Jan;14(1):68–85.

    Article  CAS  Google Scholar 

  13. Chen W, Wang S, Adhikari S, Deng Z, Wang L, Chen L, et al. Simple and integrated spintip-based technology applied for deep proteome profiling. Anal Chem. 2016;88(9):4864–71.

    Article  CAS  Google Scholar 

  14. Lange V, Picotti P, Domon B, Aebersold R. Selected reaction monitoring for quantitative proteomics: a tutorial. Mol Syst Biol. 2008;4(1):222.

    Article  Google Scholar 

  15. Peterson AC, Russell JD, Bailey DJ, Westphall MS, Coon JJ. Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics. Mol Cell Proteomics. 2012;11(11):1475–88.

    Article  Google Scholar 

  16. Gillet LC, Navarro P, Tate S, Röst H, Selevsek N, Reiter L, et al. Targeted data extraction of the MS/MS spectra generated by data-independent acquisition: a new concept for consistent and accurate proteome analysis. Mol Cell Proteomics. 2012;11(6):O111.016717.

    Article  Google Scholar 

  17. Ludwig C, Gillet L, Rosenberger G, Amon S, Collins BC, Aebersold R. Data-independent acquisition-based SWATH-MS for quantitative proteomics: a tutorial. Mol Syst Biol. 2018;14(8).

  18. Li W, Chi H, Salovska B, Wu C, Sun L, Rosenberger G, et al. Assessing the relationship between mass window width and retention time scheduling on protein coverage for data-independent acquisition. J Am Soc Mass Spectrom. 2019;30(8):1396–405.

    Article  CAS  Google Scholar 

  19. Mittelbrunn M, Sánchez-Madrid F. Intercellular communication: diverse structures for exchange of genetic information. Nat Rev Mol Cell Biol. 2012;13(5):328–35.

    Article  CAS  Google Scholar 

  20. Simons M, Raposo G. Exosomes–vesicular carriers for intercellular communication. Curr Opin Cell Biol. 2009;21(4):575–81.

    Article  CAS  Google Scholar 

  21. Théry C, Zitvogel L, Amigorena S. Exosomes: composition, biogenesis and function. Nat Rev Immunol. 2002;2(8):569–79.

    Article  Google Scholar 

  22. Simpson RJ, Lim JW, Moritz RL, Mathivanan S. Exosomes: proteomic insights and diagnostic potential. Expert Rev Proteomics. 2009;6(3):267–83.

    Article  CAS  Google Scholar 

  23. Hou R, Li Y, Sui Z, Yuan H, Yang K, Liang Z, et al. Advances in exosome isolation methods and their applications in proteomic analysis of biological samples. Anal Bioanal Chem. 2019;411(21):5351–61.

    Article  CAS  Google Scholar 

  24. Fricke F, Lee J, Michalak M, Warnken U, Hausser I, Suarez-Carmona M, et al. TGFBR2-dependent alterations of exosomal cargo and functions in DNA mismatch repair-deficient HCT116 colorectal cancer cells. Cell Commun Signal. 2017;15(1):14.

    Article  Google Scholar 

  25. Mitchell MI, Ben‐Dov IZ, Liu C, Ye K, Chow K, Kramer Y, et al. Extracellular vesicle capture by AnTibody of CHoice and Enzymatic Release (EV‐CATCHER): a customizable purification assay designed for small‐RNA biomarker identification and evaluation of circulating small‐EVs. J Extracell Vesicles. 2021;10(8).

  26. Aldeghaither DS, Zahavi DJ, Murray JC, Fertig EJ, Graham GT, Zhang Y-W, et al. A mechanism of resistance to antibody-targeted immune attack. Cancer Immunol Res. 2019;7(2):230–43.

    Article  CAS  Google Scholar 

  27. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B Methodol. 1995;57(1):289–300.

    Google Scholar 

  28. Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4(1):44–57.

    Article  CAS  Google Scholar 

  29. Galperin MY, Cochrane GR. Nucleic acids research annual database issue and the NAR online Molecular Biology Database Collection in 2009. Nucleic Acids Res. 2009 Jan 1;37(Database):D1–4.

  30. Ludwig KR, Schroll MM, Hummon AB. Comparison of in-solution, FASP, and S-Trap based digestion methods for bottom-up proteomic studies. J Proteome Res. 2018;17(7):2480–90.

    Article  CAS  Google Scholar 

  31. Keerthikumar S, Chisanga D, Ariyaratne D, Saffar H, Anand S, Zhao K, Samuel M, Pathan M, Jois M, Chilamkurti N, Gangoda L, Mathivanan S. ExoCarta: a web-based compendium of exosomal cargo. J Mol Biol. 2016;428(4):688–92.

    Article  CAS  Google Scholar 

  32. Risha Y, Minic Z, Ghobadloo SM, Berezovski MV. The proteomic analysis of breast cell line exosomes reveals disease patterns and potential biomarkers. Sci Rep. 2020 Dec;10(1):13572.

    Article  CAS  Google Scholar 

  33. Cho K-C, Clark DJ, Schnaubelt M, Teo GC, Leprevost F da V, Bocik W, et al. Deep proteomics using two dimensional data independent acquisition mass spectrometry. Anal Chem. 2020;92(6):4217–25.

    Article  CAS  Google Scholar 

  34. Yamaguchi H, Condeelis J. Regulation of the actin cytoskeleton in cancer cell migration and invasion. Biochim Biophys Acta BBA - Mol Cell Res. 2007;1773(5):642–52.

    Article  CAS  Google Scholar 

  35. Kanojia D, Morshed RA, Zhang L, Miska JM, Qiao J, Kim JW, et al. βIII-Tubulin regulates breast cancer metastases to the brain. Mol Cancer Ther. 2015;14(5):1152–61.

    Article  CAS  Google Scholar 

  36. Hoshino A, Costa-Silva B, Shen T-L, Rodrigues G, Hashimoto A, Tesic Mark M, et al. Tumour exosome integrins determine organotropic metastasis. Nature. 2015;527(7578):329–35.

    Article  CAS  Google Scholar 

  37. Tang H, Wang Y, Zhang B, Xiong S, Liu L, Chen W, et al. High brain acid soluble protein 1(BASP1) is a poor prognostic factor for cervical cancer and promotes tumor growth. Cancer Cell Int. 2017;17(1):97.

    Article  Google Scholar 

  38. Hsiao K-C, Shih N-Y, Fang H-L, Huang T-S, Kuo C-C, Chu P-Y, et al. Surface α-enolase promotes extracellular matrix degradation and tumor metastasis and represents a new therapeutic target. Karamanos NK, editor. PLoS ONE. 2013;8(7):e69354.

  39. Tu S-H, Chang C-C, Chen C-S, Tam K-W, Wang Y-J, Lee C-H, et al. Increased expression of enolase α in human breast cancer confers tamoxifen resistance in human breast cancer cells. Breast Cancer Res Treat. 2010 Jun;121(3):539–53.

    Article  CAS  Google Scholar 

  40. Li S, Li X, Yang S, Pi H, Li Z, Yao P, et al. Proteomic landscape of exosomes reveals the functional contributions of CD151 in triple-negative breast cancer. Mol Cell Proteomics. 2021;20:100121.

    Article  CAS  Google Scholar 

  41. Cascone T, McKenzie JA, Mbofung RM, Punt S, Wang Z, Xu C, et al. Increased tumor glycolysis characterizes immune resistance to adoptive T cell therapy. Cell Metab. 2018;27(5):977-987.e4.

    Article  CAS  Google Scholar 

  42. Wortzel I, Dror S, Kenific CM, Lyden D. Exosome-mediated metastasis: communication from a distance. Dev Cell. 2019;49(3):347–60.

    Article  CAS  Google Scholar 

  43. Costa-Silva B, Aiello NM, Ocean AJ, Singh S, Zhang H, Thakur BK, et al. Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat Cell Biol. 2015;17(6):816–26.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work is partially supported by NIH/NCI grant P30 CA051008 and GUMC institutional support. The Orbitrap Lumos Tribrid mass spectrometer is partially supported by Dekelbaum Foundation.

Author information

Authors and Affiliations

  1. Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, 20007, USA

    Ci Wu, Shiyun Zhou, Stephen Byers, Olivier Loudig & Junfeng Ma

  2. Center for Discovery and Innovation, Hackensack Meridian Health, Nutley, NJ, 07110, USA

    Megan I. Mitchell & Olivier Loudig

  3. Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, Liaoning, China

    Chunyan Hou

Authors
  1. Ci Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shiyun Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Megan I. Mitchell
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chunyan Hou
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Stephen Byers
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Olivier Loudig
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Junfeng Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Junfeng Ma.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wu, C., Zhou, S., Mitchell, M.I. et al. Coupling suspension trapping–based sample preparation and data-independent acquisition mass spectrometry for sensitive exosomal proteomic analysis. Anal Bioanal Chem 414, 2585–2595 (2022). https://doi.org/10.1007/s00216-022-03920-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-022-03920-z

Keywords

Search

Navigation