Biobanking pp 403-416 | Cite as

Shotgun Proteomic Profiling of Bloodborne Nanoscale Extracellular Vesicles

  • Pete HeinzelmanEmail author
  • David N. Powers
  • James A. Wohlschlegel
  • Varghese John
Part of the Methods in Molecular Biology book series (MIMB, volume 1897)


Analyses of bloodborne nanoscale extracellular vesicles (nsEVs) have shown tremendous promise in enabling the development of noninvasive blood-based clinical diagnostic tests, predicting and monitoring the efficacy of treatment programs, and identifying new drug targets in the context of health conditions such as cancer and Alzheimer’s disease. In this chapter we present a protocol for generating global nsEV proteomic profiles that can further the utility of nsEV analysis for the above biomedical applications by enlightening us of differences in protein abundance across normal and disease state nsEVs. This protocol features the use of magnetic particle-based immunoprecipitation to enrich highly purified populations of nsEVs directly from plasma or serum samples. The constituent proteins of these vesicles are subsequently characterized using a comparative shotgun proteomics approach that entails bottom-up, tandem mass spectrometric analysis of peptides generated by proteolytic digestion of nsEV-derived proteins. The methods described here are compatible with parallel processing of dozens of plasma or serum samples and can be valuable tools in enabling nsEV biomarker discoveries that have high translational relevance in the development of both novel therapeutics and blood sample diagnostic assays.

Key words

Proteomics Extracellular vesicle Ectosome Exosome Mass spectrometry Diagnostics Liquid biopsy Immunoprecipitation Drug discovery 


  1. 1.
    Cocucci E, Meldolesi J (2015) Ectosomes and exosomes: shedding the confusion between extracellular vesicles. Trends Cell Biol 25:364–372CrossRefGoogle Scholar
  2. 2.
    Shi M, Liu C, Cook TJ, Bullock KM, Zhao Y, Ginghina C, Li Y, Aro P, Dator R, He C, Hipp MJ, Zabetian CP, Peskind ER, Hu SC, Quinn JF, Galasko DR, Banks WA, Zhang J (2014) Plasma exosomal α-synuclein is likely CNS-derived and increased in Parkinson’s disease. Acta Neuropathol 128:639–650CrossRefGoogle Scholar
  3. 3.
    Yoshioka Y et al (2014) Ultra-sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen. Nat Commun 7(5):3591CrossRefGoogle Scholar
  4. 4.
    He M, Crow J, Roth M, Zeng Y, Godwin AK (2014) Integrated immunoisolation and protein analysis of circulating exosomes using microfluidic technology. Lab Chip 14:3773–3780CrossRefGoogle Scholar
  5. 5.
    Fiandaca MS, Kapogiannis D, Mapstone M, Boxer A, Eitan E, Schwartz JB, Abner EL, Petersen RC, Federoff HJ, Miller BL, Goetzl EJ (2015) Identification of preclinical Alzheimer’s disease by a profile of pathogenic proteins in neurally derived blood exosomes: a case-control study. Alzheimers Dement 11:600–607CrossRefGoogle Scholar
  6. 6.
    Goetzl EJ, Boxer A, Schwartz JB, Abner EL, Petersen RC, Miller BL, Kapogiannis D (2015) Altered lysosomal proteins in neural-derived plasma exosomes in preclinical Alzheimer disease. Neurology 85(1):40–47CrossRefGoogle Scholar
  7. 7.
    Goetzl EJ, Boxer A, Schwartz JB, Abner EL, Petersen RC, Miller BL, Carlson OD, Mustapic M, Kapogiannis D (2015) Low neural exosomal levels of cellular survival factors in Alzheimer’s disease. Ann Clin Transl Neurol 2:769–773CrossRefGoogle Scholar
  8. 8.
    Kapogiannis D, Boxer A, Schwartz JB, Abner EL, Biragyn A, Masharani U, Frassetto L, Petersen RC, Miller BL, Goetzl EJ (2015) Dysfunctionally phosphorylated type 1 insulin receptor substrate in neural-derived blood exosomes of preclinical Alzheimer’s disease. FASEB J 29(2):589–596CrossRefGoogle Scholar
  9. 9.
    Caradec J, Kharmate G, Hosseini-Beheshti E, Adomat H, Gleave M, Guns E (2014) Reproducibility and efficiency of serum-derived exosome extraction methods. Clin Biochem 47:1286–1292CrossRefGoogle Scholar
  10. 10.
    Kanninen KM, Bister N, Koistinaho J, Malm T (2015) Exosomes as new diagnostic tools in CNS diseases. Biochim Biophys Acta S09:292–296Google Scholar
  11. 11.
    Webber J, Clayton A (2013) How pure are your vesicles? J Extracell Vesicles 2.
  12. 12.
    Xie H, Griffin TJ (2006) Trade-off between high sensitivity and increased potential for false positive peptide sequence matches using a two-dimensional linear ion trap for tandem mass spectrometry-based proteomics. J Proteome Res 5(4):1003–1009CrossRefGoogle Scholar
  13. 13.
    Rudnick SI, Adams GP (2007) Affinity and avidity in antibody-based tumor targeting. Cancer Biother Radiopharm 24:155–161CrossRefGoogle Scholar
  14. 14.
    Liao L, McClatchy DB, Yates JR (2009) Shotgun proteomics in neuroscience. Neuron 63:12–26CrossRefGoogle Scholar
  15. 15.
    Elias JE, Gygi SP (2010) Target-decoy search strategy for mass spectrometry-based proteomics. Methods Mol Biol 604:55–71CrossRefGoogle Scholar
  16. 16.
    He L, Diedrich J, Chu YY, Yates JR 3rd (2015) Extracting accurate precursor information for tandem mass spectra by RawConverter. Anal Chem 87:11361–11367CrossRefGoogle Scholar
  17. 17.
    Kelstrup CD, Young C, Lavallee R, Nielsen ML, Olsen JV (2012) Optimized fast and sensitive acquisition methods for shotgun proteomics on a quadrupole orbitrap mass spectrometer. J Proteome Res 11:3487–3497CrossRefGoogle Scholar
  18. 18.
    Michalski A, Damoc E, Hauschild JP, Lange O, Wieghaus A, Makarov A, Nagaraj N, Cox J, Mann M, Horning S (2011) Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer. Mol Cell Proteomics 10(9):M111CrossRefGoogle Scholar
  19. 19.
    Xu T, Park SK, Venable JD, Wohlschlegel JA, Diedrich JK, Cociorva D, Lu B, Liao L, Hewel J, Han X, Wong CC, Fonslow B, Delahunty C, Gao Y, Shah H, Yates JR 3rd (2015) ProLuCID: an improved SEQUEST-like algorithm with enhanced sensitivity and specificity. J Proteome 129:16–24CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Pete Heinzelman
    • 1
    Email author
  • David N. Powers
    • 2
  • James A. Wohlschlegel
    • 2
  • Varghese John
    • 1
  1. 1.Drug Discovery Laboratory, Department of Neurology, Mary S. Easton Center for Alzheimer’s Disease ResearchUniversity of California, Los AngelesLos AngelesUSA
  2. 2.Department of Biological Chemistry, Proteome Research CenterUniversity of California, Los AngelesLos AngelesUSA

Personalised recommendations