Autophagy pp 341-357 | Cite as

Identification and Regulation of Multimeric Protein Complexes in Autophagy via SILAC-Based Mass Spectrometry Approaches

  • Stéphanie Kaeser-Pebernard
  • Britta Diedrich
  • Jörn DengjelEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1880)


Mass spectrometry (MS)-based identification and characterization of protein complexes is becoming a prerequisite for in-depth biochemical analyses of intracellular processes. Here, we describe two state-of-the-art MS-based approaches to characterize protein-protein interactions and multi-protein complexes involved in autophagy in mammalian cells. The combination of affinity purification (AP)-MS, which identifies binary protein-protein interactions, with size-exclusion chromatography (SEC)-protein correlation profiling (PCP), which helps monitor protein complex assemblies, is a powerful tool to acquire a full overview of the interlinkage and regulation of novel multi-protein complexes that might play a role in autophagy.

Key words

Macroautophagy Autophagy Starvation SILAC AP-MS SEC-PCP-SILAC Proteomics Mass spectrometry 



This work was generously supported by the Swiss National Science Foundation (grants 31003A-166482/1, 316030-177088 ), by the Novartis Foundation for Medical-Biological Research (grant 16C221), by TRANSAUTOPHAGY (COST Action CA15138), and by the Alexander von Humboldt Foundation.


  1. 1.
    Ktistakis NT, Tooze SA (2016) Digesting the Expanding Mechanisms of Autophagy. Trends Cell Biol 26(8):624–635CrossRefGoogle Scholar
  2. 2.
    Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, Adachi H, Adams CM, Adams PD, Adeli K et al (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12(1):1–222CrossRefGoogle Scholar
  3. 3.
    Ryter SW, Cloonan SM, Choi AM (2013) Autophagy: a critical regulator of cellular metabolism and homeostasis. Mol Cells 36(1):7–16CrossRefGoogle Scholar
  4. 4.
    Choi AM, Ryter SW, Levine B (2013) Autophagy in human health and disease. N Engl J Med 368(7):651–662CrossRefGoogle Scholar
  5. 5.
    Blagoev B, Kratchmarova I, Ong SE, Nielsen M, Foster LJ, Mann M (2003) A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling. Nat Biotechnol 21(3):315–318CrossRefGoogle Scholar
  6. 6.
    Kristensen AR, Foster LJ (2014) Protein correlation profiling-SILAC to study protein-protein interactions. Methods Mol Biol 1188:263–270CrossRefGoogle Scholar
  7. 7.
    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):376–386CrossRefGoogle Scholar
  8. 8.
    Kuttner V, Mack C, Gretzmeier C, Bruckner-Tuderman L, Dengjel J (2014) Loss of collagen VII is associated with reduced transglutaminase 2 abundance and activity. J Invest Dermatol 134(9):2381–2389CrossRefGoogle Scholar
  9. 9.
    Diedrich B, Rigbolt KT, Roring M, Herr R, Kaeser-Pebernard S, Gretzmeier C, Murphy RF, Brummer T, Dengjel J (2017) Discrete cytosolic macromolecular BRAF complexes exhibit distinct activities and composition. EMBO J 36(5):646–663CrossRefGoogle Scholar
  10. 10.
    Becker AC, Dengjel J (2014) Autophagosomal proteome analysis by protein correlation profiling-SILAC. Methods Mol Biol 1188:271–279CrossRefGoogle Scholar
  11. 11.
    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):1896–1906CrossRefGoogle Scholar
  12. 12.
    Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26(12):1367–1372CrossRefGoogle Scholar
  13. 13.
    Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, Mann M, Cox J (2016) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 13(9):731–740CrossRefGoogle Scholar
  14. 14.
    Rigbolt KT, Vanselow JT, Blagoev B (2011) GProX, a user-friendly platform for bioinformatics analysis and visualization of quantitative proteomics data. Mol Cell Proteomics 10(8):O110 007450CrossRefGoogle Scholar
  15. 15.
    Selbach M, Mann M (2006) Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK). Nat Methods 3(12):981–983CrossRefGoogle Scholar
  16. 16.
    Gretzmeier C, Eiselein S, Johnson GR, Engelke R, Nowag H, Zarei M, Kuttner V, Becker AC, Rigbolt KTG, Hoyer-Hansen M et al (2017) Degradation of protein translation machinery by amino acid starvation- induced macroautophagy. Autophagy 13(6):1064–1075CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Stéphanie Kaeser-Pebernard
    • 1
  • Britta Diedrich
    • 2
  • Jörn Dengjel
    • 1
    • 3
    Email author
  1. 1.Department of BiologyUniversity of FribourgFribourgSwitzerland
  2. 2.Agilent TechnologiesWaldbronnGermany
  3. 3.Department of DermatologyMedical Center—University of FreiburgFreiburgGermany

Personalised recommendations