A Novel 2-DE-Based Proteomic Analysis to Identify Multiple Substrates for Specific Protease in Neuronal Cells

  • Chiho Kim
  • Young J. OhEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1598)


Proteolysis is a process where proteins are broken down into smaller polypeptides or amino acids, comprising one of the important posttranslational modifications of proteins. Since this process is exquisitely achieved by specialized enzymes called proteases under physiological conditions, abnormal protease activity and dysregulation of their substrate proteins are closely associated with a progression of several neurodegenerative diseases including Alzheimer disease, Parkinson disease, stroke, and spinal cord injury. Thus, it is important to identify the specific substrates of proteases with nonbiased high-throughput screenings to understand how proteolysis contributes to neurodegeneration. Here, we described a so-called gel-based protease proteomic approach. Critical steps of our novel strategy consist of two-dimensional polyacrylamide gel electrophoresis (2-DE)-based protein separation and in vitro incubation with the specific protease of interest. As a prototypic example, cellular lysates obtained from neuronal cells are separated by an isoelectric focusing, and the resulting immobilized proteins on a gel strip are incubated with a predetermined amount of a recombinant or a purified protease. By densitometric analysis of the Coomassie Brilliant Blue-stained gel images following separation by 2-DE, significantly altered protein spots are subjected to a mass spectral analysis for protein identification. Interestingly, the concepts of our strategy can be applied to any proteases, and to any neural cells or neural tissues of one’s interest. Since the immobilized protein spots are exposed to the purified protease, this protocol ensures the identification of only substrates that are directly cleaved by specific protease. This protocol ensures to avoid the possibility of identifying substrates that may be cleaved by combinatorial or sequential activation of proteolytic enzymes present in a liquid state of the lysates. We propose that our strategy can be effectively utilized to provide meaningful insights into newly identified protease substrates and to decipher molecular mechanisms critically involved in neurodegenerative processes.

Key words

Two-dimensional polyacrylamide gel electrophoresis Mass spectrometry Proteome analysis Proteolysis Cleavage Caspase Calpain Neurodegeneration 



This work was supported by the Ministry for Health, Welfare and Family Affairs (A111382 to YJO) and in part by the Yonsei Intramural Grant (year of 2000 to YJO). We thank Dr. Chung Ju at Korea University for helpful discussion.


  1. 1.
    Shahinian H, Tholen S, Schilling O (2013) Proteomic identification of protease cleavage sites: cell-biological and biomedical applications. Expert Rev Proteomics 10:421–433CrossRefPubMedGoogle Scholar
  2. 2.
    Wojcik C, DeMartino GN (2003) Intracellular localization of proteasomes. Int J Biochem Cell Biol 35:579–589CrossRefPubMedGoogle Scholar
  3. 3.
    Camins A, Verdaguer E, Folch J, Pallas M (2006) Involvement of calpain activation in neurodegenerative processes. CNS Drug Rev 12:135–148CrossRefPubMedGoogle Scholar
  4. 4.
    Puente XS, Sanchez LM, Overall CM, Lopez-Otin C (2003) Human and mouse proteases: a comparative genomic approach. Nat Rev Genet 4:544–558CrossRefPubMedGoogle Scholar
  5. 5.
    Samara C, Tavernarakis N (2003) Calcium-dependent and aspartyl proteases in neurodegeneration and ageing in C. elegans. Ageing Res Rev 2:451–471CrossRefPubMedGoogle Scholar
  6. 6.
    Wang KK (2000) Calpain and caspase: can you tell the difference? Trends Neurosci 23:20–26CrossRefPubMedGoogle Scholar
  7. 7.
    Choi WS, Lee EH, Chung CW, Jung YK, Jin BK, Kim SU, Oh TH, Saido TC, Oh YJ (2001) Cleavage of Bax is mediated by caspase-dependent or -independent calpain activation in dopaminergic neuronal cells: protective role of Bcl-2. J Neurochem 77:1531–1541CrossRefPubMedGoogle Scholar
  8. 8.
    Han BS, Hong HS, Choi WS, Markelonis GJ, Oh TH, Oh YJ (2003) Caspase-dependent and -independent cell death pathways in primary cultures of mesencephalic dopaminergic neurons after neurotoxin treatment. J Neurosci 23:5069–5078PubMedGoogle Scholar
  9. 9.
    Choi WS, Yoon SY, Oh TH, Choi EJ, O'Malley KL, Oh YJ (1999) Two distinct mechanisms are involved in 6-hydroxydopamine- and MPP + −induced dopaminergic neuronal cell death: role of caspases, ROS, and JNK. J Neurosci Res 57:86–94CrossRefPubMedGoogle Scholar
  10. 10.
    Han BS, Noh JS, Gwag BJ, Oh YJ (2003) A distinct death mechanism is induced by 1-methyl-4-phenylpyridinium or by 6-hydroxydopamine in cultured rat cortical neurons: degradation and dephosphorylation of tau. Neurosci Lett 341:99–102CrossRefPubMedGoogle Scholar
  11. 11.
    Kang H, Han BS, Kim SJ, Oh YJ (2012) Mechanisms to prevent caspase activation in rotenone-induced dopaminergic neurodegeneration: role of ATP depletion and procaspase-9 degradation. Apoptosis 17:449–462CrossRefGoogle Scholar
  12. 12.
    Cryns VL, Byun Y, Rana A, Mellor H, Lustig KD, Ghanem L, Parker PJ, Kirschner MW, Yuan J (1997) Specific proteolysis of the kinase protein kinase C-related kinase 2 by caspase-3 during apoptosis. Identification by a novel, small pool expression cloning strategy. J Biol Chem 272:29449–29453CrossRefPubMedGoogle Scholar
  13. 13.
    Kamada S, Kusano H, Fujita H, Ohtsu M, Koya RC, Kuzumaki N, Tsujimoto Y (1998) A cloning method for caspase substrates that uses the yeast two-hybrid system: cloning of the antiapoptotic gene gelsolin. Proc Natl Acad Sci USA 95:8532–8537CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Agard NJ, Wells JA (2009) Methods for the proteomic identification of protease substrates. Curr Opin Chem Biol 13:503–509CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Mahrus S, Trinidad JC, Barkan DT, Sali A, Burlingame AL, Wells JA (2008) Global sequencing of proteolytic cleavage sites in apoptosis by specific labeling of protein N termini. Cell 134:866–876CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Van Damme P, Martens L, Van Damme J, Hugelier K, Staes A, Vandekerckhove J, Gevaert K (2005) Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis. Nat Methods 2:771–777CrossRefPubMedGoogle Scholar
  17. 17.
    Celis JE, Ostergaard M, Jensen NA, Gromova I, Rasmussen HH, Gromov P (1998) Human and mouse proteomic databases: novel resources in the protein universe. FEBS Lett 430:64–72CrossRefPubMedGoogle Scholar
  18. 18.
    O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021PubMedPubMedCentralGoogle Scholar
  19. 19.
    O'Farrell PZ, Goodman HM, O'Farrell PH (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12:1133–1141CrossRefPubMedGoogle Scholar
  20. 20.
    Klose J (1975) Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues A novel approach to testing for induced point mutations in mammals. Humangenetik 26:231–243PubMedGoogle Scholar
  21. 21.
    Gorg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W (2000) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21:1037–1053CrossRefPubMedGoogle Scholar
  22. 22.
    Klaiman G, Petzke TL, Hammond J, Leblanc AC (2008) Targets of caspase-6 activity in human neurons and Alzheimer disease. Mol Cell Proteomics 7:1541–1555CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Lamkanfi M, Kanneganti TD, Van Damme P, Vanden Berghe T, Vanoverberghe I, Vandekerckhove J, Vandenabeele P, Gevaert K, Nunez G (2008) Targeted peptidecentric proteomics reveals caspase-7 as a substrate of the caspase-1 inflammasomes. Mol Cell Proteomics 7:2350–2363CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Xu D, Suenaga N, Edelmann MJ, Fridman R, Muschel RJ, Kessler BM (2008) Novel MMP-9 substrates in cancer cells revealed by a label-free quantitative proteomics approach. Mol Cell Proteomics 7:2215–2228CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Wood DE, Newcomb EW (1999) Caspase-dependent activation of calpain during drug-induced apoptosis. J Biol Chem 274:8309–8315CrossRefPubMedGoogle Scholar
  26. 26.
    Choi HK, Won LA, Kontur PJ, Hammond DN, Fox AP, Wainer BH, Hoffmann PC, Heller A (1991) Immortalization of embryonic mesencephalic dopaminergic neurons by somatic cell fusion. Brain Res 552:67–76CrossRefPubMedGoogle Scholar
  27. 27.
    Kim C, Yun N, Lee YM, Jeong JY, Baek JY, Song HY, Ju C, Youdim MB, Jin BK, Kim WK, Oh YJ (2013) Gel-based protease proteomics for identifying the novel calpain substrates in dopaminergic neuronal cell. J Biol Chem 288:36717–36732CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Yun N, Lee YM, Kim C, Shibayama H, Tanimura A, Hamanaka Y, Kanakura Y, Park IS, Jo A, Shin JH, Ju C, Kim WK, Oh YJ (2014) Anamorsin, a novel caspase-3 substrate in neurodegeneration. J Biol Chem 289:22183–22195CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Chevalier F (2010) Standard dyes for total protein staining in gel-based proteomic analysis. Materials 3:4784–4792CrossRefGoogle Scholar
  30. 30.
    Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422:198–207CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Systems BiologyYonsei University College of Life Science and BiotechnologySeoulSouth Korea

Personalised recommendations