Lys-C/Trypsin Tandem-Digestion Protocol for Gel-Free Proteomic Analysis of Colon Biopsies

  • Armin Schniers
  • Yvonne Pasing
  • Terkel HansenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1959)


The protocol presented was specifically optimized for in-depth analysis of the human colon mucosa proteome. After cell lysis in a sodium deoxycholate/urea buffer, a tandem digestion with Lys-C and trypsin was performed. Prior to LC-MS/MS analysis, peptides were TMT-labeled and fractionated by high pH reversed-phase spin columns. This protocol is a powerful, reproducible, sample-saving, and cost-effective option when an in-depth quantitative proteome analysis is desired.

Key words

Colon mucosa proteome Sodium deoxycholate Lys-C TMT labeling High pH reversed-phase fractionation 



We thank Ilona Urbarova and Jack-Ansgar Bruun for fruitful discussions, as well as Prof Jon Florholmen and Rasmus Goll for supplying colon biopsies. Our work was supported by a grant from the North Norway Regional Health Authorities.


  1. 1.
    Sandle G (1998) Salt and water absorption in the human colon: a modern appraisal. Gut 43(2):294–299. Scholar
  2. 2.
    Shi N, Li N, Duan X et al (2017) Interaction between the gut microbiome and mucosal immune system. Mil Med Res 4:14. Scholar
  3. 3.
    Kasper LH, Buzoni-Gatel D (2001) Ups and downs of mucosal cellular immunity against protozoan parasites. Infect Immun 69(1):1–8. Scholar
  4. 4.
    Curtis MM, Sperandio V (2011) A complex relationship: the interaction among symbiotic microbes, invading pathogens, and their mammalian host. Mucosal Immunol 4(2):133–138. Scholar
  5. 5.
    Baumgart DC, Carding SR (2007) Inflammatory bowel disease: cause and immunobiology. Lancet 369(9573):1627–1640. Scholar
  6. 6.
    Glatter T, Ludwig C, Ahrné E et al (2012) Large-scale quantitative assessment of different in-solution protein digestion protocols reveals superior cleavage efficiency of tandem Lys-C/Trypsin proteolysis over trypsin digestion. J Proteome Res 11(11):5145–5156. Scholar
  7. 7.
    Pasing Y, Colnoe S, Hansen T (2017) Proteomics of hydrophobic samples: Fast, robust and low-cost workflows for clinical approaches. Proteomics 17(6).
  8. 8.
    Pasing Y, Schniers A, Hansen T (2018) Straightforward protocol for gel-free proteomic analysis of adipose tissue. Methods Mol Biol 1788:289–296. Scholar
  9. 9.
    Schniers A, Anderssen E, Fenton CG et al (2017) The proteome of ulcerative colitis in colon biopsies from adults - optimized sample preparation and comparison with healthy controls. Proteomics Clin Appl 11(11–12).
  10. 10.
    Nakamura T, Kuromitsu J, Oda Y (2008) Evaluation of comprehensive multidimensional separations using reversed-phase, reversed-phase liquid chromatography/mass spectrometry for shotgun proteomics. J Proteome Res 7(3):1007–1011. Scholar
  11. 11.
    Gilar M, Olivova P, Chakraborty AB et al (2009) Comparison of 1-D and 2-D LC MS/MS methods for proteomic analysis of human serum. Electrophoresis 30(7):1157–1167. Scholar
  12. 12.
    Bennike TB, Carlsen TG, Ellingsen T (2017) Proteomics dataset: the colon mucosa from inflammatory bowel disease patients, gastrointestinal asymptomic rheumatoid arthritis patients, and controls. Data Brief 15:511–516. Scholar
  13. 13.
    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):1367–1372. Scholar
  14. 14.
    Tyanova S, Temu T, Sinitcyn P et al (2016) The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat Methods 13(9):731–740Google Scholar
  15. 15.
    Tyanova S, Cox J (2018) Perseus: a bioinformatics platform for integrative analysis of proteomics data in cancer research. In: von Stechow L. (eds) Cancer systems biology. Methods in molecular biology, vol 1711. Humana Press, New York, NY, p 133–148. doi:
  16. 16.
    Scopes RK (1974) Measurement of protein by spectrophotometry at 205 nm. Anal Biochem 59(1):277–282. Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Natural Products and Medicinal Chemistry Research Group, Department of PharmacyUiT—The Arctic University of NorwayTromsøNorway
  2. 2.Tromsø Endocrine Research Group, Department of Clinical MedicineUiT—The Arctic University of NorwayTromsøNorway
  3. 3.Division of Internal MedicineUniversity Hospital of North NorwayTromsøNorway

Personalised recommendations