Trypsin digestion
-Structural characterization of proteins by using enzymatic reactor
E.Grifnée1, G.Mazzucchelli1, N.Smargiasso1, E. Hanozin1, F. Baumans1, C. Hage2, A.Sinz2, L.Quinton1and E.Depauw11Mass spectrometry laboratory, GIGA-R University of Liège 2Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther University of Halle-Wittenberg, Germany
Introduction
The tridimensional structure of proteins and the mapping of protein-protein interactions are precious sources of information for the understanding of their function. The goal of this study is to obtain structural information concerning native, partially or completely denatured proteins or interacting with a ligand using digestion kinetic data. Our work hypothesis is based on the observation of digestion kinetic variation (peptide nature and digestion rate) resulting from protection effects by comparing digestion in native or denatured conditions. Protection of cleavage zones can be induced by
Concept 2 : Ion mobility
400 600 800 1000 1200 1400 1600 2000 4000 6000 8000 10000 12000 C C S ( Å 2) Mass (Da) Cytochrome-C peptides z = 6+ z = 4+
-Trypsin digestionResults part 2 : Residual structure analyses by ion mobility
Loss of structuring domains or total destructuration ?
Separation of ions based on their size, shape andcharge under the influence of an electric field and a buffer gas. K o n ij n e n b e rg e t a l., 2 0 1 3 Information on the Collision Cross Section,
Ω Waters, Manchester, UK Mobility law: v = KE Mason-Shamp law:
Overview
Purpose : Structural characterization of proteins by using enzymatic reactor coupled to mass spectrometry.
Tools :Enzymatic reactor coupled to mass spectrometry, traveling-wave ion mobility spectrometry (TWIMS), chemical cross linking.
Main results : - Information on the accessibility of peptides to digestion in native conditions
- Presence of both structured and deployed protein domains during digestion.
Target residues :
Cross linker
Residual structure m = 5557,068 DaResidual structure m = 5759,244 Da Contact : elodie.grifnee@ulg.ac.be 5 10 15 20 25 30 35 40Time [min] 0.0 0.5 1.0 1.5 7x10 Intens. 5 10 15 20 25 30 35 40Time [min] 0.0 0.5 1.0 1.5 7x10 Intens.
Signal profil over time N N D D BSA 3D structure 999. 089 999.357 999.587 999. 859 1000. 359 1000.808 +MS, 0.4-15. 1min #(2-77) 2 4 6 8 6 x10 Intens. 999. 0 999. 2 999. 4 999.6 999.8 1000. 0 1000. 2 1000.4 1000.6 1000.8m/z 633.035 633. 370 633. 703 634.038 +MS, 0. 4-15.1min #(2-77) 1 2 3 4 6 x10 Intens. 633.0 633.2 633.4 633. 6 633.8 634.0 634. 2 m/z M/z: 633,035 z: +3 M: 1896,098 Da TRKVPQVSTPTLVEVSR T1 ~ 9 min T2 > 20 min T1 ~ 4,5 min T2 ~ 7 min M/z: 999,357 z: +2 M: 1996,723 Da ADLAKYICDNQDTISSKL native or denatured conditions. Protection of cleavage zones can be induced by intermolecular interactions (complexes formation) or by intramolecular interactions (protein folding). Special attention regarding residual structure conservation for protection effect must be considered when using peptides apparition times. In parallel, chemical cross-linking mass spectrometry (CX-MS) methods are currently used to probe the protein-protein interaction area. Here we first investigate the 3D structure of the well-known Cab-HuL6 Lysozyme complex based on CX-MS.
Enzymatic reactor
Formic Acid 0.1% Variable and controlled T°
Digestion or Mixing chamber Acidification Quenching Mass spectrometer
Concept 1
Results part 1 : Structural characterization thanks to online digestion
Conclusions
1.Thanks to online enzymatic digestion on native and denatured protein coupled to mass spectrometry, it is possible to obtain information on the accessibility of peptides to digestion in native conditions. From those data it is possible to locate peptides in the protein 3D structure.
2.Ion mobility (IMS) analyses of residual structures of protein allowed to highlight the presence of both structured and deployed protein domains. However, IMS only gives insights into ion density but not the actual degree of protection. Therefore, an independant characterization technique is required to further confirm these preliminary results.
Loss of structuring domains or total destructuration ?
Non-specific interactions betweencAb-HuL6andlysozyme?
Most CX residues are located on loops and flexible elements. The flexible character of these parts may allow the access to CX positions theoretically forbidden based on distance restrains.
Example of possible XL
Results part 3 : Study of Cab-HuL6 Lysozyme complex
1All AA involved in XL Another epitope ?
α < 25,3 Å α > 25,3 Å Conditions : pH = 7,5 ; T = 4°C ; t = 1h, excess XL = 5X m/z Native Time Denatured Time 3D Structure Sequence 633,04 20 9,2 Buried TRKVPQVSTPTLVEVSR 756,284 7,3 5,3 Surface TPDETYVPKAFDE 831,473 23,7 7,1 Surface DKLKHLVDEPQNLI 837,312 6,9 4,7 Surface TKCCTESL-RPCFSAL 839,79 7,5 5,7 Surface CVLHE-VTKCCTESLV 918,34 6,1 4,9 Surface CVLHEK-KVTKCCTESL 982,052 25,5 11,8 Buried FKDLGEEHFKGLVLIAF 999,369 6,7 4,5 Surface ADLAKYICDNQDTISSKL 1080,4 6,9 4,7 Surface RETYGDMADCCEKQEPER 1242,459 6,9 4,9 Surface QEPERNECFLSHKDDSPDLPK K13 Nter S60 K62 S82 K84 Nter K73 T11 K13 K1 K84 K13 PDB : 1OP9 PDB : 1OP9 = CID cleavable bonds
Target residues : K and N-terminus (also S,T and Y)
Lysines K have a dynamic lateral side chain length of 6,4 Å
Maximum distance (α) between two lysines K : Cα = 25,3 Å 6,4 Å NHS-BuUrBu-NHS
Cross linker
BSA 3D structure Residual structure m = 5557,068 Da More deployedthan m = 5759,244 Da ?Enzymatic digestion Trypsin / Glu-C /Chymotrypsin
