Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry

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Author(s) Cubrilovic, Dragana, Biela, Adam, Sielaff, Frank, Steinmetzer, Torsten, Klebe, Gerhard, Zenobi, Renato
Publication Type Journal Items, Publication Status: Published
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Title Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry
Subtitle A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors
Author(s) Cubrilovic, Dragana
Biela, Adam
Sielaff, Frank
Steinmetzer, Torsten
Klebe, Gerhard
Zenobi, Renato
Journal or Series Title Journal of the American Society for Mass Spectrometry
Volume Number 23
Issue Number 10
Start Page 1768
End Page 1777
ISSN 1044-0305
Publisher Springer
Publication Place New York
Publication Date 2012-10
Keyword(s) Noncovalent interactions
Electrospray ionization mass spectrometry
Binding affinity
Protein-ligand complexes
Hydrophobic effect
Abstract NanoESI-MS is used for determining binding strengths of trypsin in complex with two different series of five congeneric inhibitors, whose binding affinity in solution depends on the size of the P3 substituent. The ligands of the first series contain a 4-amidinobenzylamide as P1 residue, and form a tight complex with trypsin. The inhibitors of the second series have a 2-aminomethyl-5-chloro-benzylamide as P1 group, and represent a model system for weak binders. The five different inhibitors of each group are based on the same scaffold and differ only in the length of the hydrophobic side chain of their P3 residue, which modulates the interactions in the S3/4 binding pocket of trypsin. The dissociation constants (K-D) for high affinity ligands investigated by nanoESI-MS ranges from 15 nM to 450 nM and decreases with larger hydrophobic P3 side chains. Collision-induced dissociation (CID) experiments of five trypsin and benzamidine-based complexes show a correlation between trends in K-D and gas-phase stability. For the second inhibitor series we could show that the effect of imidazole, a small stabilizing additive, can avoid the dissociation of the complex ions and as a result increases the relative abundance of weakly bound complexes. Here the K-D values ranging from 2.9 to 17.6 mu M, some 1-2 orders of magnitude lower than the first series. For both ligand series, the dissociation constants (K-D) measured via nanoESI-MS were compared with kinetic inhibition constants (K-i) in solution.
DOI 10.1007/s13361-012-0451-6
Additional Notes Received 29 March 2012, Accepted 17 July 2012, Published online 7 August 2012
Document Type Article
Publication Status Published
Language English
Assigned Organisational Unit(s) 03430
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NEBIS System Number 000541631
Source Database ID WOS-000309236900015
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  author = "Cubrilovic, Dragana and Biela, Adam and Sielaff, Frank and Steinmetzer, Torsten and Klebe, Gerhard and Zenobi, Renato",
  title = "{Q}uantifying {P}rotein-{L}igand {B}inding {C}onstants using {E}lectrospray {I}onization {M}ass {S}pectrometry: {A} {S}ystematic {B}inding {A}ffinity {S}tudy of a {S}eries of {H}ydrophobically {M}odified {T}rypsin {I}nhibitors",
  journal = "Journal of the American Society for Mass Spectrometry",
  year = 2012,
  volume = "23",
  number = "10",
  pages = "1768--1777",
  month = oct,

E-Citations record created: Mon, 05 Nov 2012, 08:42:52 CET