Improved Accuracy of Low Affinity Protein–Ligand Equilibrium Dissociation Constants Directly Determined by Electrospray Ionization Mass Spectrometry

Abstract

There is continued interest in the determination by ESI-MS of equilibrium dissociation constants (K(D)) that accurately reflect the affinity of a protein-ligand complex in solution. Issues in the measurement of K(D) are compounded in the case of low affinity complexes. Here we present a K(D) measurement method and corresponding mathematical model dealing with both gas-phase dissociation (GPD) and aggregation. To this end, a rational mathematical correction of GPD (f(sat)) is combined with the development of an experimental protocol to deal with gas-phase aggregation. A guide to apply the method to noncovalent protein-ligand systems according to their kinetic behavior is provided. The approach is validated by comparing the K(D) values determined by this method with in-solution K(D) literature values. The influence of the type of molecular interactions and instrumental setup on f(sat) is examined as a first step towards a fine dissection of factors affecting GPD. The method can be reliably applied to a wide array of low affinity systems without the need for a reference ligand or protein.

SSPaQ: A Subtractive Segmentation Approach for the Exhaustive Parallel Quantification of the Extent of Protein Modification at Every Possible Site

“…Recombinant hPEBP1 overexpressed in BL21DE3 E.coli was purified without tag using QAE Sephadex A-50 chromatography, isoelectrofocusing, and Blue-Sepharose chromatography, as previously described [16]. The purified protein was stored at −80°C in ultrapure water with two equivalents of TCEP.…”

“…The fact that the LocMin f bound shown for these flanking nonoverlapping sides cannot be unambiguously distributed between sides is represented by a small linking curve between the corresponding sequences in Supplementary Figure S8. In this dataset, an example of such potential problem areas are [4][5][6][7][8] and [13][14][15][16][17] with f bound around zero. LocMin f bound values generated by class C groups can also be overestimated because modifications leading to these f bound values can be located outside the common sequence for all segments in the group.…”

Nanofluidics-Based Mass Spectrometry. Applications for Biomarker Discovery in Lysosomal Storage Diseases

“…The second advantage of multiple charging is the formation of ions with reduced m/z ratio measurable with good resolution by almost any type of analyzer with which ESI has been interfaced: magnetic sector, single or triple quadrupole, time-of-flight (TOF), quadrupole ion trap (QIT), Fourier-transform ion cyclotron resonance (FTICR) or hybrid quadrupole time-of-flight analyzer (QTOF). All these make ESI the method of choice for large biopolymers and molecular aggregates or complexes that only have weak non-covalent interactions, such as protein-protein, enzyme-substrate or protein-ligand complexes [13][14][15][16].…”

Native ESI Mass Spectrometry Can Help to Avoid Wrong Interpretations from Isothermal Titration Calorimetry in Difficult Situations

“…The latter method was thought to be misleading for aggregation resulting from strong electrostatic interactions and was thus modified by using a reference cognate ligand competing with a putative ligand for the same site [12]. Finally, a processing method taking into account directly the amount of GPD (in conjunction with a micro gel filtration step to remove excess of ligand) was proposed [13]. …”

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Figure 1Illustration of the ring species R 0 , R 1 and R 2 . Structure of the E.coli processivity factor [13]. Each specifically-bound peptide is represented as a sphere at the position known from X-ray crystallography [16–18].

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