Amr El-Hawiet

Reliable determinations of protein-ligand interactions by direct ESI-MS measurements: Are we there yet?

The association-dissociation of noncovalent interactions between protein and ligands, such as other proteins, carbohydrates, lipids, DNA, or small molecules, are critical events in many biological processes. The discovery and characterization of these interactions is essential to a complete understanding of biochemical reactions and pathways and to the design of novel therapeutic agents that may be used to treat a variety of diseases and infections. Over the last 20 y, electrospray ionization mass spectrometry (ESI-MS) has emerged as a versatile tool for the identification and quantification of protein–ligand interactions in vitro. Here, we describe the implementation of the direct ESI-MS assay for the determination of protein–ligand binding stoichiometry and affinity. Additionally, we outline common sources of error encountered with these measurements and various strategies to overcome them. Finally, we comment on some of the outstanding challenges associated with the implementation of the assay and highlight new areas where direct ESI-MS measurements are expected to make significant contributions in the future.

Gas Phase Stabilization of Noncovalent Protein Complexes Formed by Electrospray Ionization

The use of gas phase additives to stabilize noncovalent protein complexes in electrospray ionization mass spectrometry (ES-MS) is demonstrated for two protein-ligand interactions, an enzyme-small molecule inhibitor complex, and a protein-disaccharide complex. It is shown that the introduction of gas phase imidazole into the ES ion source effectively protects gas phase protein-ligand complexes against in-source dissociation. The stabilizing effect of imidazole vapor is comparable to that observed upon addition of imidazole to the ES solution. The introduction of sulfur hexafluoride, at high partial pressure, into the source region also effectively suppresses in-source dissociation of protein complexes. It is proposed that evaporative cooling is the primary mechanism responsible for the stabilizing effects observed for the gas phase additives.

Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry

A new electrospray ionization mass spectrometry (ES-MS) approach for quantifying protein—ligand complexes that are prone to in-source (gas-phase) dissociation is described. The method, referred to here as the reference ligand ES-MS method, is based on the direct ES-MS assay and competitive ligand binding. A reference ligand (Lref), which binds specifically to the protein (P), at the same binding site as the ligand (L) of interest, with known affinity and forms a stable protein—ligand complex in the gas phase, is added to the solution. The fraction of P bound to Lref, which is determined directly from the ES mass spectrum, is sensitive to the fraction of P bound to L in solution and enables the affinity of P for L to be determined. A mathematical framework for the implementation of the method in cases where P has one or two specific ligand binding sites is given. Affinities of two carbohydrate-binding proteins, a single chain fragment of a monoclonal antibody and the lectin concanavalin A, for monosaccharide ligands are reported and the results are shown to agree with values obtained using isothermal titration calorimetry.

Quantifying Ligand Binding to Large Protein Complexes Using Electrospray Ionization Mass Spectrometry

An electrospray ionization mass spectrometry (ESI-MS) method for quantifying protein-ligand complexes that cannot be directly detected by ESI-MS is described. The proxy protein ESI-MS method combines direct ESI-MS binding measurements with competitive protein-ligand binding. To implement the method, a proxy protein (P(proxy)), which interacts specifically with the ligand of interest with known affinity and can be detected directly by ESI-MS, is used to quantitatively monitor the extent of ligand binding to the protein of interest. A mathematical framework for establishing the association constant (K(a)) for protein-ligand binding by the proxy protein ESI-MS method, implemented with a P(proxy) containing a single ligand binding site, is given. A modified form of the proxy protein ESI-MS method, which accounts for real-time changes in ligand concentration, is also described. The reliability of these methods is demonstrated for the interactions between the 180 kDa wildtype homotrimeric tailspike protein of the bacteriophage P22 and its endorhamnosidase point mutant (D392N) with its ligands comprising two and three O-antigen repeats from Salmonella enterica serovar Typhimurium: octasaccharide ([α-Gal-(1→2)-[α-Abe-(1→3)]-α-Man-(1→4)-α-Rha](2)) and dodecasaccharide ([α-Gal-(1→2)-[α-Abe-(1→3)]-α-Man-(1→4)-α-Rha](3)). A 27 kDa single chain antibody, which binds to both ligands, served as P(proxy). The results of binding measurements performed at 10 and 25 °C are in excellent agreement with K(a) values measured previously using a fluorescence quenching assay.

Applications of a catch and release electrospray ionization mass spectrometry assay for carbohydrate library screening.

Applications of a catch and release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for screening carbohydrate libraries against target proteins are described. Direct ESI-MS measurements were performed on solutions containing a target protein (a single chain antibody, an antigen binding fragment, or a fragment of a bacterial toxin) and a library of carbohydrates containing multiple specific ligands with affinities in the 10(3) to 10(6) M(-1) range. Ligands with moderate affinity (10(4) to 10(6) M(-1)) were successfully detected from mixtures containing >200 carbohydrates (at concentrations as low as 0.25 μM each). Additionally, the absolute affinities were estimated from the abundance of free and ligand-bound protein ions determined from the ESI mass spectrum. Multiple low affinity ligands (~10(3) M(-1)) were successfully detected in mixtures containing >20 carbohydrates (at concentrations of ~10 μM each). However, identification of specific interactions required the use of the reference protein method to correct the mass spectrum for the occurrence of nonspecific carbohydrate-protein binding during the ESI process. The release of the carbohydrate ligands, as ions, was successfully demonstrated using collision-induced dissociation performed on the deprotonated ions of the protein-carbohydrate complexes. The use of ion mobility separation, performed on deprotonated carbohydrate ions following their release from the complex, allowed for the positive identification of isomeric ligands.

Screening carbohydrate libraries for protein interactions using the direct ESI-MS assay: Applications to libraries of unknown concentration

AbstractA semiquantitative electrospray ionization mass spectrometry (ESI-MS) binding assay suitable for analyzing mixtures of oligosaccharides, at unknown concentrations, for interactions with target proteins is described. The assay relies on the differences in the ratio of the relative abundances of the ligand-bound and free protein ions measured by ESI-MS at two or more initial protein concentrations to distinguish low affinity (≤103 M–1) ligands from moderate and high affinity (>105 M–1) ligands present in the library and to rank their affinities. Control experiments were performed on solutions of a single chain antibody and a mixture of synthetic oligosaccharides, with known affinities, in the absence and presence of a 40-component carbohydrate library to demonstrate the implementation and reliability of the assay. The application of the assay for screening natural libraries of carbohydrates against proteins is also demonstrated using mixtures of human milk oligosaccharides, isolated from breast milk, and fragments of a bacterial toxin and human galectin 3. Graphical Abstractᅟ

Quantifying Protein Interactions with Isomeric Carbohydrate Ligands Using a Catch and Release Electrospray Ionization-Mass Spectrometry Assay

The application of a catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay to quantify interactions between proteins and isomeric carbohydrate ligands is described. Absolute affinities for each ligand are determined from the abundance ratio of ligand-bound to free protein measured directly by ESI-MS and the relative abundances of the individual isomeric ligands, which are established by releasing the ligands, in their deprotonated form, from the protein using collision-induced dissociation (CID) and subjecting them to ion mobility separation (IMS) or another stage of CID to fragment the ions. Using Gaussian functions to represent the contributions of individual ligands to the arrival time distributions (ATDs) measured by IMS, the relative abundance of each ligand bound to the protein can be established. A modification of this method, suitable for cases where nonspecific ligand-protein binding occurs during the ESI process, is also described. In cases where the ATDs are not sufficiently different to distinguish the isomeric ligands, CID can establish the relative abundance of each ligand bound to the protein from the relative abundance of the resulting fragment ions. The implementation and reliability of the CaR-ESI-MS assay for the analysis of isomeric carbohydrate ligands is demonstrated using three carbohydrate-binding proteins, a single chain antibody, an antigen binding fragment, and a fragment of a bacterial toxin, and their interactions with isomeric carbohydrate ligands with affinities ranging from 10(3) to 10(5) M(-1).

High-Throughput Label- and Immobilization-Free Screening of Human Milk Oligosaccharides Against Lectins

The intense interest in the mechanisms responsible for the beneficial effects of breast-feeding on infant health has created a significant need for analytical methods capable of rapidly identifying interactions between human milk oligosaccharides (HMOs) and their protein receptors. Currently, there are no established, high-throughput assays for the screening libraries of free (unmodified) HMOs against lectins. The present work describes a rapid and label- and immobilization-free assay, based on catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS), capable of simultaneously screening mixtures of free HMOs of known concentration for binding to lectins in vitro. Ligand identification relies on the molecular weights (MWs), ion mobility separation arrival times, and collision-induced dissociation fingerprints of HMO anions released from the target protein in the gas phase. To establish the reliability of the assay, a library of 31 free HMOs, ranging in size from tri- to octasaccharide, was screened against three human galectin (hGal) proteins (a stable mutant of hGal1 (hGal-1), a C-terminal fragment of hGal-3 (hGal-3C) and hGal-7), with known HMO affinities. When implemented using an equimolar concentration library, the CaR-ESI-MS assay identified 100% of ligands with affinities >500 M-1 and ≥93% of all HMO ligands (hGal-1-31 of 31 ligands; hGal-3C-25 of 25; hGal-7-28 of 30); no false positives were detected. The assay also successfully identified the majority of the highest affinity HMO ligands (or isomer sets that contain the highest affinity ligands) in the library for each of the three hGal. Notably, for each lectin, CaR-ESI-MS screening required <1 h to complete and consumed <5 ng of each HMO and <0.5 μg of protein.

Mycobacteriophage cell binding proteins for the capture of mycobacteria

Slow growing Mycobacterium avium subsp. paratuberculosis (MAP) causes a deadly condition in cattle known as Johnes disease where asymptomatic carriers are the major source of disease transmission. MAP was also shown to be associated with chronic Crohns disease in humans. Mycobacterium smegmatis is a model mycobacterium that can cause opportunistic infections in a number of human tissues and, rarely, a respiratory disease. Currently, there are no rapid, culture-independent, reliable and inexpensive tests for the diagnostics of MAP or M. smegmatis infections. Bacteriophages are viruses producing a number of proteins that effectively and specifically recognize the cell envelopes of their bacterial hosts. We demonstrate that the mycobacterial phage L5 minor tail protein Gp6 and lysin Gp10 are useful tools for the rapid capture of mycobacteria. Immobilized Gp10 was able to bind both MAP and M. smegmatis cells whereas Gp6 was M. smegmatis specific. Neither of the 2 proteins was able to capture E. coli, salmonella, campylobacter or Mycobacterium marinum cells. Gp6 was detected previously as a component of the phage particle and shows no homology to proteins with known function. Therefore, electrospray ionization mass spectrometry was used to determine whether recombinant Gp6 could bind to a number of chemically synthesized fragments of mycobacterial surface glycans. These findings demonstrate that mycobacteriophage proteins could be used as a pathogen capturing platform that can potentially improve the effectiveness of existing diagnostic methods.