Quantifying Protein–Ligand Interactions by Direct Electrospray Ionization-MS Analysis: Evidence of Nonuniform Response Factors Induced by High Molecular Weight Molecules and Complexes

The deleterious effects of high molecular weight (MW) solute (polymers and noncovalent assemblies) on protein–ligand (PL) affinity measurements carried out using the direct electrospray ionization mass spectrometry (ESI-MS) assay are described. The presence of high MW solute, that do not interact with the protein (P) or ligand (L) of interest, is shown to result in a decrease in the abundance (Ab) ratio (<i>R</i>) of ligand-bound to free protein ions (i.e., Ab­(PL)/Ab­(P)) measured for protein–carbohydrate complexes. This effect, which can reduce the apparent association constant by more than 60%, is found to be more pronounced as the differences in the surface properties of P and PL become more significant. It is proposed that the decrease in <i>R</i> reflects a reduction in the number of available surface sites in the ESI droplets upon introduction of large solute and increased competition between P and the more hydrophilic PL for these sites. That a similar decrease in <i>R</i> is observed upon introduction of surfactants to solution provides qualitative support for this hypothesis

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 10³ to 10⁵ M^–1

Nanodiscs and Electrospray Ionization Mass Spectrometry: A Tool for Screening Glycolipids Against Proteins

Electrospray ionization-mass spectrometry (ESI-MS) is extensively employed to detect and quantify protein–carbohydrate interactions <i>in vitro</i> and is increasingly used to screen carbohydrate libraries against target proteins. However, current ESI-MS methods are limited to carbohydrate ligands that are relatively soluble in water and are, therefore, not generally suitable for studying protein interactions with glycolipids, an important class of cellular receptors. Here, we describe a catch-and-release (CaR)-ESI-MS assay, which exploits nanodiscs (NDs) to solubilize glycolipids and mimic their natural cellular environment, suitable for screening libraries of glycosphingolipids (GSL) against proteins to identify specific interactions and to rank their relative affinities. Using the B subunit homopentamers of cholera toxin and heat labile toxin as model GSL-binding proteins, the CaR-ESI-MS was applied to NDs containing mixtures of gangliosides. The results demonstrate that the CaR-ESI-MS assay can simultaneously detect both high and low affinity GSL ligands using either a library of NDs that each contains one GSL or incorporating a mixture of GSLs into a single ND. Moreover, the relative abundances of the released ligands appear to reflect their relative affinities in solution. Application of the CaR-ESI-MS assay using NDs containing gangliosides extracted from porcine brain led to the discovery of a neolacto GSL as a cholera toxin ligand, highlighting the power of the assay for identifying specific protein–glycolipid interactions from biologically relevant mixtures

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 10³ to 10⁶ M^–1 range. Ligands with moderate affinity (10⁴ to 10⁶ 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 (∼10³ 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

Gangliosides are Ligands for Human Noroviruses

Human noroviruses (NoVs) are known to recognize histo-blood group antigens (HBGAs) as attachment factors. We report the first experimental evidence that sialic acid-containing glycosphingolipids (gangliosides) are also ligands for human NoVs. Electrospray ionization mass spectrometry-based carbohydrate binding measurements performed on assemblies (P dimer, P particle, and virus-like particle) of recombinant viral capsid proteins of two NoV strains, VA387 (GII.4) and VA115 (GI.3), identified binding to the oligosaccharides of mono-, di-, and trisialylated gangliosides. The intrinsic (per binding site) affinities measured for these ligands are similar in magnitude (10²–10³ M^–1) to those of human HBGAs. Binding of NoV VLPs, P particles, and glutathione S-transferase (GST)-P domain fusion proteins to sialic acid-containing glycoconjugates, observed in enzyme-linked immunosorbent assays, provided additional confirmation of the NoV–ganglioside interactions

Investigating the Influence of Membrane Composition on Protein–Glycolipid Binding Using Nanodiscs and Proxy Ligand Electrospray Ionization Mass Spectrometry

This work describes a versatile analytical approach, which combines the <i>proxy ligand</i> electrospray ionization mass spectrometry (ESI-MS) assay and model membranes of defined composition, to quantify the influence of lipid bilayer composition on protein–glycolipid binding <i>in vitro</i>. To illustrate the implementation of the assay (experimental design and data analysis), affinities of the monosialoganglioside ligand GM1, incorporated into nanodiscs (NDs), for cholera toxin B subunit homopentamer (CTB₅) were measured. A series of NDs containing GM1 and cholesterol were prepared using three different phospholipids (1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-<i>sn</i>-glycero-3-phosphocholine (DPPC), and 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine (POPC)), and the average GM1 and cholesterol content of each ND were determined. The intrinsic affinities of GM1-containing NDs prepared with the three phospholipids are found to be similar in magnitude, indicating that small differences in the fatty acid chain length and the number of unsaturated bonds do not significantly affect the CTB₅–GM1 interaction. Moreover, the measured affinities are similar to the value measured for GM1 pentasaccharide, indicating that neither the ceramide moiety nor the surface of the phospholipid membrane plays a significant role in CTB₅ binding. The intrinsic (per binding site) affinity of the CTB₅–GM1 interaction was found to decrease with increasing GM1 content of the ND, consistent with the occurrence of GM1 clustering in the membrane, which sterically hinders binding to CTB₅. Notably, the addition of cholesterol to GM1-containing NDs did not have a significant effect on the strength of the CTB₅–GM1 interaction. This result, which is at odds with the findings of a previous study of CTB₅ binding to GM1 in vesicles, suggests that cholesterol does not “mask” GM1, at least not in NDs. These data, in addition to providing new insights into the influence of membrane composition on CTB₅–GM1 binding, demonstrate the potential of the <i>proxy ligand</i> ESI-MS approach for comprehensive and quantitative studies of lectin interactions with glycolipids in native-like, membrane environments

Silent Encoding of Chemical Post-Translational Modifications in Phage-Displayed Libraries

<i>In vitro</i> selection of chemically modified peptide libraries presented on phage, while a powerful technology, is limited to one chemical post-translational modification (cPTM) per library. We use unique combinations of redundant codons to encode cPTMs with “silent barcodes” to trace multiple modifications within a mixed modified library. As a proof of concept, we produced phage-displayed peptide libraries Ser-[X]₄-Gly-Gly-Gly, with Gly and Ser encoded using unique combinations of codons (TCA-[X]₄-GGAGGAGGA, AGT-[X]₄-GGTGGTGGT, etc., where [X]₄ denotes a random NNK library). After separate chemical modification and pooling, mixed-modified libraries can be panned and deep-sequenced to identify the enriched peptide sequence and the accompanying cPTM simultaneously. We panned libraries bearing combinations of modifications (sulfonamide, biotin, mannose) against matched targets (carbonic anhydrase, streptavidin, concanavalin A) to identify desired ligands. Synthesis and validation of sequences identified by deep sequencing revealed that specific cPTMs are significantly enriched in panning against the specific targets. Panning on carbonic anhydrase yielded a potent ligand, sulfonamide–WIVP, with <i>K</i>_d = 6.7 ± 2.1 nM, a 20-fold improvement compared with the control ligand sulfonamide–GGGG. Silent encoding of multiple cPTMs can be readily incorporated into other <i>in vitro</i> display technologies such as bacteriophage T7 or mRNA display

Silent Encoding of Chemical Post-Translational Modifications in Phage-Displayed Libraries

<i>In vitro</i> selection of chemically modified peptide libraries presented on phage, while a powerful technology, is limited to one chemical post-translational modification (cPTM) per library. We use unique combinations of redundant codons to encode cPTMs with “silent barcodes” to trace multiple modifications within a mixed modified library. As a proof of concept, we produced phage-displayed peptide libraries Ser-[X]₄-Gly-Gly-Gly, with Gly and Ser encoded using unique combinations of codons (TCA-[X]₄-GGAGGAGGA, AGT-[X]₄-GGTGGTGGT, etc., where [X]₄ denotes a random NNK library). After separate chemical modification and pooling, mixed-modified libraries can be panned and deep-sequenced to identify the enriched peptide sequence and the accompanying cPTM simultaneously. We panned libraries bearing combinations of modifications (sulfonamide, biotin, mannose) against matched targets (carbonic anhydrase, streptavidin, concanavalin A) to identify desired ligands. Synthesis and validation of sequences identified by deep sequencing revealed that specific cPTMs are significantly enriched in panning against the specific targets. Panning on carbonic anhydrase yielded a potent ligand, sulfonamide–WIVP, with <i>K</i>_d = 6.7 ± 2.1 nM, a 20-fold improvement compared with the control ligand sulfonamide–GGGG. Silent encoding of multiple cPTMs can be readily incorporated into other <i>in vitro</i> display technologies such as bacteriophage T7 or mRNA display

Characterizing the Size and Composition of Saposin A Lipoprotein Picodiscs

Saposin A (SapA) lipoprotein discs, also known as picodiscs (PDs), represent an attractive method to solubilize glycolipids for protein interaction studies in aqueous solution. Recent electrospray ionization mass spectrometry (ESI-MS) data suggest that the size and composition of 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine (POPC)-containing PDs at neutral pH differs from those of <i>N</i>,<i>N</i>-dimethyldodecylamine <i>N</i>-oxide determined by X-ray crystallography. Using high-resolution ESI-MS, multiangle laser light scattering (MALLS), and molecular dynamics (MD) simulations, the composition, heterogeneity, and structure of POPC–PDs in aqueous ammonium acetate solutions at pH 4.8 and 6.8 were investigated. The ESI-MS and MALLS data revealed that POPC–PDs consist predominantly of (SapA dimer + <i>i</i>POPC) complexes, with <i>i</i> = 23–29, and have an average molecular weight (MW) of 38.2 ± 3.3 kDa at pH 4.8. In contrast, in freshly prepared solutions at pH 6.8, POPC–PDs are composed predominantly of (SapA tetramer + <i>i</i>POPC) complexes, with <i>i</i> = 37–60, with an average MW of 68.0 ± 2.7 kDa. However, the (SapA tetramer + <i>i</i>POPC) complexes are unstable at neutral pH and convert, over a period of hours, to (SapA trimer + <i>i</i>POPC) complexes, with <i>i</i> = 29–36, with an average MW of 51.1 ± 2.9 kDa. The results of molecular modeling suggest spheroidal structures for the (SapA dimer + <i>i</i>POPC), (SapA trimer + <i>i</i>POPC), and (SapA tetramer + <i>i</i>POPC) complexes in solution. Comparison of measured collision cross sections (Ω) with values calculated for gaseous (SapA dimer + 26POPC)⁸⁺, (SapA trimer + 33POPC)¹²⁺, and (SapA tetramer + 42POPC)¹⁶⁺ ions produced from modeling suggests that the solution structures are largely preserved in the gas phase, although the lipids do not maintain regular bilayer orientations

Apparent association constants (<i>K</i>_a) for <i>Pa</i>AlgX_27–474 and <i>Pa</i>AlgJ_79–379 for short polymannuronic oligosaccharides at 298 K and pH 7 determined by the direct ESI-MS assay.

<p>NB: No Binding.</p><p>*Ligand name as referenced in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004334#ppat.1004334-Walvoort2" target="_blank">[78]</a>.</p><p>Apparent association constants (<i>K</i>_a) for <i>Pa</i>AlgX_27–474 and <i>Pa</i>AlgJ_79–379 for short polymannuronic oligosaccharides at 298 K and pH 7 determined by the direct ESI-MS assay.</p