LC–MS bioanalysis of intact proteins and peptides

Bioanalysis assays that reliably quantify biotherapeutics and biomarkers in biological samples play pivotal roles in drug discovery and development. Liquid chromatography coupled with mass spectrometry (LC–MS), owing to its superior specificity, faster method development and multiplex capability, has evolved as one of the most important platforms for bioanalysis of biotherapeutics, particularly new scaffolds such as half-life extension platforms for proteins and peptides, as well as antibody drug conjugates. Intact LC–MS analysis is orthogonal to bottom-up surrogate peptide approach by providing whole molecule quantitation and high-level sequence and structure information. Here we review the latest development in LC–MS bioanalysis of intact proteins and peptides by summarizing recent publications and discussing the important topics such as the comparison between top-down intact analysis and bottom-up surrogate peptide approach, as well as simultaneous quantitation and catabolite identification. Key bioanalytical issues around intact protein bioanalysis such as sensitivity, data processing strategies, specificity, sample preparation and LC condition are elaborated. For peptides, topics including quantitation of intact peptide vs. digested surrogate peptide, metabolites, sensitivity, LC condition, assay performance, internal standard and sample preparation are discussed.     

Stafia-1: a STAT5a-Selective Inhibitor Developed via Docking-Based Screening of in Silico O-Phosphorylated Fragments

We present a new approach for the identification of inhibitors of phosphorylation-dependent protein–protein interaction domains, in which phenolic fragments are adapted by in silico O-phosphorylation before docking-based screening. From a database of 10 369 180 compounds, we identified 85 021 natural product-derived phenolic fragments, which were virtually O-phosphorylated and screened for in silico binding to the STAT3 SH2 domain. Nine screening hits were then synthesized, eight of which showed a degree of in vitro inhibition of STAT3. After analysis of its selectivity profile, the most potent inhibitor was then developed to Stafia-1, the first small molecule shown to preferentially inhibit the STAT family member STAT5a over the close homologue STAT5b. A phosphonate prodrug based on Stafia-1 inhibited STAT5a with selectivity over STAT5b in human leukemia cells, providing the first demonstration of selective in vitro and intracellular inhibition of STAT5a by a small-molecule inhibitor.     

Synthesis of C12-Keto Saxitoxin Derivatives with Unusual Inhibitory Activity Against Voltage-Gated Sodium Channels

A novel series of C12-keto-type saxitoxin (STX) derivatives bearing an unusual nonhydrated form of the ketone at C12 has been synthesized, and their Na_V-inhibitory activity has been evaluated in a cell-based assay as well as whole-cell patch-clamp recording. Among these compounds, 11-benzylidene STX ( 3 a ) showed potent inhibitory activity against neuroblastoma Neuro 2A in both cell-based and electrophysiological analyses, with EC₅₀ and IC₅₀ values of 8.5 and 30.7 nm , respectively. Interestingly, the compound showed potent inhibitory activity against tetrodotoxin-resistant subtype of Na_V1.5, with an IC₅₀ value of 94.1 nm . Derivatives 3 a – d and 3 f showed low recovery rates from Na_V1.2 subtype (ca 45–79 %) compared to natural dcSTX ( 2 ), strongly suggesting an irreversible mode of interaction. We propose an interaction model for the C12-keto derivatives with Na_V in which the enone moiety in the STX derivatives 3 works as Michael acceptor for the carboxylate of Asp¹⁷¹⁷.     

Synergistic Biophysical Techniques Reveal Structural Mechanisms of Engineered Cationic Antimicrobial Peptides in Lipid Model Membranes

In the quest for new antibiotics, two novel engineered cationic antimicrobial peptides (eCAPs) have been rationally designed. WLBU2 and D8 (all 8 valines are the d -enantiomer) efficiently kill both Gram-negative and -positive bacteria, but WLBU2 is toxic and D8 nontoxic to eukaryotic cells. We explore protein secondary structure, location of peptides in six lipid model membranes, changes in membrane structure and pore evidence. We suggest that protein secondary structure is not a critical determinant of bactericidal activity, but that membrane thinning and dual location of WLBU2 and D8 in the membrane headgroup and hydrocarbon region may be important. While neither peptide thins the Gram-negative lipopolysaccharide outer membrane model, both locate deep into its hydrocarbon region where they are primed for self-promoted uptake into the periplasm. The partially α-helical secondary structure of WLBU2 in a red blood cell (RBC) membrane model containing 50 % cholesterol, could play a role in destabilizing this RBC membrane model causing pore formation that is not observed with the D8 random coil, which correlates with RBC hemolysis caused by WLBU2 but not by D8.     

Extracellular Matrix Proteins Involved in Alzheimer's Disease

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases and characterized by cognitive and memory impairments. Emerging evidence suggests that the extracellular matrix (ECM) in the brain plays an important role in the etiology of AD. It has been detected that the levels of ECM proteins have changed in the brains of AD patients and animal models. Some ECM components, for example, elastin and heparan sulfate proteoglycans, are considered to promote the upregulation of extracellular amyloid-beta (Aβ) proteins. In addition, collagen VI and laminin are shown to have interactions with Aβ peptides, which might lead to the clearance of those peptides. Thus, ECM proteins are involved in both amyloidosis and neuroprotection in the AD process. However, the molecular mechanism of neuronal ECM proteins on the pathophysiology of AD remains elusive. More investigation of ECM proteins with AD pathogenesis is needed, and this may lead to novel therapeutic strategies and biomarkers for AD.     

Activity-Directed Synthesis of Inhibitors of the p53/hDM2 Protein–Protein Interaction

Protein–protein interactions (PPIs) provide a rich source of potential targets for drug discovery and biomedical science research. However, the identification of structural-diverse starting points for discovery of PPI inhibitors remains a significant challenge. Activity-directed synthesis (ADS), a function-driven discovery approach, was harnessed in the discovery of the p53/hDM2 PPI. Over two rounds of ADS, 346 microscale reactions were performed, with prioritisation on the basis of the activity of the resulting product mixtures. Four distinct and novel series of PPI inhibitors were discovered that, through biophysical characterisation, were shown to have promising ligand efficiencies. It was thus shown that ADS can facilitate ligand discovery for a target that does not have a defined small-molecule binding site, and can provide distinctive starting points for the discovery of PPI inhibitors.     

O6-Alkylguanine DNA Alkyltransferase Mediated Disassembly of a DNA Tetrahedron

Tetrahedron DNA structures were formed by the assembly of three-way junction ( TWJ ) oligonucleotides containing O⁶-2′-deoxyguanosine-alkylene-O⁶-2′-deoxyguanosine (butylene and heptylene linked) intrastrand cross-links (IaCLs) lacking a phosphodiester group between the 2′-deoxyribose residues. The DNA tetrahedra containing TWJs were shown to undergo an unhooking reaction by the human DNA repair protein O⁶-alkylguanine DNA alkyltransferase (hAGT) resulting in structure disassembly. The unhooking reaction of hAGT towards the DNA tetrahedra was observed to be moderate to virtually complete depending on the protein equivalents. DNA tetrahedron structures have been explored as drug delivery platforms that release their payload in response to triggers, such as light, chemical agents or hybridization of release strands. The dismantling of DNA tetrahedron structures by a DNA repair protein contributes to the armamentarium of approaches for drug release employing DNA nanostructures.     

Tailoring a Dress to Single Protein Molecules: Proteins Can Do It Themselves through Localized Photo-Polymerization and Molecular Imprinting

Molecularly imprinted polymer nanoparticles (MIP NPs) are antibody-like recognition materials prepared by a template-assisted synthesis. MIP NPs able to target biomolecules, like proteins, are under the spotlight for their great potential in medicine, but efficiently imprinting biological templates is still very challenging. Here we propose generating a molecular imprint in single NPs, by photochemically initiating the polymerization from individual protein templates. In this way, each protein molecule tailors itself its own “polymeric dress”. For this, the template protein is covalently coupled with a photoinitiator, Eosin Y. Irradiated with light at 533 nm, the Eosin moiety acts as an antenna and transfers energy to a co-initiator (an amine), which generates a radical and initiates polymerization. As a result, a polymer network is forming only around the very template molecule, producing cross-linked NPs of 50 nm, with single binding sites showing high affinity (K_D 10⁻⁹ m ) for their biological target, and selectivity over other proteins.     

Electrospinnability study of pea (Pisum sativum) and common bean (Phaseolus vulgaris L.) using the conformational and rheological behavior of their protein isolates

Pea protein isolate (PPI) and bean protein concentrate (BPC) were evaluated as fiber-forming vegetal source materials through electrospinning using various solvents. The effects of hexafluoroisopropanol (HFIP), trifluoroethanol (TFE), trifluoroacetic acid (TFA), formic acid (FA) and water on rheological and conformational properties of the protein solutions were determined. The morphology and molecular organization of the electrospun structures were studied. All PPI and BPC solutions displayed pseudoplastic behavior. Circular dichroism spectroscopy revealed that β-type turns and β-sheets were the dominant protein conformations in water, HFIP, and TFE. After electrospinning, most of the solutions afforded beads. Fiber-like morphologies were only obtained when BPC was dissolved in HFIP. BPC demonstrated better performance in the electrospinning process than PPI. Denaturation of the protein isolates was not sufficient to form fibers, the viscosity of the solution as well as the vapor pressure of the solvents played an important role in defining the morphology.