Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N﻿- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved 'high-coverage' and 'high-accuracy' glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.

Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis.

Severe acute respiratory syndrome coronavirus 2 is the causative pathogen of the COVID-19 pandemic which as of March 29, 2021, has claimed 2 776 175 lives worldwide Vaccine development efforts focus on the viral trimeric spike glycoprotein as the main target of the humoral immune response Viral spikes carry glycans that facilitate immune evasion by shielding specific protein epitopes from antibody neutralization, and antigen efficacy is influenced by spike glycoprotein production in vivo Therefore, immunogen integrity is important for glycoprotein-based vaccine candidates Here, we show how site-specific glycosylation differs between virus-derived spikes, wild-type, non-stabilized spikes expressed from a plasmid with a CMV promoter and tPA signal sequence, and commonly used recombinant, engineered spike glycoproteins Furthermore, we show that their distinctive cellular secretion pathways result in different protein glycosylation and secretion patterns, including shedding of spike monomeric subunits for the non-stabilized wild-type spike tested, which may have implications for the resulting immune response and vaccine design

Assessing Antigen Structural Integrity through Glycosylation Analysis of the SARS-CoV-2 Viral Spike

Protein misfolding and aggregation are characteristic of a wide range of neurodegenerative disorders, including Alzheimer and Parkinson diseases. A hallmark of these diseases is the aggregation of otherwise soluble and functional proteins into amyloid aggregates. Although for many decades such amyloid deposits have been thought to be responsible for disease progression, it is now increasingly recognized that the misfolded protein oligomers formed during aggregation are, instead, the main agents causing pathological processes. These oligomers are transient and heterogeneous, which makes it difficult to detect and quantify them, generating confusion about their exact role in disease. The lack of suitable methods to address these challenges has hampered efforts to investigate the molecular mechanisms of oligomer toxicity and to develop oligomer-based diagnostic and therapeutic tools to combat protein misfolding diseases. In this Review, we describe methods to quantify misfolded protein oligomers, with particular emphasis on diagnostic applications as disease biomarkers and on therapeutic applications as target biomarkers. The development of these methods is ongoing, and we discuss the challenges that remain to be addressed to establish measurement tools capable of overcoming existing limitations and to meet present needs. Alzheimer and Parkinson diseases involve protein oligomers that are dilute, transient and heterogeneous. This Review describes techniques to characterize these challenging analytes.

Quantifying misfolded protein oligomers as drug targets and biomarkers in Alzheimer and Parkinson diseases

The quantification of membrane-associated biomolecular interactions is crucial to our understanding of various cellular processes. State-of-the-art single-molecule approaches rely largely on the addition of fluorescent labels, which complicates the quantification of the involved stoichiometries and dynamics because of low temporal resolution and the inherent limitations associated with labeling efficiency, photoblinking and photobleaching. Here, we demonstrate dynamic mass photometry, a method for label-free imaging, tracking and mass measurement of individual membrane-associated proteins diffusing on supported lipid bilayers. Application of this method to the membrane remodeling GTPase, dynamin-1, reveals heterogeneous mixtures of dimer-based oligomers, oligomer-dependent mobilities, membrane affinities and (dis)association of individual complexes. These capabilities, together with assay-based advances for studying integral membrane proteins, will enable the elucidation of biomolecular mechanisms in and on lipid bilayers. Dynamic mass photometry allows label-free tracking and mass measurement of individual membrane-associated proteins diffusing on supported lipid bilayers. The approach can be used to monitor dynamic (dis)assembly of protein complexes.

/pdf/mass-photometry-enables-label-free-tracking-and-mass-4m5l395nq1.pdf

Mass photometry enables label-free tracking and mass measurement of single proteins on lipid bilayers.

Our ability to detect, image, and quantify nanoscopic objects and molecules with visible light has undergone dramatic improvements over the past few decades. While fluorescence has historically been the go-to contrast mechanism for ultrasensitive light microscopy due to its superior background suppression and specificity, recent developments based on light scattering have reached single-molecule sensitivity. They also have the advantages of universal applicability and the ability to obtain information about the species of interest beyond its presence and location. Many of the recent advances are driven by novel approaches to illumination, detection, and background suppression, all aimed at isolating and maximizing the signal of interest. Here, we review these developments grouped according to the basic principles used, namely darkfield imaging, interferometric detection, and surface plasmon resonance microscopy.

Scattering-based Light Microscopy: From Metal Nanoparticles to Single Proteins.

Interactions between biomolecules control the processes of life in health and their malfunction in disease, making their characterization and quantification essential. Immobilization- and label-free analytical techniques are desirable because of their simplicity and minimal invasiveness, but they struggle with quantifying tight interactions. Here, we show that mass photometry can accurately count, distinguish by molecular mass, and thereby reveal the relative abundances of different unlabelled biomolecules and their complexes in mixtures at the single-molecule level. These measurements determine binding affinities over four orders of magnitude at equilibrium for both simple and complex stoichiometries within minutes, as well as the associated kinetics. These results introduce mass photometry as a rapid, simple and label-free method for studying sub-micromolar binding affinities, with potential for extension towards a universal approach for characterizing complex biomolecular interactions.

https://onlinelibrary.wiley.com/doi/epdf/10.1002/anie.202001578

Quantifying protein‐protein interactions by molecular counting with mass photometry

Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase.

Interactions between biomolecules control the processes of life in health, and their malfunction in disease, making their characterization and quantification essential. Immobilization- and label-free analytical techniques are particular desirable because of their simplicity and minimal invasiveness, but struggle to quantify tight interactions. Here, we show that we can accurately count, distinguish by molecular mass, and thereby reveal the relative abundances of different un-labelled biomolecules and their complexes in mixtures at the single-molecule level by mass photometry. These measurements enable us to quantify binding affinities over four orders of magnitude at equilibrium for both simple and complex stoichiometries within minutes, as well as to determine the associated kinetics. Our results introduce mass photometry as a rapid, simple and label-free method for studying sub-μM binding affinities, with potential to be extended towards a universal approach for characterising complex biomolecular interactions.

/pdf/quantifying-protein-protein-interactions-by-molecular-4eicflj47u.pdf

Quantifying protein-protein interactions by molecular counting with mass photometry

Protein–protein interactions are involved in the regulation and function of the majority of cellular processes. As a result, much effort has been aimed at the development of methodologies capable of quantifying protein–protein interactions, with label-free methods being of particular interest due to the associated simplified workflows and minimisation of label-induced perturbations. Here, we review recent advances in optical technologies providing label-free in vitro measurements of affinities and kinetics. We provide an overview and comparison of existing techniques and their principles, discussing advantages, limitations, and recent applications.

/pdf/label-free-methods-for-optical-in-vitro-characterization-of-pzuju4954h.pdf

Fabian Soltermann

Papers

Quantifying protein‐protein interactions by molecular counting with mass photometry

Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase.

Quantifying protein-protein interactions by molecular counting with mass photometry

Label-free methods for optical in vitro characterization of protein–protein interactions

Frontispiece: Quantifying Protein–Protein Interactions by Molecular Counting with Mass Photometry