The Minimum Information Required for a Glycomics Experiment (MIRAGE) project: improving the standards for reporting glycan microarray-based data

MIRAGE (Minimum Information Required for A Glycomics Experiment) is an initiative that was created by experts in the fields of glycobiology, glycoanalytics, and glycoinformatics to produce guidelines for reporting results from the diverse types of experiments and analyses used in structural and functional studies of glycans in the scientific literature. As a sequel to the guidelines for sample preparation (Struwe et al. 2016, Glycobiology, 26, 907-910) and mass spectrometry (MS) data (Kolarich et al. 2013, Mol. Cell Proteomics. 12, 991-995), here we present the first version of guidelines intended to improve the standards for reporting data from glycan microarray analyses. For each of eight areas in the workflow of a glycan microarray experiment, we provide guidelines for the minimal information that should be provided in reporting results. We hope that the MIRAGE glycan microarray guidelines proposed here will gain broad acceptance by the community, and will facilitate interpretation and reproducibility of the glycan microarray results with implications in comparison of data from different laboratories and eventual deposition of glycan microarray data in international databases

A genetic cause of Alzheimer disease: mechanistic insights from Down syndrome

Down syndrome, caused by an extra copy of chromosome 21, is associated with a greatly increased risk of early onset Alzheimer disease. It is thought that this risk is conferred by the presence of three copies of the gene encoding amyloid precursor protein (APP), an Alzheimer risk factor, although the possession of extra copies of other chromosome 21 genes may also play a role. Further study of the mechanisms underlying the development of Alzheimer disease in Down syndrome could provide insights into the mechanisms that cause dementia in the general population

Relating glycoprotein structural heterogeneity to function – insights from native mass spectrometry

Glycosylation is the most complex and prevalent protein modification that influences attributes ranging from cellular localization and signaling to half-life and proteolysis. Glycoconjugates are fundamental for cellular function and alterations in their structure are often observed in pathological states. Most biotherapeutic proteins are glycosylated, which influences drug safety and efficacy. Therefore, the ability to characterize glycoproteins is important in all areas of biomolecular and medicinal research. Here we discuss recent advances in native mass spectrometry that have significantly improved our ability to characterize heterogeneous glycoproteins and to relate glycan structure to protein function

Relating glycoprotein structural heterogeneity to function – insights from native mass spectrometry

Glycosylation is the most complex and prevalent protein modification that influences attributes ranging from cellular localization and signaling to half-life and proteolysis. Glycoconjugates are fundamental for cellular function and alterations in their structure are often observed in pathological states. Most biotherapeutic proteins are glycosylated, which influences drug safety and efficacy. Therefore, the ability to characterize glycoproteins is important in all areas of biomolecular and medicinal research. Here we discuss recent advances in native mass spectrometry that have significantly improved our ability to characterize heterogeneous glycoproteins and to relate glycan structure to protein function

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

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

Single molecule mass photometry of nucleic acids

Mass photometry is a recently developed methodology capable of measuring the mass of individual proteins under solution conditions. Here, we show that this approach is equally applicable to nucleic acids, enabling their facile, rapid and accurate detection and quantification using sub-picomoles of sample. The ability to count individual molecules directly measures relative concentrations in complex mixtures without need for separation. Using a dsDNA ladder, we find a linear relationship between the number of bases per molecule and the associated imaging contrast for up to 1200 bp, enabling us to quantify dsDNA length with up to 2 bp accuracy. These results introduce mass photometry as an accurate, rapid and label-free single molecule method complementary to existing DNA characterization techniques

Native mass spectrometry: towards high-throughput structural proteomics.

Native mass spectrometry (MS) has become a sensitive method for structural proteomics, allowing practitioners to gain insight into protein self-assembly, including stoichiometry and three-dimensional architecture, as well as complementary thermodynamic and kinetic aspects. Although MS is typically performed in vacuum, a body of literature has described how native solution-state structure is largely retained on the timescale of the experiment. Native MS offers the benefit that it requires substantially smaller quantities of a sample than traditional structural techniques such as NMR and X-ray crystallography, and is therefore well suited to high-throughput studies. Here we first describe the native MS approach and outline the structural proteomic data that it can deliver. We then provide practical details of experiments to examine the structural and dynamic properties of protein assemblies, highlighting potential pitfalls as well as principles of best practice

Probing N-glycoprotein microheterogeneity by lectin affinity purification-mass spectrometry analysis

Lectins are carbohydrate binding proteins that recognize specific epitopes present on target glycoproteins. Changes in lectin-reactive carbohydrate repertoires are related to many biological signaling pathways and recognized as hallmarks of several pathological processes. Consequently, lectins are valuable probes, commonly used for examining glycoprotein structural and functional microheterogeneity. However, the molecular interactions between a given lectin and its preferred glycoproteoforms are largely unknown due to the inherent complexity and limitations of methods used to investigate intact glycoproteins. Here, we apply a lectin-affinity purification procedure coupled with native mass spectrometry to characterize lectin-reactive glycoproteoforms at the intact protein level. We investigate the interactions between the highly fucosylated and highly branched glycoproteoforms of haptoglobin and α1-acid glycoprotein using two different lectins Aleuria aurantia lectin (AAL) and Phaseolus vulgaris leucoagglutinin (PHA-L), respectively. Firstly we show a co-occurrence of fucosylation and N-glycan branching on haptoglobin, particularly among highly fucosylated glycoproteoforms. Secondly, we analyze the global heterogeneity of highly branched glycoproteoforms of haptoglobin and α1-acid glycoprotein and reveal that while multi-fucosylation attenuates the lectin PHA-L binding to haptoglobin, it has no impact on AGP. Taken together, our lectin affinity purification native MS approach elucidates lectin specificities between intact glycoproteins, not achievable by other methods. Moreover, since aberrant glycosylation of Hp and AGP are potential markers for many diseases, including pancreatic, hepatic and ovarian cancers, understanding their interactions with lectins will help the development of carbohydrate-centric monitoring methods to understand their pathophysiological implications

Immune recruitment or suppression by glycan engineering of endogenous and therapeutic antibodies

Human serum IgG contains multiple glycoforms which exhibit a range of binding properties to effector molecules such as cellular Fc receptors. Emerging knowledge of how the Fc glycans contribute to the antibody structure and effector functions has opened new avenues for the exploitation of defined antibody glycoforms in the treatment of diseases. Here, we review the structure and activity of antibody glycoforms and highlight developments in antibody glycoengineering by both the manipulation of the cellular glycosylation machinery and by chemoenzymatic synthesis. We discuss wide ranging applications of antibody glycoengineering in the treatment of cancer, autoimmunity and inflammation