Glycoproteomic Sample Processing, LC-MS, and Data Analysis Using GlycReSoft.

Identification of N- and O-glycosylation on specific sites of proteins, along with glycan structural information, is necessary to determine the roles glycoproteins play in normal and pathologic cellular functions. Because such glycosylation is macro- and micro-heterogeneous and alters the dissociation behavior of glycopeptides, specific sample preparation, mass spectrometry, and data analysis techniques are required. Advanced tandem mass spectrometry-based glycoproteomics coupled with powerful data mining algorithms are key elements for characterization of protein glycosylation. This article includes the detailed, streamlined sample preparation method for liquid chromatography-mass spectrometry data acquisition and subsequent bioinformatics-based data annotation using the publicly available GlycReSoft program for highly efficient identification and quantification of glycoprotein glycosylation. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Characterization of glycans and site occupancy on purified glycoprotein Support Protocol 1: In-gel digestion of glycoproteins Support Protocol 2: Detection of glycoproteins from cells/tissue through glycopeptide enrichment Basic Protocol 2: Acquisition of glycopeptides through high-resolution nano-LC-MS/MS Basic Protocol 3: Identification and quantification of glycopeptides using GlycReSoft.

Steroidal saponins and sapogenins from fenugreek and their inhibitory activity against α-glucosidase.

The seed of Trigonella foenum-graecum L. (fenugreek) has been reported to be rich in saponins, especially the dioscin or diosgenin, which are natural anti-diabetic agents with relatively low toxicity. Thus, the present study was to purify the saponins and sapogenins from fenugreek and to evaluate their α-glucosidase inhibitory activity in vitro. As a result, 33 steroidal saponins and sapogenins were isolated, including six undescribed ones and 27 previously known molecules. Among them, compounds 10, 12, 17, 22 and 29 were five 25R and 25S isomer mixtures of spirostanol saponins or sapogenins. The structures of compound 1-6 were established by 1D and 2D NMR spectroscopic analyses, high-resolution mass spectrometry, and chemical evidence. Compared to the positive control, sapogenins 26, 27, 14 and saponins 18 and 23 considerably inhibited α-glucosidase at IC50 values of 15.16, 8.98, 7.26, 5.49 and 14.01 µM, respectively. These results support the therapeutic potential of fenugreek in the treatment of diabetes with saponins and sapogenins as the active constituents.

Efficient enrichment of glycopeptides by supramolecular nanoassemblies that use proximity-assisted covalent binding.

Mass spectrometry (MS)-based analysis of glycoproteins and glycopeptides requires selective separation strategies to eliminate interferences from more abundant non-glycosylated biomolecules. In this work, we describe a two-phase liquid-liquid extraction method using supramolecular polymeric nanoassemblies that can selectively and efficiently enrich glycopeptides for enhanced MS detection. The polymeric nanoassemblies are made selective for glycopeptides via the incorporation of hydrazide functional groups that covalently bind to glycans. The enrichment efficiency is further enhanced via the incorporation of acidic functional groups that lead to a proximity-assisted catalysis of the hydrazide-glycan conjugation reaction. Our results further demonstrate the value of designer supramolecular nanomaterials for the selective enrichment of modified peptides from complicated mixtures.

Sequential Nucleophilic "Click" Reactions for Functional Amphiphilic Homopolymers.

Amphiphilic homopolymers with high densities of functional groups are synthetically challenging. Thiol-yne nucleophilic click reactions have been investigated to introduce multiple functional groups in polymers with high density. An electron deficient alkyne group bearing methacrylate monomer was polymerized using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Subsequently, the electron deficient alkyne group on polymer side chain was readily reacted with a thiol reagent using triethylamine (TEA) as the organocatalyst. This reaction was found to be very efficient under mild conditions. The resultant homopolymer bearing thiol vinyl ether functional groups could perform a second thiol addition with a stronger base, such as triazabicyclodecene (TBD), to prepare multifunctional homopolymers. This stepwise addition process was monitored by 1H NMR as well as gel permeation chromatography. The fidelity of this method was demonstrated by attaching four different functionalities, including both hydrophobic and hydrophilic moieties. Furthermore, these dual functionalized polymers bearing dithio-acetal groups are sensitive to reactive oxygen species (ROS), which compromises the host-guest properties of the assembly in response to this stimulus. The ROS responsive polymers reported here may have potential use in therapeutic delivery.

Improved mass spectrometric detection of acidic peptides by variations in the functional group pKa values of reverse micelle extraction agents.

Polymeric reverse micelles can be used to selectively extract peptides from complex mixtures via a two-phase extraction approach. In previous work, we have shown that the charge polarity of the hydrophilic functional group that is in the interior of the reverse micelle dictates the extraction selectivity. To investigate how the extraction is influenced by the inherent pKa of the functional group, we designed and tested a series of polymeric reverse micelles with variations in the hydrophilic functional group. From this series of polymers, we find that the extraction capability of the reverse micelles in an apolar phase is directly related to the aqueous phase pKa of the interior functional group, suggesting that the functional groups maintain their inherent chemistry even in the confined environment of the reverse micelle interior. Because these functional groups maintain their inherent pKa in the reverse micelle interior, they provide predictable extraction selectivity upon changes in aqueous phase pH. We exploit this finding to demonstrate that sulfonate-containing polymers can be used to remove basic peptides from complex mixtures, thereby allowing the improved detection of acidic peptides. Using these new materials, we also demonstrate a new means of isoelectric point (pI) bracketing that allows the mass spectrometric detection of peptides with a defined and narrow range of pI values.

Supramolecular Assemblies for Transporting Proteins Across an Immiscible Solvent Interface.

Polymeric supramolecular assemblies that can effectively transport proteins across an incompatible solvent interface are described. We show that electrostatics and ligand-protein interactions can be used to selectively transport proteins from an aqueous phase to organic phase. These transported proteins have been shown to maintain their tertiary structure and function. This approach opens up new possibilities for application of supramolecular assemblies in sensing, diagnostics and catalysis.

Altering the Peptide Binding Selectivity of Polymeric Reverse Micelle Assemblies via Metal Ion Loading.

Supramolecular reverse micelle assemblies, formed by amphiphilic copolymers, can selectively encapsulate molecules in their interiors depending on the functional groups present in the polymers. Altering the binding selectivity of these materials typically requires the synthesis of alternate functional groups. Here, we demonstrate that the addition of Zr(IV) ions to the interiors of reverse micelles having phosphonate functional groups transforms the supramolecular materials from ones that selectively bind positively charged peptides into materials that selectively bind phosphorylated peptides. We also show that the binding selectivity of these reverse micelle assemblies can be further tuned by varying the fractions of phosphonate groups in the copolymer structure. The optimized reverse micelle materials can selectively transfer and bind phosphorylated peptides from aqueous solutions over a wide range of pH conditions and can selectively enrich phosphorylated peptides even in complicated mixtures.