The in-depth characterization of glycan structures is crucial to understanding their structure−function relationships and their effects on health and various diseases. Despite advances in rapid analysis, the utility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is limited for complex mixtures of carbohydrates due to their low ionization efficiency and the difficulty in separating oligosaccharides because of their high structural similarity. In this study, we developed an ionic liquid (IL)-stabilized, nanomatrix-decorated, thin-layer chromatography (TLC)-MALDI MS method for simultaneous and rapid separation, detection, and identification of oligosaccharides to achieve efficient profiling. The IL demonstrated good dispersion and stabilization for the spin coating of dihydroxybenzoic acid-functionalized magnetic nanoparticles (DHB@MNPs) on the TLC plate with spot homogeneity, which contributed to the observed high reproducibility (<20% CV) and 12-and 28-fold signal enhancement. Although the TLC was not able to separate isomeric glycans, the DHB@MNPs generate diagnostic glycosidic and cross-ring cleavage ions, enabling on-spot structural elucidation of composition, sequence, branching, and linkage of glycans in each separated spot. Without chemical derivatization of glycan samples, glycan visualization by TLC and tandem MS, our integrated platform, allowed the identification of 25 oligosaccharides from human milk, and heatmap analysis revealed the variability in the oligosaccharide abundance in samples from individual donors at different lactation times, which may provide insight into the microbiota and immunity of infants. With the demonstrated simplicity of our sample preparation method along with the achieved separation and in-depth structural characterization, our approach can be used for the rapid screening of other oligosaccharide-rich samples.
Due to their unique glycan composition and linkage, protein glycosylation plays significant roles in cellular function and is associated with various diseases. For comprehensive characterization of their extreme structural complexity occurring in >50% of human proteins, time-consuming multi-step enrichment of glycopeptides is required. Here we report zwitterionic n-dodecylphosphocholine-functionalized magnetic nanoparticles (ZIC-cHILIC@MNPs) as a highly efficient affinity nanoprobe for large-scale enrichment of glycopeptides. We demonstrate that ZIC-cHILIC@MNPs possess excellent affinity, with 80–91% specificity for glycopeptide enrichment, especially for sialylated glycopeptide (90%) from biofluid specimens. This strategy provides rapidity (~10 min) and high sensitivity (<1 μL serum) for the whole enrichment process in patient serum, likely due to the rapid separation using magnetic nanoparticles, fast reaction, and high performance of the affinity nanoprobe at nanoscale. Using this strategy, we achieved personalized profiles of patients with hepatitis B virus (HBV, n = 3) and hepatocellular carcinoma (HCC, n = 3) at the depth of >3000 glycopeptides, especially for the large-scale identification of under-explored sialylated glycopeptides. The glycoproteomics atlas also revealed the differential pattern of sialylated glycopeptides between HBV and HCC groups. The ZIC-cHILIC@MNPs could be a generic tool for advancing the glycoproteome analysis, and contribute to the screening of glycoprotein biomarkers.
Phospholipids are the major components of cellular membranes and possess important biological roles. Despite their significance in diagnosis and therapeutic application in various diseases, systematic profiling of phospholipids remains challenging due to time‐consuming sample preparation and their inherent structure complexity. Taking advantages of complementary, simple, and fast structural analysis by matrix‐assisted laser desorption ionization‐time of flight mass spectrometry (MALDI‐TOF MS) and Fourier transform infrared spectroscopy (FTIR), we reported a nanoprobe‐based dual detection strategy for large‐scale phospholipid profiling. Based on electrostatic interaction between nitrilotriacetic acid (NTA) chelated iron ion and phosphate head on phospholipids, we developed an NTA‐decorated magnetic nanoparticle (MNP)‐based strategy for enrichment of phospholipids from lung cancer cell lines. Compared to the conventional liquid–liquid extraction, NTA@MNP nanoprobe enrichment demonstrated 2.6‐fold more identified phospholipids. By direct on‐particle analysis, FTIR confirmed the P‐O‐C and phosphate stretching characteristic of the phospholipid head group, and MALDI‐TOF MS identified a total of 59 phospholipids from PC9 and A549 lung cancer cells. Comparing the variation of phospholipids between two cell lines, we identified significantly more phospholipids in the PC9 cells (49) compared to 35 phospholipids in the A549 cells. The comparison also revealed 24 and 10 cell line‐specific phospholipids in the PC9 and A549 cells, respectively. Given the demonstrated rapid enrichment and unambiguous identification of phospholipids at cellular levels, the developed approach can provide systematic profiling to study the under‐explored phospholipid species.
Ultra-low molecular weight heparins (ULMWHs) are being used as anticoagulant drugs that exhibit a low risk, long lifetime and high efficacy. However, a robust and cost-effective purification methodology to isolate ULMWHs from naturally occurring heparins (HPs) is lacking. Vaccinia virus envelope protein A27 and a truncated variant (sA27-aa) bind specifically to HPs for virus/cell attachment. Here, we reported that using a high-affinity bioengineered sA27-aa for heparinase depolymerisation enables extraction of size-specific oligosaccharides, such as tetra-, hexa- and octa-saccharides (HP-4, -6 and − 8, respectively), with high purity and yield. Molecular interactions of the HP-4•viral protein complex and HP-4 sugar composition were resolved by mass and NMR, with data from both spectrometry in good agreement. As demonstrated by in vivo and in vitro bioassays, HP-4 possesses superior antithrombotic efficacy, stable properties and a low bleeding risk. Our methodology facilitates unprecedented extraction of unique ULMWHs that could benefit patients suffering from thrombosis.
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