Comprehensive characterization of complex glycosphingolipids in human pancreatic cancer tissues.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes of cancer-related deaths worldwide, accounting for 90% of primary pancreatic tumors with an average 5-year survival rate of less than 10%. PDAC exhibits aggressive biology, which, together with late detection, results in most PDAC patients presenting with unresectable, locally advanced, or metastatic disease. In-depth lipid profiling and screening of potential biomarkers currently appear to be a promising approach for early detection of PDAC or other cancers. Here, we isolated and characterized complex glycosphingolipids (GSL) from normal and tumor pancreatic tissues of patients with PDAC using a combination of TLC, chemical staining, carbohydrate-recognized ligand-binding assay, and LC/ESI-MS2. The major neutral GSL identified were GSL with the terminal blood groups A, B, H, Lea, Leb, Lex, Ley, P1, and PX2 determinants together with globo- (Gb3 and Gb4) and neolacto-series GSL (nLc4 and nLc6). We also revealed that the neutral GSL profiles and their relative amounts differ between normal and tumor tissues. Additionally, the normal and tumor pancreatic tissues differ in type 1/2 core chains. Sulfatides and GM3 gangliosides were the predominant acidic GSL along with the minor sialyl-nLc4/nLc6 and sialyl-Lea/Lex. The comprehensive analysis of GSL in human PDAC tissues extends the GSL coverage and provides an important platform for further studies of GSL alterations; therefore, it could contribute to the development of new biomarkers and therapeutic approaches.

Recognition of CD1d-sulfatide mediated by a type II natural killer T cell antigen receptor.

Natural killer T cells (NKT cells) are divided into type I and type II subsets on the basis of differences in their T cell antigen receptor (TCR) repertoire and CD1d-antigen specificity. Although the mode by which type I NKT cell TCRs recognize CD1d-antigen has been established, how type II NKT cell TCRs engage CD1d-antigen is unknown. Here we provide a basis for how a type II NKT cell TCR, XV19, recognized CD1d-sulfatide. The XV19 TCR bound orthogonally above the A' pocket of CD1d, in contrast to the parallel docking of type I NKT cell TCRs over the F' pocket of CD1d. At the XV19 TCR-CD1d-sulfatide interface, the TCRα and TCRß chains sat centrally on CD1d, where the malleable CDR3 loops dominated interactions with CD1d-sulfatide. Accordingly, we highlight the diverse mechanisms by which NKT cell TCRs can bind CD1d and account for the distinct antigen specificity of type II NKT cells.

Sulfatides are endogenous ligands for the TLR4-MD-2 complex.

Many endogenous molecules, mostly proteins, purportedly activate the Toll-like receptor 4 (TLR4)-myeloid differentiation factor-2 (MD-2) complex, the innate immune receptor for lipopolysaccharide (LPS) derived from gram-negative bacteria. However, there is no structural evidence supporting direct TLR4-MD-2 activation by endogenous ligands. Sulfatides (3-O-sulfogalactosylceramides) are natural, abundant sulfated glycolipids that have variously been shown to initiate or suppress inflammatory responses. We show here that short fatty acid (FA) chain sulfatides directly activate mouse TLR4-MD-2 independent of CD14, trigger MyD88- and TRIF-dependent signaling, and stimulate tumor necrosis factor α (TNFα) and type I interferon (IFN) production in mouse macrophages. In contrast to the agonist activity toward the mouse receptor, the tested sulfatides antagonize TLR4-MD-2 activation by LPS in human macrophage-like cells. The agonistic and antagonistic activities of sulfatides require the presence of the sulfate group and are inversely related to the FA chain length. The crystal structure of mouse TLR4-MD-2 in complex with C16-sulfatide revealed that three C16-sulfatide molecules bound to the MD-2 hydrophobic pocket and induced an active dimer conformation of the receptor complex similar to that induced by LPS or lipid A. The three C16-sulfatide molecules partially mimicked the detailed interactions of lipid A to achieve receptor activation. Our results suggest that sulfatides may mediate sterile inflammation or suppress LPS-stimulated inflammation, and that additional endogenous negatively charged lipids with up to six lipid chains of limited length might also bind to TLR4-MD-2 and activate or inhibit this complex.

Crystal structure of Vδ1 T cell receptor in complex with CD1d-sulfatide shows MHC-like recognition of a self-lipid by human γδ T cells.

The nature of the antigens recognized by γδ T cells and their potential recognition of major histocompatibility complex (MHC)-like molecules has remained unclear. Members of the CD1 family of lipid-presenting molecules are suggested ligands for Vδ1 TCR-expressing γδ T cells, the major γδ lymphocyte population in epithelial tissues. We crystallized a Vδ1 TCR in complex with CD1d and the self-lipid sulfatide, revealing the unusual recognition of CD1d by germline Vδ1 residues spanning all complementarity-determining region (CDR) loops, as well as sulfatide recognition separately encoded by nongermline CDR3δ residues. Binding and functional analysis showed that CD1d presenting self-lipids, including sulfatide, was widely recognized by gut Vδ1+ γδ T cells. These findings provide structural demonstration of MHC-like recognition of a self-lipid by γδ T cells and reveal the prevalence of lipid recognition by innate-like T cell populations.

Leukocyte and Dried Blood Spot Arylsulfatase A Assay by Tandem Mass Spectrometry.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed to measure arylsulfatase A (ARSA) activity in leukocytes and dried blood spots (DBS) using deuterated natural sulfatide substrate. These new assays were highly specific and sensitive. Patients with metachromatic leukodystrophy (MLD) and multiple sulfatase deficiency (MSD) displayed a clear deficit in the enzymatic activity and could be completely distinguished from normal controls. The leukocyte assay reported here will be important for diagnosing MLD and MSD patients and for monitoring the efficacy of therapeutic treatments. ARSA activity was measured in DBS for the first time without an antibody. This new ARSA DBS assay can serve as a second-tier test following the sulfatide measurement in DBS for newborn screening of MLD. This leads to an elimination of most of the false positives identified by the sulfatide assay.

The Role of Sulfatides in Axon-Glia Interactions.

Myelin is heavily enriched in lipids (comprising approximately 70% of its dry weight), and the amount of cholesterol and glycolipids is higher than in any other cell membrane. Galactocerebroside (GalC) and its sulfated form, sulfatide, comprise the major glycolipid components of myelin. Their functional significance has been extensively studied using membrane models, cell culture, and in vivo experiments in which either GalC/sulfatide or sulfatide is deficient. From these studies, GalC and sulfatide have been distinctly localized within oligodendrocytes and their specific function in myelin has been elucidated. Here, the function of sulfatide in axo-glial interactions in myelin-forming cells as well as within myelin and its potential mechanisms of action are discussed.

Sulfatide isoform pattern in cerebrospinal fluid discriminates progressive MS from relapsing-remitting MS.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Several biomarkers including proteins and lipids have been reported in MS cerebrospinal fluid (CSF), reflecting different aspects of the pathophysiology particularly of relapsing-remitting MS (RRMS). Sulfatide, abundant in the myelin sheath and a proposed target for autoimmune attack in MS, has been reported altered in MS CSF. Here, we investigated the potential of CSF sulfatide and its isoforms as biomarkers in MS. A highly sensitive and quantitative mass spectrometry method was employed to determine levels of sulfatide isoforms in CSF from RRMS and progressive MS (PMS) patients, and healthy donors (HD). We demonstrate that levels of total CSF sulfatide and C24:1, C26:1, and C26:1-OH isoforms were significantly increased in PMS compared with RRMS patients and HD, while C23:0-OH was significantly decreased in CSF from PMS patients compared to the other two groups. Multivariate discriminant analysis showed that CSF sulfatide isoform pattern in PMS patients was distinct and non-overlapping with that of RRMS patients and HD. Sulfatide levels did not correlate with tested biomarkers or clinical parameters. The results suggest that CSF sulfatide isoform levels may be used to discriminate the phenotype of MS and might play a role in the progression of the disease.

Direct visualization of the lateral structure of giant vesicles composed of pseudo-binary mixtures of sulfatide, asialo-GM1 and GM1 with POPC.

We compared the lateral structure of giant unilamellar vesicles (GUVs) composed of three pseudo binary mixtures of different glycosphingolipid (GSL), i.e. sulfatide, asialo-GM1 or GM1, with POPC. These sphingolipids possess similar hydrophobic residues but differ in the size and charge of their polar head group. Fluorescence microscopy experiments using LAURDAN and DiIC18 show coexistence of micron sized domains in a molar fraction range that depends on the nature of the GSLs. In all cases, experiments with LAURDAN show that the membrane lateral structure resembles the coexistence of solid ordered and liquid disordered phases. Notably, the overall extent of hydration measured by LAURDAN between the solid ordered and liquid disordered membrane regions show marked similarities and are independent of the size of the GSL polar head group. In addition, the maximum amount of GSL incorporated in the POPC bilayer exhibits a strong dependence on the size of the GSL polar head group following the order sulfatide>asialo-GM1>GM1. This observation is in full harmony with previous experiments and theoretical predictions for mixtures of these GSL with glycerophospholipids. Finally, compared with previous results reported in GUVs composed of mixtures of POPC with the sphingolipids cerebroside and ceramide, we observed distinctive curvature effects at particular molar fraction regimes in the different mixtures. This suggests a pronounced effect of these GSL on the spontaneous curvature of the bilayer. This observation may be relevant in a biological context, particularly in connection with the highly curved structures found in neural cells.

Interaction of lysozyme with a tear film lipid layer model: A molecular dynamics simulation study.

The tear film is a thin multilayered structure covering the cornea. Its outermost layer is a lipid film underneath of which resides on an aqueous layer. This tear film lipid layer (TFLL) is itself a complex structure, formed by both polar and nonpolar lipids. It was recently suggested that due to tear film dynamics, TFLL contains inhomogeneities in the form of polar lipid aggregates. The aqueous phase of tear film contains lachrymal-origin proteins, whereby lysozyme is the most abundant. These proteins can alter TFLL properties, mainly by reducing its surface tension. However, a detailed nature of protein-lipid interactions in tear film is not known. We investigate the interactions of lysozyme with TFLL in molecular details by employing coarse-grained molecular dynamics simulations. We demonstrate that lysozyme, due to lateral restructuring of TFLL, is able to penetrate the tear lipid film embedded in inverse micellar aggregates.

Oligodendroglial membrane dynamics in relation to myelin biogenesis.

In the central nervous system, oligodendrocytes synthesize a specialized membrane, the myelin membrane, which enwraps the axons in a multilamellar fashion to provide fast action potential conduction and to ensure axonal integrity. When compared to other membranes, the composition of myelin membranes is unique with its relatively high lipid to protein ratio. Their biogenesis is quite complex and requires a tight regulation of sequential events, which are deregulated in demyelinating diseases such as multiple sclerosis. To devise strategies for remedying such defects, it is crucial to understand molecular mechanisms that underlie myelin assembly and dynamics, including the ability of specific lipids to organize proteins and/or mediate protein-protein interactions in healthy versus diseased myelin membranes. The tight regulation of myelin membrane formation has been widely investigated with classical biochemical and cell biological techniques, both in vitro and in vivo. However, our knowledge about myelin membrane dynamics, such as membrane fluidity in conjunction with the movement/diffusion of proteins and lipids in the membrane and the specificity and role of distinct lipid-protein and protein-protein interactions, is limited. Here, we provide an overview of recent findings about the myelin structure in terms of myelin lipids, proteins and membrane microdomains. To give insight into myelin membrane dynamics, we will particularly highlight the application of model membranes and advanced biophysical techniques, i.e., approaches which clearly provide an added value to insight obtained by classical biochemical techniques.

Metabolism and functions of lipids in myelin.

Rapid conduction of nerve impulses requires coating of axons by myelin sheaths, which are lipid-rich and multilamellar membrane stacks. The lipid composition of myelin varies significantly from other biological membranes. Studies in mutant mice targeting various lipid biosynthesis pathways have shown that myelinating glia have a remarkable capacity to compensate the lack of individual lipids. However, compensation fails when it comes to maintaining long-term stability of myelin. Here, we summarize how lipids function in myelin biogenesis, axon-glia communication and in supporting long-term maintenance of myelin. We postulate that change in myelin lipid composition might be relevant for our understanding of aging and demyelinating diseases. This article is part of a Special Issue titled Brain Lipids.

Activation of PPARα by Fatty Acid Accumulation Enhances Fatty Acid Degradation and Sulfatide Synthesis.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first reaction in the mitochondrial fatty acid ß-oxidation pathway. VLCAD deficiency is associated with the accumulation of fat in multiple organs and tissues, which results in specific clinical features including cardiomyopathy, cardiomegaly, muscle weakness, and hepatic dysfunction in infants. We speculated that the abnormal fatty acid metabolism in VLCAD-deficient individuals might cause cell necrosis by fatty acid toxicity. The accumulation of fatty acids may activate peroxisome proliferator-activated receptor (PPAR), a master regulator of fatty acid metabolism and a potent nuclear receptor for free fatty acids. We examined six skin fibroblast lines, derived from VLCAD-deficient patients and identified fatty acid accumulation and PPARα activation in these cell lines. We then found that the expression levels of three enzymes involved in fatty acid degradation, including long-chain acyl-CoA synthetase (LACS), were increased in a PPARα-dependent manner. This increased expression of LACS might enhance the fatty acyl-CoA supply to fatty acid degradation and sulfatide synthesis pathways. In fact, the first and last reactions in the sulfatide synthesis pathway are regulated by PPARα. Therefore, we also measured the expression levels of enzymes involved in sulfatide metabolism and the regulation of cellular sulfatide content. The levels of these enzymes and cellular sulfatide content both increased in a PPARα-dependent manner. These results indicate that PPARα activation plays defensive and compensative roles by reducing cellular toxicity associated with fatty acids and sulfuric acid.

HspA1A, a 70-kDa heat shock protein, differentially interacts with anionic lipids.

HspA1A, a 70-kDa heat shock protein, binds to specific lipids. This interaction allows HspA1A to associate with the plasma and other cellular membranes, where it regulates many vital functions like immunity, membrane stabilization, autophagy, and apoptosis. However, the molecular mechanism of the HspA1A-lipid interactions has yet to be fully characterized. Therefore, in this study, we characterized the interaction of HspA1A with three lipids, bis-(monoacylglycero)-phosphate, cardiolipin, and sulfatide. Our results revealed that, first, HspA1A embeds in membranes when bound to liposomes composed of cardiolipin and sulfatide. Second, the binding of HspA1A to lipids is complex and although important, electrostatic interactions alone cannot fully explain the observed binding. Third, the two HspA1A domains, the nucleotide-binding domain and the substrate-binding domain, differentially bind to lipids in a lipid-specific manner. Fourth, HspA1A lipid-binding is reduced by the presence of nucleotides, but it is unaffected by the presence of a peptide-substrate. These observations suggest that HspA1A binds to lipids via a multi-step mechanism and this interaction depends on the specific physicochemical properties of the lipid. We speculate that the association of HspA1A with lipids like the mitochondrial cardiolipin, which is an organelle marker, may facilitate the translocation and localized function of the molecular chaperone to particular sub-cellular compartments.

Quantitative imaging mass spectrometry of renal sulfatides: validation by classical mass spectrometric methods.

Owing to its capability of discriminating subtle mass-altering structural differences such as double bonds or elongated acyl chains, MALDI-based imaging MS (IMS) has emerged as a powerful technique for analysis of lipid distribution in tissue at moderate spatial resolution of about 50 µm. However, it is still unknown if MS(1)-signals and ion intensity images correlate with the corresponding apparent lipid concentrations. Analyzing renal sulfated glycosphingolipids, sulfatides, we validate for the first time IMS-signal identities using corresponding sulfatide-deficient kidneys. To evaluate the extent of signal quenching effects interfering with lipid quantification, we surgically dissected the three major renal regions (papillae, medulla, and cortex) and systematically compared MALDI IMS of renal sulfatides with quantitative analyses of corresponding lipid extracts by on-target MALDI TOF-MS and by ultra-performance LC-ESI-(triple-quadrupole)tandem MS. Our results demonstrate a generally strong correlation (R(2) > 0.9) between the local relative sulfatide signal intensity in MALDI IMS and absolute sulfatide quantities determined by the other two methods. However, high concentrations of sulfatides in the papillae and medulla result in an up to 4-fold signal suppression. In conclusion, our study suggests that MALDI IMS is useful for semi-quantitative dissection of relative local changes of sulfatides and possibly other lipids in tissue.

Renal sulfatides: sphingoid base-dependent localization and region-specific compensation of CerS2-dysfunction.

Mammalian kidneys are rich in sulfatides. Papillary sulfatides, especially, contribute to renal adaptation to chronic metabolic acidosis. Due to differences in their cer-amide (Cer) anchors, the structural diversity of renal sulfatides is large. However, the underling biological function of this complexity is not understood. As a compound's function and its tissue location are intimately connected, we analyzed individual renal sulfatide distributions of control and Cer synthase 2 (CerS)2-deficient mice by imaging MS (IMS) and by LC-MS(2) (in controls for the cortex, medulla, and papillae separately). To explain locally different structures, we compared our lipid data with regional mRNA levels of corresponding anabolic enzymes. The combination of IMS and in source decay-LC-MS(2) analyses revealed exclusive expression of C20-sphingosine-containing sulfatides within the renal papillae, whereas conventional C18-sphingosine-containing compounds were predominant in the medulla, and sulfatides with a C18-phytosphingosine were restricted to special cortical structures. CerS2 deletion resulted in bulk loss of sulfatides with C23/C24-acyl chains, but did not lead to decreased urinary pH, as previously observed in sulfatide-depleted kidneys. The reasons may be the almost unchanged C22-sulfatide levels and constant total renal sulfatide levels due to compensation with C16- to C20-acyl chain-containing compounds. Intriguingly, CerS2-deficient kidneys were completely depleted of phytosphingosine-containing cortical sulfatides without any compensation.

A new analytical bench assay for the determination of arylsulfatase a activity toward galactosyl-3-sulfate ceramide: implication for metachromatic leukodystrophy diagnosis.

Here, we present the design and validation of a new assay for the diagnosis of metachromatic leukodystrophy. The method is highly specific, simple, reproducible, and straightforward. In our spectrophotometric method, the determination of arylsulfatase A (ARSA) activity toward the natural substrate, galactosyl-3-sulfate ceramide (or sulfatide), is performed using neat sulfatide without chemical modification. This confers to the assay high analytical specificity. The hydrolyzed sulfatide is monitored upon inclusion of the colorimetric reagent Azure A. The nonhydrolyzed sulfatide-Azure A is recovered and measured at a wavelength of λ = 650 nm. Thus, ARSA activity toward the sulfatide is obtained by subtracting the nonhydrolyzed sulfatide from the total sulfatide used in the enzyme reaction (sulfatide-Azure A present in a parallel assay performed in the absence of ARSA). Within a clinical context, our method definitely discriminated between healthy subject samples and metachromatic leukodystrophy patient samples, and, therefore, it is suitable for diagnostic applications and for monitoring the efficacy of therapeutic treatments in patients or animal models.

Ultra-performance liquid chromatography/tandem mass spectrometry for determination of sulfatides in dried blood spots from patients with metachromatic leukodystrophy.

RATIONALE: Metachromatic leukodystrophy (MLD) is a genetic autosomal recessive disease caused by a deficiency in arylsulfatase A. Accumulated sulfatides can be detected in the urine and detection of sulfatiduria is a useful test for diagnosis and monitoring. To our knowledge, no studies have explored the accumulation of sulfatides in dried blood spots (DBSs). We developed an ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method for measuring sulfatides in DBSs from patients with MLD. METHODS: DBSs were eluted with internal standard. After mixing and centrifugation, the organic layer was transferred to a 96-well microplate and dried, then resuspended in methanol/propanol solution. Samples were analyzed on an UPLC system. Total running time was 4 min. Quantification was achieved by multiple reaction monitoring using a tandem mass spectrometer. We evaluated the precision, linearity, and ion suppression of the method and analyzed sulfatide concentrations in DBS specimens from MLD patients (n = 9), pseudodeficiency (PD) patient (n = 1), obligate heterozygotes (OH) (n = 2) and normal controls (n = 124). RESULTS: In negative-ion mode, sulfatides species subjected to collision-induced dissociation readily fragment to produce an intense ion at m/z 96.8 (HSO4(-)). The precisions of low and high concentration controls ranged from 5.4 to 19.9%. The sulfatides produced linear responses. Molecular species of sulfatides were barely detected in DBSs from normal individuals and the PD-OH group [mean (range), 0.07 (<0.05-0.34) and 0.13 (<0.05-0.22) µg/mL, respectively]. In contrast, the DBSs from MLD patients showed a marked increase in several molecular species of sulfatide [mean (range), 2.02 (1.18-3.89) µg/mL]. CONCLUSIONS: Simultaneous detection for sulfatides using UPLC/MS/MS can be successfully applied to DBS analysis. This method provides a fast and effective screening and monitoring tool for the diagnosis and treatment of MLD.

Structure, sulfatide binding properties, and inhibition of platelet aggregation by a disabled-2 protein-derived peptide.

Disabled-2 (Dab2) targets membranes and triggers a wide range of biological events, including endocytosis and platelet aggregation. Dab2, through its phosphotyrosine-binding (PTB) domain, inhibits platelet aggregation by competing with fibrinogen for α(IIb)ß(3) integrin receptor binding. We have recently shown that the N-terminal region, including the PTB domain (N-PTB), drives Dab2 to the platelet membrane surface by binding to sulfatides through two sulfatide-binding motifs, modulating the extent of platelet aggregation. The three-dimensional structure of a Dab2-derived peptide encompassing the sulfatide-binding motifs has been determined in dodecylphosphocholine micelles using NMR spectroscopy. Dab2 sulfatide-binding motif contains two helices when embedded in micelles, reversibly binds to sulfatides with moderate affinity, lies parallel to the micelle surface, and when added to a platelet mixture, reduces the number and size of sulfatide-induced aggregates. Overall, our findings identify and structurally characterize a minimal region in Dab2 that modulates platelet homotypic interactions, all of which provide the foundation for rational design of a new generation of anti-aggregatory low-molecular mass molecules for therapeutic purposes.

Levels of plasma sulfatides C18 : 0 and C24 : 1 correlate with disease status in relapsing-remitting multiple sclerosis.

Multiple sclerosis (MS) is considered an autoimmune demyelinating disease of the CNS and myelin-derived glycolipids are one of the targets of this autoimmune attack. In this study, we examined for the first time the plasma distribution of sulfatide isoforms. Sulfatides with long-chain (C24 : 0 or C24 : 1) and short-chain (C16 : 0 or C18 : 0) fatty acids were quantified in plasma of relapsing­remitting MS patients by ultra-high-performance liquid chromatography tandem mass spectrometry. We found that C18 : 0 and C24 : 1 sulfatide plasma levels positively correlated with the Expanded Disability Status Scale. C16/C18 : 0 and C16/C24 : 0 ratios also correlated with the age and the time since last relapse. Healthy women showed higher levels of C16 : 0 sulfatide than healthy men; however, this gender difference disappeared in MS patients. Our data underline the potential use of sulfatides as biomarkers in relapsing­remitting MS and points to a possible association with the higher susceptibility of women to develop MS.Sulfatides are glycolipids highly enriched in myelin that have been associated with multiple sclerosis (MS). In this study, we have found a positive correlation between levels of specific sulfatides in plasma and increased disability in patients with relapsing-remitting MS. These findings underline the potential use of these molecules as biomarkers for MS.

Robust quantitation of gangliosides and sulfatides in human brain using UHPLC-MRM-MS: Method development and application in Alzheimer's disease.

Gangliosides (GAs) and sulfatides (STs) are acidic glycosphingolipids that are particularly abundant in the nervous system and are closely related to aging and neurodegenerative disorders. To explore their roles in brain diseases, in-depth molecular profiling, including structural variations of sphingoid backbone, fatty acyl group, and sugar chain of GAs and STs was performed. A total of 210 GAs and 38 STs were characterized in the inferior frontal gyrus (IFG) of human brain, with 90 GAs discovered in brain tissues for the first time. Influential MS parameters for detecting GAs and STs in multiple reaction monitoring (MRM) mode were systematically examined and optimized to minimize in-source fragmentation, resulting in remarkable signal intensity enhancement for GAs and STs, especially for polysialylated species. To eliminate analytical variations, isotopic interference-free internal standards were prepared by simple and fast reduction reaction. The final established method facilitated the simultaneous quantitation of 184 GAs and 30 STs from 25 subtypes, which represents the highest number of GAs quantitated among all quantitation methods recorded in literature so far. The method was further validated and applied to reveal the aberrant change of GAs and STs in the IFG of 12 Alzheimer's disease (AD) patients. Four GAs exhibited high classification capacity for AD (AUC ≥0.80) and were thereby considered the most promising signatures for AD. These findings suggested the close correlation between GAs and the pathogenesis of AD, highlighting the achievements of our robust method for investigating the roles of GAs and STs in various physiological states and diseases.