Differential analysis of site-specific glycans on plasma and cellular fibronectins: application of a hydrophilic affinity method for glycopeptide enrichment

Isolation of glycopeptides utilizing hydrogen bonding between glycopeptide glycans and a carbohydrate-gel matrix in the organic phase is useful for site-specific characterization of oligosaccharides of glycoproteins, when combined with mass spectrometry. In this study, recovery of glycopeptides was improved by including divalent cations or increasing the organic solvent in the binding solution, without losing specificity, whereas it was still less effective for those with a long peptide backbone exceeding 50 amino acid residues. The method was then applied to the analysis of glycan heterogeneities at seven N-glycosylation sites in each of the plasma and cellular fibronectins (FNs). There was a remarkable site-specific difference in fucosylation between these isoforms; Asn1244 selectively escaped the global fucosylation of cellular FN, whereas only Asn1007 and Asn2108 of the plasma isoform underwent modification. In addition, a new O-glycosylation site was identified at Thr279 in the connecting segment between the fibrin- and heparin-binding domain and the collagen-binding domain, and the glycopeptide was reactive to a peanut agglutinin lectin. Considering that another mucin-type O-glycosylation site lies within a different connecting segment, the O-glycosylation of FN was suggested to play a significant role in segregating the neighboring domains and thus maintaining the topology of FN and the domain functions. In addition, the method was applied to apolipoprotein B-100 (apoB100) whose N-glycan structures at 17 of 19 potential sites have been reported, and characterized the remaining sites. The results also demonstrated that the enriched glycopeptide provides resources for site-specific analysis of oligosaccharides in glycoproteomics.     

Targeted analysis of ganglioside and sulfatide molecular species by LC/ESI-MS/MS with theoretically expanded multiple reaction monitoring

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has been applied primarily to the analysis of glycosphingolipids separated from other complex mixtures by TLC, but it is difficult to obtain quantitative profiling of each glycosphingolipid among the different spots on TLC by MALDI-MS. Thus, the development of a convenient approach that utilizes liquid chromatography/electrospray ionization (LC/ESI)-MS has received interest. However, previously reported methods have been insufficient to separate and distinguish each ganglioside class. Here we report an effective method for the targeted analysis of theoretically expected ganglioside molecular species by LC/ESI tandem mass spectrometry (LC/ESI-MS/MS) in combination with multiple reaction monitoring (MRM). MRM detection specific for sialic acid enabled us to analyze ganglioside standards such as GM1, GM2, GM3, GD1, and GT1 at picomolar to femtomolar levels. Furthermore, other gangliosides, such as GD2, GD3, GT2, GT3, and GQ1, were also detected in glycosphingolipid standard mixtures from porcine brain and acidic glycolipid extract from mouse brain by theoretically expanded MRM. We found that this approach was also applicable to sulfatides contained in the glycosphingolipid mixtures. In addition, we established a method to separate and distinguish regioisomeric gangliosides, such as GM1a and -1b, GD1a, -1b, and -1c, and GT1a, -1b, and -1c with diagnostic sugar chains in the MRM.     

Improved analysis of bile acids in tissues and intestinal contents of rats using LC/ESI-MS

To evaluate bile acid (BA) metabolism in detail, we established a method for analyzing BA composition in various tissues and intestinal contents using ultra performance liquid chromatography/electrospray ionization mass spectrometry (UPLC/ESI-MS). Twenty-two individual BAs were determined simultaneously from extracts. We applied this method to define the differences in BA metabolism between two rat strains, WKAH and DA. The amount of total bile acids (TBAs) in the liver was significantly higher in WKAH than in DA rats. In contrast, TBA concentration in jejunal content, cecal content, colorectal content, and feces was higher in DA rats than in WKAH rats. Nearly all BAs in the liver were in the taurine- or glycine-conjugated form in DA rats, and the proportion of conjugated liver BAs was up to 75% in WKAH rats. Similar trends were observed for the conjugation rates in bile. The most abundant secondary BA in cecal content, colorectal content, and feces was hyodeoxycholic acid in WKAH rats and omega-muricholic acid in DA rats. Analyzing detailed BA profiles, including conjugation status, in a single run is possible using UPLC/ESI-MS. This method will be useful for investigating the roles of BA metabolism under physiological and pathological conditions.     

Analysis of vitamin E metabolites including carboxychromanols and sulfated derivatives using LC/MS/MS

Tocopherols and tocotrienols are metabolized via hydroxylation and oxidation of their hydrophobic side chain to generate 13′-hydroxychromanols (13′-OHs) and various carboxychromanols, which can be further metabolized by conjugation including sulfation. Recent studies indicate that long-chain carboxychromanols, especially 13′-carboxychromanol (13′-COOH), appear to be more bioactive than tocopherols in anti-inflammatory and anticancer actions. To understand the potential contribution of metabolites to vitamin E-mediated effects, an accurate assay is needed to evaluate bioavailability of these metabolites. Here we describe an LC/MS/MS assay for quantifying vitamin E metabolites using negative polarity ESI. This assay includes a reliable sample extraction procedure with efficacy of ≥ 89% and interday/intraday variation of 3–11% for major metabolites. To ensure accurate quantification, short-chain, long-chain, and sulfated carboxychromanols are included as external/internal standards. Using this assay, we observed that sulfated carboxychromanols are the primary metabolites in the plasma of rodents fed with γ-tocopherol or δ-tocopherol. Although plasma levels of 13′-COOHs and 13′-OHs are low, high concentrations of these compounds are found in feces. Our study demonstrates an LC/MS/MS assay for quantitation of sulfated and unconjugated vitamin E metabolites, and this assay will be useful for evaluating the role of these metabolites in vivo.     

Rapid measurement of plasma free fatty acid concentration and isotopic enrichment using LC/MS

Measurements of plasma free fatty acids (FFA) concentration and isotopic enrichment are commonly used to evaluate FFA metabolism. Until now, gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) was the best method to measure isotopic enrichment in the methyl derivatives of ¹³C-labeled fatty acids. Although IRMS is excellent for analyzing enrichment, it requires time-consuming derivatization steps and is not optimal for measuring FFA concentrations. We developed a new, rapid, and reliable method for simultaneous quantification of ¹³C-labeled fatty acids in plasma using high-performance liquid chromatography-mass spectrometry (HPLC/MS). This method involves a very quick Dole extraction procedure and direct injection of the samples on the HPLC system. After chromatographic separation, the samples are directed to the mass spectrometer for electrospray ionization (ESI) and analysis in the negative mode using single ion monitoring. By employing equipment with two columns connected parallel to a mass spectrometer, we can double the throughput to the mass spectrometer, reducing the analysis time per sample to 5 min. Palmitate flux measured using this approach agreed well with the GC/C/IRMS method. This HPLC/MS method provides accurate and precise measures of FFA concentration and enrichment.     

Structure and conformational analysis of lipid-associating peptides of apolipoprotein B-100 produced by trypsinolysis

Apolipoprotein B-100 (apo B-100) contains putative lipid-associating regions that are, in part, responsible for its overall structure in human plasma low-density lipoproteins. Some of these regions have been identified by reassembly of the total tryptic peptides of apo B-100 with bovine brain sphingomyelin, 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and dimyristoylphos-phatidylcholine (DPMC). Although more than 500 tryptic peptides are predicted from the known number of arginines and lysines in apo B-100, significant amounts of only 13 peptides spontaneously associate with all three phospholipids. These peptides share some structural characteristics, as predicted by several algorithms, that distinguish them from the water-soluble apolipoproteins. Most apolipoproteins associate with lipids via amphipathic helices and are highly helical in native and reassembled lipoproteins. Analysis of all apo B-100 lipophilic peptides by circular dichroism and by use of a predictive algorithm reveals no evidence of amphipathic helices. Although the predictive algorithm suggested that the lipophilic peptides of apo B-100 contain the sequence determinants for -sheet, no spectroscopic evidence for this structure was found. We conclude that the lipophilic regions of apo B-100 liberated by trypsinolysis are highly hydrophobic, although their secondary structures do not fit any simple model.     

LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery

Ceramides (CERs) in the upper layer of the skin, the stratum corneum (SC), play a key role in the skin barrier function. In human SC, the literature currently reports 11 CER subclasses that have been identified. In this paper, a novel quick and robust LC/MS method is presented that allows the separation and analysis of all known human SC CER subclasses using only limited sample preparation. Besides all 11 known and identified subclasses, a 3D multi-mass chromatogram shows the presence of other lipid subclasses. Using LC/MS/MS with an ion trap (IT) system, a Fourier transform-ion cyclotron resonance system, and a triple quadrupole system, we were able to identify one of these lipid subclasses as a new CER subclass: the ester-linked ω-hydroxy fatty acid with a dihydrosphingosine base (CER [EOdS]). Besides the identification of a new CER subclass, this paper also describes the applicability and robustness of the developed LC/MS method by analyzing three (biological) SC samples: SC from human dermatomed skin, human SC obtained by tape stripping, and SC from full-thickness skin explants. All three biological samples showed all known CER subclasses and slight differences were observed in CER profile.     

Differentiation of isomeric malonylated flavonoid glyconjugates in plant extracts with UPLC-ESI/MS/MS

Introduction: Glycosylation at different hydroxyl groups of flavonoids and acylation of sugar moieties are ubiquitous modifications observed in plants. These modifications give rise to simultaneous presence of numerous isomeric and isobaric compounds in tissues and extracts thereof. Objective: To develop UPLC‐MS method capable for resolution of isomeric malonylated glycoconjugates of flavonoids and recognition of structural differences. Methodology: Flavonoid glycoconjugates were extracted from leaves of blue lupin (Lupinus angustifolius L.) plants with 80% methanol. Extracts were analysed using ultraperformance liquid chromatography (UPLC) combined with tandem (quadrupole–time of flight, QToF) mass spectrometry. Results: Differentiation of malonylated glycosides of isoflavones and flavones is demonstrated in this paper. The use of UPLC‐MS/MS enabled 38 flavonoid conjugates to be distinguished, including the discrimination of five different isomers of a single 3′‐O‐methylluteolin glycoside. Additionally, pseudo MS³ experiments (CID spectra registered at high cone voltages) enabled confirmation of the aglycone structures by comparison of their spectra with those obtained from aglycone standards. Conclusions: Application of UPLC‐MS/MS allows separation and identification numerous positional isomers of malonylated glycosides of flavonoids and isoflavonoids in plant material. Provided there is strict control of the MS ionisation parameters, this method may be useful for preparation of a flavonoids spectra database, enabling the inter‐laboratory comparison of analytical results. Copyright © 2008 John Wiley & Sons, Ltd.     

基于GC-MS和UPLC-QTOF/MS技术的灵芝孢子粉化学成分分析

采用极性不同的6种溶剂（石油醚、乙酸乙酯、丙酮、乙醇、甲醇和水）、按索氏提取法逐级萃取破壁灵芝孢子粉,并同时运用气相色谱-质谱联用（GC/MS）和超高效液相串联四极杆飞行时间质谱（UPLC-QTOF/MS）技术对各萃取物进行化学成分分析与鉴定。结果表明：GC/MS共鉴定出101种化合物,其中酸类10种、酯类40种、醇类7种、酮类6种、酚类2种、烃类18种、甾类9种和杂原子化合物9种;UPLC-Q-TOF/MS共推断出40种化合物,其中倍半萜类1种、二萜类1种、三萜类9种、生物碱类4种、酰胺类7种、有机酸类9种以及其他化合物9种。两种测定方法间共有化合物仅1种,仅存在于5种有机溶剂（石油醚、乙酸乙酯、丙酮、乙醇和甲醇）萃取物之一的化合物共105种,2种或2种以上萃取物共有的化合物共31种,实验方法较好地实现了样品中化合物组分的充分分离,扩大了可检测化合物的范围。研究结果为灵芝孢子粉中化学成分的系统分析与鉴定、及灵芝孢子粉的化合物谱图库的完善提供了基础资料,为相关药理、药效分析及灵芝的药用模式真菌研究提供参考。     

Identification of the major constituents of Hypericum perforatum by LC/SPE/NMR and/or LC/MS

The newly established hyphenated instrumentation of LC/DAD/SPE/NMR and LC/UV/(ESI)MS techniques have been applied for separation and structure verification of the major known constituents present in Greek Hypericum perforatum extracts. The chromatographic separation was performed on a C18 column. Acetonitrile-water was used as a mobile phase. For the on-line NMR detection, the analytes eluted from column were trapped one by one onto separate SPE cartridges, and hereafter transported into the NMR flow-cell. LC/DAD/SPE/NMR and LC/UV/MS allowed the characterization of constituents of Greek H. perforatum, mainly naphtodianthrones (hypericin, pseudohypericin, protohypericin, protopseudohypericin), phloroglucinols (hyperforin, adhyperforin), flavonoids (quercetin, quercitrin, isoquercitrin, hyperoside, astilbin, miquelianin, I3,II8-biapigenin) and phenolic acids (chlorogenic acid, 3-O-coumaroylquinic acid). Two phloroglucinols (hyperfirin and adhyperfirin) were detected for the first time, which have been previously reported to be precursors in the biosynthesis of hyperforin and adhyperforin.     

Quantitative analysis of adenosine using liquid chromatography/atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS)

Adenosine-secreting cellular brain implants constitute a promising therapeutic approach for the treatment of epilepsy. To engineer neural stem cells for therapeutic adenosine delivery, a reliable and fast analytical method is necessary to quantify cell-based adenosine release. Here we describe the development, optimization and validation of adenosine measurement using liquid chromatography–atmospheric pressure chemical ionization-tandem mass spectrometry (LC–APCI-MS/MS). LC–MS/MS in positive ion mode used selected reaction monitoring at m/z of 268.2/136.1 and 302.2/170.0 for adenosine and the internal standard, respectively. The bias was within 15% of the nominal value and evaluation of precision showed a relative standard deviation lower than 15% for all measured concentrations. The lower limit of quantification of adenosine was 15.6 ng/ml. Freeze and thaw stability and processed sample stability also fulfilled the acceptance criteria. Evaluation of the matrix effect showed that the method is not affected by relative matrix effects. The major advantages of this method are the absence of an extraction phase and the combination of the high selectivity and sensitivity characteristic for the LC–MS/MS technique, with a short run time of 4.5 min. These results demonstrate that this method is a useful tool to measure adenosine concentrations in culture medium released from stem cells in vitro.     

Site-specific N-glycosylation analysis of human plasma ceruloplasmin using liquid chromatography with electrospray ionization tandem mass spectrometry

Ceruloplasmin has ferroxidase activity and plays an essential role in iron metabolism. In this study, a site-specific glycosylation analysis of human ceruloplasmin (CP) was carried out using reversed-phase high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). A tryptic digest of carboxymethylated CP was subjected to LC-ESI-MS/MS. Product ion spectra acquired data-dependently were used for both distinction of the glycopeptides from the peptides using the carbohydrate B-ions, such as m/z 204 (HexNAc) and m/z 366 (HexHexNAc), and identification of the peptide moiety of the glycopeptide based on the presence of the b- and y-series ions derived from the peptide. Oligosaccharide composition was deduced from the molecular weight calculated from the observed mass of the glycopeptide and theoretical mass of the peptide. Of the seven potential N-glycosylation sites, four (Asn119, Asn339, Asn378, and Asn743) were occupied by a sialylated biantennary or triantennary oligosaccharide with fucose residues (0, 1, or 2). A small amount of sialylated tetraantennary oligosaccharide was detected. Exoglycosidase digestion suggested that fucose residues were linked to reducing end GlcNAc in biantennary oligosaccharides and to reducing end and/or alpha1-3 to outer arms GlcNAc in triantennary oligosaccharides and that roughly one of the antennas in triantennary oligosaccharides was alpha2-3 sialylated and occasionally alpha1-3 fucosylated at GlcNAc.     

Thermospray liquid chromatography/mass spectrometry for the analysis of l-carnitine and its short-chain acyl derivatives

A method utilizing thermospray high-performance liquid chromatography/mass spectrometry for the separation and direct analysis of carnitine, acetylcarnitine, and propionylcarnitine is described. On-column analysis of mixtures of the acylcarnitines with their corresponding stable, isotope-labeled analogs at nanomolar concentrations has indicated that isotope dilution assays can be applied towards the analysis of carnitine and short-chain acylcarnitines present in biological samples.     

Effects of ezetimibe and simvastatin on apolipoprotein B metabolism in males with mixed hyperlipidemia

Sixteen hyperlipidemic men were enrolled in a randomized, placebo-controlled, double-blind, cross-over study to evaluate the effect of ezetimibe 10 mg and simvastatin 40 mg, coadministered and alone, on the in vivo kinetics of apolipoprotein (apo) B-48 and B-100 in humans. Subjects underwent a primed-constant infusion of a stable isotope in the fed state. The coadministration of simvastatin and ezetimibe significantly reduced plasma concentrations of cholesterol (−43.0%), LDL-C (−53.6%), and triglycerides (−44.0%). Triglyceride-rich lipoproteins (TRL) apoB-48 pool size (PS) was significantly decreased (−48.9%) following combination therapy mainly through a significant reduction in TRL apoB-48 production rate (PR) (−38.0%). The fractional catabolic rate (FCR) of VLDL and LDL apoB-100 were significantly increased with all treatment modalities compared with placebo, leading to a significant reduction in the PS of these fractions. We also observed a positive correlation between changes in TRL apoB-48 PS and changes in TRL apoB-48 PR (r = 0.85; P < 0.0001) with combination therapy. Our results indicate that treatment with simvastatin plus ezetimibe is effective in reducing plasma TRL apoB-48 levels and that this effect is most likely mediated by a reduction in the intestinal secretion of TRL apoB-48. Our study also indicated that the reduction in LDL-C concentration following combination therapy is mainly driven by an increase in FCR of apoB-100 containing lipoproteins.     

Detection and quantification of (R) and (S)-dechloroethylifosfamide metabolites in plasma from children by enantioselective LC/MS/MS

Ifosfamide (IF), a potent chemotherapeutic agent for solid tumors, is known to cause high rates of nephrotoxicity in children with cancer, which is most likely due to the renal production of the metabolite chloroacetaldehyde. Using plasma samples obtained from pediatric oncology patients, we developed a simple nonderivatizing enantioselective liquid chromatography mass spectrometry method to detect the (R) and (S)-2- and 3-dechloroethylifosfamide metabolites. The (R) and (S)-enantiomers of the 2- and 3-DCEIF (N-3-dechlroethylifosfamide) were detectable in all 22 patients' samples with levels ranging from 9.9 to 238.7 ng/ml for (R)-2-DCEIF, 15.8 to 663.0 ng/ml for (S)-2-DCEIF, 20.8 to 852.8 ng/l for (R)-3-DCEIF and 28.0 to 862.0 ng/ml for (S)-3-DCEIF. In addition, the lower limit of quantification for this method is 1 ng/ml. Future studies should concentrate on (R) or (S) production of the 2-DCEIF and 3-DCEIF and subsequently chloroacetaldehyde formation with the aim of considering the administration of only the (R)-IF as its metabolism results in a lower production of chloroacetaldehyde.     

Identification and characterization of glycosylation sites in human serum clusterin.

Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation. Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 +/- 250 and 63,507 +/- 200). Mass spectrometry also revealed molecular heterogeneity associated with both the alpha and beta subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17-27% carbohydrate by weight, the alpha subunit contains 0-30% carbohydrate and the beta subunit contains 27-30% carbohydrate. Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide. A total of six N-linked glycosylation sites were identified, three in the alpha subunit (alpha 64N, alpha 81N, alpha 123N) and three in the beta subunit (beta 64N, beta 127N, and beta 147N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialobiantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure. Site beta 64N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site beta 147N exhibited the greatest diversity, with five or six detected types of oligosaccharides. Overall, the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennary with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated alpha and beta subunits, including all Ser- and Thr-containing peptides. No evidence was found for the presence of O-linked or sulfated oligosaccharides. The results provide a molecular basis for developing a better understanding of clusterin structure-function relationships and the role clusterin glycosylation plays in physiological function.