MALDI-TOF Fingerprinting of Seminal Plasma Lipids in the Study of Human Male Infertility

This study proposed lipid fingerprinting of human seminal plasma by mass spectrometry as an analytical method to differentiate biological conditions. For this purpose, we chose infertile men as a model to study specific conditions, namely: high and low seminal plasma lipid peroxidation levels (sub‐study 1.1), high and low sperm nuclear DNA fragmentation (sub‐study 1.2), and intervention status: before and after subinguinal microsurgical varicocelectomy (study 2). Study 1 included 133 patients, of which 113 were utilized for sub‐study 1.1 and 89 for sub‐study 1.2. Study 2 included 17 adult men submitted to subinguinal varicocelectomy, before and 90 days after varicocelectomy. Lipids were extracted from seminal plasma and submitted to Matrix‐Assisted Laser Desorption Ionization Quadrupole‐Time‐of‐Flight Mass Spectrometry in the positive ionization mode. Spectra were processed using Waters^® MassLynx, and MetaboAnalyst online software was used for statistical analyses. For sub‐studies 1.1 and 1.2, and study 2, univariate analysis revealed 8, 87 and 34 significant ions, respectively. Multivariate analysis was performed through PCA and PLS‐DA. PCA generated 56, 32 and 34 components respectively for each study and these were submitted to logistic regression. A ROC curve was plotted and the area under the curve was equal to 97.4, 92.5 and 96.5 %. PLS‐DA generated a list of 19, 24 and 23 VIP ions for sub‐studies 1.1 and 1.2, and study 2, respectively. Therefore, this study established the lipid profile and comparison of patterns altered in response to specific biological conditions.     

Suppression of sialylated by sulfated oligosaccharides in negative MALDI-FTMS

Sialylated oligosaccharides are suppressed by sulfated oligosaccharides during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The sialylated oligosaccharide may be suppressed by as much as a factor of ten to twenty. This suppression factor is constant over a large concentration range. As both sialylated and sulfated (or anionic) oligosaccharides are found together in many biological systems, the suppression has important consequences in the analysis of these compounds. Methods for overcoming the effect of suppression without laborious separation are provided.     

Detection of Serum Protein Biomarkers for the Diagnosis and Staging of Hepatoblastoma

The present study aimed to identify serum biomarkers for the detection of hepatoblastoma (HB). Serum samples were collected from 71 HB patients (stage I, n = 19; stage II, n = 19, stage III, n = 19; and stage IV, n = 14) and 23 age- and sex-matched healthy children. Differential expression of serum protein markers were screened using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), and the target proteins were isolated and purified using HPLC and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), SEQUEST, and bioinformatics analysis. Differential protein expression was confirmed by enzyme-linked immunosorbent analysis (ELISA). SELDI-TOF-MS screening identified a differentially expressed protein with an m/z of 9348 Da, which was subsequently identified as Apo A–I; its expression was significantly lower in the HB group as compared to the normal control group (1546.67 ± 757.81 vs. 3359.21 ± 999.36, respectively; p < 0.01). Although the expression level decreased with increasing disease stage, pair-wise comparison revealed significant differences in Apo A–I expression between the normal group and the HB subgroups (p < 0.01). ELISA verified the reduced expression of Apo A–I in the HB group. Taken together, these results suggest that Apo A–I may represent a serum protein biomarker of HB. Further studies will assess the value of using Apo A–I expression for HB diagnosis and staging.     

Plasma lipid profiling by liquid chromatography with chloride-attachment mass spectrometry

A sensitive high-performance liquid chromatographic assay was developed using chloride attachment negative chemical ionization mass spectrometry for detection of glyceryl esters and ceramides, and positive chemical ionization mass spectrometry for detection of free cholesterol and cholesteryl esters in minimal quantities of plasma. The novel technique was validated by high temperature gas-liquid chromatography with flame ionization detection. Sample preparation was achieved by phospholipase C digestion of whole plasma, total lipid extraction and derivatization of any free carboxyl and hydroxyl groups by trimethyl- ortert-butyldimethyl-chlorosilane. The lipids were separated by reverse phase HPLC with 20–90% propionitrile in acetonitrile containing 1% dichloromethane, which served as the reagent and the source of chloride. Negative chemical ionization with chloride attachment is estimated to provide about 100 times higher response for the triacylglycerols and the trimethylsilyl ortert-butyldimethylsilyl ethers of diacylglycerols, and about 500 times higher response for the trimethylsilyl ortert-butyldimethylsilyl ethers of ceramides than positive chemical ionization mass spectrometry. Determination of the full negative chemical ionization mass spectra showed that each glycerolipid and ceramide species yielded a single ionic species corresponding to the chloride-attachment product of the parent ion. The cholesteryl esters and ethers failed to attach chloride and remained undetected by negative chemical ionization. However, the cholesteryl esters and ethers gave a high response for the steroid nucleus in positive chemical ionization mass spectrometry. Chloride attachment negative chemical ionization mass spectrometry is suitable for the unequivocal identification of plasma glycerolipids and ceramides in high-performance liquid chromatography and for the quantitation of molecular species in any unresolved peaks following appropriate calibration of the instrument response.     

Elucidating the Function of Complex-Type Oligosaccharides by Use of Chemical Synthesis of Homogeneous Glycoproteins

Protein glycosylation is a major post-translational modification. To elucidate the effect of this modification on protein function, homogeneous glycoproteins are required. Because glycoproteins isolated from biological sources contain glycoforms, a mixture of a single protein chain with several different oligosaccharides appended, homogeneous glycoproteins obtained through chemical synthesis offer a better solution. In this review, several methods used by our group for the chemical synthesis of homogeneous glycoproteins are addressed. First, preparation of sufficient amounts of oligosaccharides with the desired structures was achieved using a combination of chemical protection and enzymatic digestion. Then glycopeptide-^αthioesters were prepared by incorporation of oligosaccharides onto the side chains of cysteine residues in peptide-^αthioesters. Finally, biologically active homogeneous glycoproteins were prepared through native chemical ligation of glycopeptide-^αthioesters and subsequent oxidative folding.     

Analysis of molecular species of peroxide adducts of triacylglycerols following treatment of corn oil with tert-butyl hydroperoxide

We recently demonstrated that exposure of synthetic mono-and diunsaturated triacylglycerols to tert-butylhy-droperoxide (TBHP) leads to formation of stable adducts of the oxidizing agent and the unsaturated esters (Sjövall, O., Kuksis, A., and Kallio, H., Reversed Phase High-Performance Liquid Chromatographic Separation of tert-Butyl Hydroperoxide Oxidation Products of Unsaturated Triacylglycerols. J. Chromatogr. A 905, 119–132, 2001). In the present study we isolated and identified the TBHP adducts of corn oil triacylglycerols. The much wider range of molecular species available in the corn oil permitted us to demonstrate that the yield of the adducts varies with the degree of unsaturation of the triacylglycerol. The highest yields were obtained for the linoleate (20% of linoleoyl-containing residual triacylglycerols) and the lowest ones for the oleate (5% of oleoyl-containing residual triacylglycerols) tria-cylglycerols, whereas the saturated triacylglycerols did not give TBHP adducts in readily detectable amounts. Normal-phase thin-layer chromatography along with reversed-phase high-performance liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization was used to isolate and separate the major molecular species of polyunsaturated triacylglycerols and corresponding TBHP adducts. As an extreme example, the dilinoleoylmonooleoylglycerol was identified as the mono-, di-, tri-, tetra-, and penta-TBHP adduct. LC/MS with electrospray ionization at elevated capillary exit voltage (pseudo tandem mass spectrometry) was used to confirm structures of the [M?RCOOH]⁺ ions and the absence of TBHP adducts of [M?RCOOH]⁺. It is concluded that stable adduct formation is an unavoidable complication of preparation of oxotriacylglycerols by oxidation with concentrated TBHP solutions and care must be taken to resolve the adducts from the desired oxidation product.     

PG-2, a Potent AMP against Pathogenic Microbial Strains,from Potato (Solanum tuberosum L cv. Gogu Valley) Tubers Not Cytotoxic against Human Cells

In an earlier study, we isolated potamin-1 (PT-1), a 5.6-kDa trypsin-chymotrypsin protease inhibitor, from the tubers of a potato strain (Solanum tuberosum L cv. Gogu Valley). We established that PT-1 strongly inhibits pathogenic microbial strains, but not human bacterial strains, and that its sequence shows 62% homology with a serine protease inhibitor. In the present study, we isolated an antifungal and antibacterial peptide with no cytotoxicity from tubers of the same potato strain. The peptide (peptide-G2, PG-2) was isolated using salt-extraction, ultrafiltration and reverse-phase high performance liquid chromatography (RP-HPLC). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) showed the protein to have a molecular mass of 3228.5 Da, while automated Edman degradation showed the N-terminal sequence of PG-2 to be LVKDNPLDISPKQVQALCTDLVIRCMCCC-. PG-2 exhibited antimicrobial activity against Candida albicans, a human pathogenic yeast strain, and Clavibacter michiganensis subsp. michiganensis, a plant late blight strain. PG-2 also showed antibacterial activity against Staphylococcus aureus, but did not lyse human red blood cells and was thermostable. Overall, these results suggest PG-2 may be a good candidate to serve as a natural antimicrobial agent, agricultural pesticide and/or food additive.     

Identification of Dermatophyte Species after Implementation of the In-House MALDI-TOF MS Database

Despite that matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool in the clinical microbiology setting, few studies have till now focused on MALDI-TOF MS-based identification of dermatophytes. In this study, we analyze dermatophytes strains isolated from clinical samples by MALDI-TOF MS to supplement the reference database available in our laboratory. Twenty four dermatophytes (13 reference strains and 11 field isolated strains), identified by both conventional and molecular standard procedures, were analyzed by MALDI-TOF MS, and the spectra obtained were used to supplement the available database, limited to a few species. To verify the robustness of the implemented database, 64 clinical isolates other than those used for the implementation were identified by MALDI-TOF MS. The implementation allowed the identification of the species not included in the original database, reinforced the identification of the species already present and correctly identified those within the Trichophyton mentagrophytes complex previously classified as Trichophyton. tonsurans by MALDI-TOF MS. The dendrogram obtained by analyzing the proteic profiles of the different species of dermatophytes reflected their taxonomy, showing moreover, in some cases, a different clusterization between the spectra already present in the database and those newly added. In this study, MALDI-TOF MS proved to be a useful tool suitable for the identification of dermatophytes for diagnostic purpose.     

Initiators of Classical and Lectin Complement Pathways Are Differently Engaged after Traumatic Brain Injury—Time-Dependent Changes in the Cortex,Striatum, Thalamus and Hippocampus in a Mouse Model

The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein—GFAP), microglia/macrophages (allograft inflammatory factor 1—IBA-1), and microglia (transmembrane protein 119—TMEM119); moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.     

Dissecting carbohydrate-Cyanovirin-N binding by structure-guided mutagenesis: functional implications for viral entry inhibition

The HIV-inactivating protein Cyanovirin-N (CV-N) is a cyanobacterial lectin that exhibits potent antiviral activity at nanomolar concentrations by interacting with high-mannose carbohydrates on viral glycoproteins. To date there is no molecular explanation for this potent virucidal activity, given the experimentally measured micromolar affinities for small sugars and the problems encountered with aggregation and precipitation of high-mannose/CV-N complexes. Here, we present results for two CV-N variants, CV-N(mutDA) and CV-N(mutDB), compare their binding properties with monomeric [P51G]CV-N (a stabilized version of wtCV-N) and test their in vitro activities. The mutations in CV-N(mutDA) and CV-N(mutDB) comprise changes in amino acids that alter the trimannose specificity of domain A(M) and abolish the sugar binding site on domain B(M), respectively. We demonstrate that carbohydrate binding via domain B(M) is essential for antiviral activity, whereas alterations in sugar binding specificity on domain A(M) have little effect on envelope glycoprotein recognition and antiviral activity. Changes in A(M), however, affect the cross-linking activity of CV-N. Our findings augment and clarify the existing models of CV-N binding to N-linked glycans on viral glycoproteins, and demonstrate that the nanomolar antiviral potency of CV-N is related to the constricted and spatially crowded arrangement of the mannoses in the glycan clusters on viral glycoproteins and not due to CV-N induced virus particle agglutination, making CV-N a true viral entry inhibitor.     

Engineered Glycosidases for the Synthesis of Analogs of Human Milk Oligosaccharides

Enzymatic synthesis is an elegant biocompatible approach to complex compounds such as human milk oligosaccharides (HMOs). These compounds are vital for healthy neonatal development with a positive impact on the immune system. Although HMOs may be prepared by glycosyltransferases, this pathway is often complicated by the high price of sugar nucleotides, stringent substrate specificity, and low enzyme stability. Engineered glycosidases (EC 3.2.1) represent a good synthetic alternative, especially if variations in the substrate structure are desired. Site-directed mutagenesis can improve the synthetic process with higher yields and/or increased reaction selectivity. So far, the synthesis of human milk oligosaccharides by glycosidases has mostly been limited to analytical reactions with mass spectrometry detection. The present work reveals the potential of a library of engineered glycosidases in the preparative synthesis of three tetrasaccharides derived from lacto-N-tetraose (Galβ4GlcNAcβ3Galβ4Glc), employing sequential cascade reactions catalyzed by β3-N-acetylhexosaminidase BbhI from Bifidobacterium bifidum, β4-galactosidase BgaD-B from Bacillus circulans, β4-N-acetylgalactosaminidase from Talaromyces flavus, and β3-galactosynthase BgaC from B. circulans. The reaction products were isolated and structurally characterized. This work expands the insight into the multi-step catalysis by glycosidases and shows the path to modified derivatives of complex carbohydrates that cannot be prepared by standard glycosyltransferase methods.     

Desorption mass spectrometry of olestra

Field desorption (FD), fast atom bombardment (FAB) and plasma desorption (PD) mass spectrometry have been used for the characterization of olestra, a mixture of octa-, hepta- and hexaesters of sucrose formed by reaction of sucrose with long-chain fatty acids (C₁₂–C₁₈). Most previous applications of desorption ionization mass spectrometry have involved polar compounds; however, the relatively low-polarity olestra is also amenable to these techniques with proper sample preparation. Field desorption provides molecular weight information, but the transience of the signals limits the usefulness for observing fragmentation and measuring ester distributions. In addition, FD may not be sensitive enough to allow characterization of fractions isolated from analytical high-performance liquid chromatography (HPLC) columns. Fast atom bombardment produces longer-lasting signals, which permit characterization of components over a wide mass range. However, signal-to-noise fluctuates substantially, depending on analyte solubility in the matrix, making the characterization of partial esters collected from HPLC uncertain and difficult. Plasma desorption mass spectrometry is the easiest and most sensitive technique for olestra characterization but provides the lowest mass resolution. Because it requires no more than a few μg of material, it is effective for the characterization of HPLC fractions. Furthermore, it is the only method, of the three investigated, that allows detection of intact dimeric species having molecular masses in the 3,000 to 5,000 dalton range.     

Metabolite Profiling of Manilkara zapota L. Leaves by High-Resolution Mass Spectrometry Coupled with ESI and APCI and In Vitro Antioxidant Activity, α-Glucosidase,and Elastase Inhibition Assays

High-resolution mass spectrometry equipped with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources was used to enhance the characterization of phytochemicals of ethanol extracts of Manilkara zapota L. leaves (ZLE). Sugar compounds, dicarboxylic acids, compounds of phenolic acids and flavonoids groups, and other phytochemicals were detected from the leaves. Antioxidant activity and inhibition potentiality of ZLE against α-glucosidase enzyme, and elastase enzyme activities were evaluated in in vitro analysis. ZLE significantly inhibited activities of α-glucosidase enzyme at a lower concentration (IC₅₀ 2.51 ± 0.15 µg/mL). Glucose uptake in C2C12 cells was significantly enhanced by 42.13 ± 0.15% following the treatment with ZLE at 30 µg/mL. It also exhibited potential antioxidant activities and elastase enzyme inhibition activity (IC₅₀ 27.51 ± 1.70 µg/mL). Atmospheric pressure chemical ionization mass spectrometry (APCI–MS) detected more m/z peaks than electrospray ionization mass spectrometry (ESI–MS), and both ionization techniques illustrated the biological activities of the detected compounds more thoroughly compared to single-mode analysis. Our findings suggest that APCI along with ESI is a potential ionization technique for metabolite profiling, and ZLE has the potential in managing diabetes by inhibiting α-glucosidase activity and enhancing glucose uptake.     

碎屑锆石U-Pb年龄学的方法和发展趋势

通过文献调研,将碎屑锆石年代学的发展做了简单介绍,同时整理了同位素稀释-热电离质谱、二次离子质谱以及激光剥蚀等离子质谱等仪器的优劣特点,以及碎屑锆石分析实验中遇到的问题:1)需要进行的分析数量;2)应如何选择晶粒进行分析。对这两问题的解决方案给出了一定的意见。最后对碎屑锆石年龄学发展趋势进行了综述。     

Measurement of Ether Phospholipids in Human Plasma with HPLC–ELSD and LC/ESI–MS After Hydrolysis of Plasma with Phospholipase A1

Ethanolamine ether phospholipid (eEtnGpl) and choline ether phospholipid (eChoGpl) are present in human plasma or serum, but the relative concentration of the ether phospholipids in plasma is very low as compared to those in other tissues. Nowadays, measurement of ether phospholipids in plasma depends on tandem mass spectrometry (LC/MS/MS), but a system for LC/MS/MS is generally too expensive for usual clinical laboratories. Treatment of plasma with phospholipase A₁ (PLA1) causes complete hydrolysis of diacylphospholipids, but ether phospholipids remain intact. After the treatment of plasma with PLA1, both eEtnGpl and eChoGpl are detected as independent peaks by high‐performance liquid chromatography with evaporative light scattering detection (HPLC–ELSD). The same sample used for HPLC–ELSD can be applied to detect eEtnGpl and eChoGpl with electrospray ionization mass spectrometry. Presence of alkylacylphospholipids in both eChoGpl and eEtnGpl in human plasma was indicated by sequential hydrolysis of plasma with PLA1 and hydrochloric acid.     

Volatile compounds from the triacylglycerol ofcis,cis 9,15-linoleic acid

cis,cis 9,15-Linoleic acid, one of the isomers produced by partial hydrogenation of linolenic acid, may be a precursor of the hydrogenation odor observed during heating of partially hydrogenated soybean oil in air. Odors of hydrogenation, for example the melon odor, are associated with foods cooked in partially hydrogenated soybean oil that has undergone oxidative deterioration during heating. To better understand this oxidation mechanism, the volatiles from decomposition of the oxidation products of a synthesized model compound, tricis,cis 9,15-linoleoylglycerol, heated in air at 192°C were collected, separated, quantitated by gas chromatography with flame ionization detection and identified by mass spectrometry. Major volatiles observed indicated that each of the isolated double bonds at carbons 9,10 and 15,16 isomerized to form an allylic radical. The allylic radical derived from the isolated double bond at carbons 9,10 reacted with oxygen to form the 8,9,10 and 11 monohydroperoxides. The allylic radical derived from the isolated double bond at carbons 15,16 produced the 14,15,16 and 17 monohydroperoxides. The 8,9,10 and 11 monohydroperoxides are the expected precursors of the following volatiles identified by mass spectrometry: 2,8-undecadienal (5.9%), 2,7-decadienal (7.8%), 6-nonenal (8.4%) and 5-octenal (1.5% of total volatiles), respectively. These four aldehydes are analogous to major volatiles obtained from thermal decomposition of autoxidized trioleoylglycerol. The 14,15,16 and 17 monohydroperoxides are the expected precursors of 2-pentenal (9.0%), 2-butenal (8.0%), propanal (12.1%) and ethanal (2.0% of total volatiles), respectively. Among the above volatiles, 6-nonenal (cucumber-melon) is reported to have the most intense odor associated with deteriorated partially hydrogenated soybean oil.     

Mycoplasma genitalium Provokes Seminal Inflammation among Infertile Males

The impact of sexually transmitted infections (STI) on male fertility is controversial. Aims: To investigate the prevalence of urethritis-associated STIs (chlamydia, gonorrhoeae, Mycoplasma genitalium, trichomoniasis) among infertile males; to analyze the effect of STIs on semen parameters and blood PSA. Case-control study. Study group (n = 2000): males with fertility problems or desire for fertility check. Control group (n = 248): male partners of pregnant women. Analyses: polymerase chain reaction for STI, seminal interleukin 6 (IL-6), semen and fractionated urine, blood analyses (PSA, reproductive hormones). The prevalence of M. genitalium and chlamydia in the study group was 1.1% and 1.2%, respectively. The prevalence of chlamydia in the control group was 1.6%, while there were no M. genitalium cases. No cases with gonorrhoeae or trichomoniasis or combined infections were observed in neither group. There was a higher seminal concentration of neutrophils and IL-6 among M. genitalium positives compared with STI negatives. There was a trend toward a lower total count of spermatozoa and progressive motility among STI positives. No impact of STIs on PSA was found. The prevalence of STIs among infertile males is low. M. genitalium is associated with seminal inflammation. The impact of STIs on semen parameters deserves further investigations.     

Identification of peptide oxidation by tandem mass spectrometry

Oxidative reactions play important roles in a variety of biochemical events ranging from normal metabolism to aging and disease processes. Proteins represent major targets for modification in these reactions, and identification of sites and structures of modifications may lead to mechanistic understanding and approaches for prevention. In this Account, the utility of mass spectrometry and its advantages are described for the identification of oxidative modifications to protein targets. A variety of examples are provided to illustrate how modifications are accurately identified and quantitated using modern methods of ionization coupled with HPLC and tandem mass spectrometry.     

Peptide Sequence Mapping around Bisecting GlcNAc-Bearing N-Glycans in Mouse Brain

N-glycosylation is essential for many biological processes in mammals. A variety of N-glycan structures exist, of which, the formation of bisecting N-acetylglucosamine (GlcNAc) is catalyzed by N-acetylglucosaminyltransferase-III (GnT-III, encoded by the Mgat3 gene). We previously identified various bisecting GlcNAc-modified proteins involved in Alzheimer’s disease and cancer. However, the mechanisms by which GnT-III acts on the target proteins are unknown. Here, we performed comparative glycoproteomic analyses using brain membranes of wild type (WT) and Mgat3-deficient mice. Target glycoproteins of GnT-III were enriched with E4-phytohemagglutinin (PHA) lectin, which recognizes bisecting GlcNAc, and analyzed by liquid chromatograph-mass spectrometry. We identified 32 N-glycosylation sites (Asn-Xaa-Ser/Thr, Xaa ≠ Pro) that were modified with bisecting GlcNAc. Sequence alignment of identified N-glycosylation sites that displayed bisecting GlcNAc suggested that GnT-III does not recognize a specific primary amino acid sequence. The molecular modeling of GluA1 as one of the good cell surface substrates for GnT-III in the brain, indicated that GnT-III acts on N-glycosylation sites located in a highly flexible and mobile loop of GluA1. These results suggest that the action of GnT-III is partially affected by the tertiary structure of target proteins, which can accommodate bisecting GlcNAc that generates a bulky flipped-back conformation of the modified glycans.