Evaluation of PDL1 positive cancer cell-specific binding activity of recombinant anti-PDL1 scFv.

Programmed cell death-ligand 1 (PDL1) is a transmembrane protein that is characterized as an immune regulatory molecule. We recently developed a recombinant single-chain fragment of variable domain (scFv) against PDL1, which showed high binding efficiency to purified recombinant PDL1 protein. However, at that time, proof-of-concept data for the effect of scFv using PDL1-expressing cells was lacking. In this study, we conducted two kinds of cell-based immunoassays, western blotting and enzyme-linked immunosorbent assay, using anti-PDL1 scFv. The results indicate that scFv can selectively and sensitively detect PDL1 from PDL1 positive human cancer cell lines. Our findings suggest that scFv could be used as a potential PDL1 inhibitor agent and probe for cell-based immunoassays to detect PDL1.

Biomimetic Silica Particles with Self-Loading BMP-2 Knuckle Epitope Peptide and Its Delivery for Bone Regeneration.

Biomimetic silica deposition is an in-situ immobilization method for bioactive molecules under biocompatible conditions. The osteoinductive P4 peptide derived from the knuckle epitope of bone morphogenetic protein (BMP), which binds to BMP receptor-II (BMPRII), has been newly found to contain silica formation ability. We found that the two lysine residues at the N-terminus of P4 played a vital role in silica deposition. The P4 peptide co-precipitated with silica during P4-mediated silicification, yielding P4/silica hybrid particles (P4@Si) with a high loading efficiency of 87%. P4 was released from P4@Si at a constant rate for over 250 h, representing a zero-order kinetic model. In flow cytometric analysis, P4@Si showed a 1.5-fold increase in the delivery capacity to MC3T3 E1 cells than the free form of P4. Furthermore, P4 was found anchored to hydroxyapatite (HA) through a hexa-glutamate tag, followed by P4-mediated silicification, yielding P4@Si coated HA. This suggested a superior osteoinductive potential compared to silica or P4 alone coated HA in the in vitro study. In conclusion, the co-delivery of the osteoinductive P4 peptide and silica by P4-mediated silica deposition is an efficient method for capturing and delivering its molecules and inducing synergistic osteogenesis.

Wide-field optical coherence tomography deviation map for early glaucoma detection.

BACKGROUND/AIMS: This study aimed to establish a wide-field optical coherence tomography (OCT) deviation map obtained from swept-source OCT (SS-OCT) scans. Moreover, it also aimed to compare the diagnostic ability of this wide-field deviation map with that of the peripapillary and macular deviation maps currently being used for the detection of early glaucoma (EG). METHODS: Four hundred eyes, including 200 healthy eyes and 200 eyes with EG were enrolled in this retrospective observational study. Patients underwent a comprehensive ocular examination, including wide-field SS-OCT (DRI-OCT Triton; Topcon, Tokyo, Japan). The individual wide-field scan was converted into a uniform template using the fovea and optic disc centres as fixed landmarks. Subsequently, the wide-field deviation map was obtained via the comparison between individual wide-field data and a normative wide-field database that had been created by combining images of healthy eyes into a uniform template in a previous study. The ability of the new wide-field deviation map to distinguish between EG and healthy eyes was assessed by comparing it with conventional deviation maps based on the area under the receiver operating characteristic curve (AUC). RESULTS: The wide-field deviation map obtained using the normative wide-field database showed the highest diagnostic ability for the diagnosis of EG (AUC=0.980 and 961 for colour-coded pixels presenting <5% and <1%, respectively) among various deviation maps. Its AUC was significantly superior to that of most conventional deviation maps (p<0.05). The wide-field deviation map demonstrated early structural glaucomatous damage well over a wider area. CONCLUSION: The wide-field SS-OCT deviation map exhibited good performance for distinguishing between eyes with EG and healthy eyes. The visualisation of the wider damaged area on the wide-field deviation map could be useful for the diagnosis of EG in clinical settings.

Immediate and Quantitative Changes in Tear Film Parameters and Meibomian Gland Structures after Warm Compression and Meibomian Gland Squeezing in Meibomian Gland Dysfunction Patients and Normal Subjects.

Meibomian gland dysfunction (MGD), a chronic abnormality of meibomian glands, causes various dry eye symptoms. Principal treatments for MGD are warm compression and mechanical squeezing of the eyelids. In this study, the immediate impact of this treatment on tear film lipid layer thickness (TFLLT) and the meibomian gland (MG) structure in MGD and normal groups was investigated to establish its efficacy and potential side effects. Nineteen MGD patients and seven normal subjects were enrolled. TFLLT and blinking parameters were evaluated before and after warm compression. Morphological changes of MG structures after mechanical squeezing were analyzed using Image J and Fiji. Differential analysis of the MGD and the normal groups of TFLLT changes after warm compression showed a significant increase in the normal group. In normal eyes, the average, maximum, and minimum TFLLT were significantly increased, and in the MGD group, only the minimum TFLLT was improved. Blinking parameters showed no significant change in either group. Morphometric analysis showed no damages of the MG after MG squeezing. A significant increase in MG length was observed in normal eyes. Warm compression immediately increased TFLLT more significantly in the normal group than in the MGD patients. Mechanical expression is a safe therapeutic option without remarkable structural MG damages.

Topographic patterns of retinal edema in eyes with branch retinal vein occlusion and their association with macular edema recurrence.

In this study, we evaluated the topographic pattern of retinal edema in eyes with macular edema (ME) secondary to branch retinal vein occlusion (BRVO) using a widefield retinal thickness map of optical coherence tomography and its association with ME recurrence. In 87 eyes with ME secondary to BRVO who were treated with anti-vascular endothelial growth factor (VEGF) injections and followed up for ≥ 1 years, 12 × 9 mm macular volume scans of swept-source optical coherence tomography (DRI-OCT Triton; Topcon Inc, Japan) were performed and retinal thickness maps were automatically generated at baseline and follow-up visits. Topographic patterns of retinal edema on the maps at baseline and 1 month after the first anti-vascular endothelial growth factor (VEGF) treatment were classified as extramacular (outside the ETDRS grid), macular (within the grid), and combined pattern and correlated with ME recurrences. Seventy-five of 87 (86.2%) eyes with BRVO ME showed combined edema at baseline. There were 4 topographic patterns of edema at 1 month following anti-VEGF injection as follows: no residual edema, extramacular only, macular only, and combined edema. In contrast to the baseline pattern, the pattern of retinal edema 1 month following anti-VEGF therapy showed significant association with 6-month recurrence, number of ME recurrences during a 1-year period, and time to first recurrence. (all P < 0.05) An automatically generated widefield retinal thickness map could be used to effectively visualize the topographic patterns of retinal edema in eyes with BRVO. The map can be used as a valuable tool for detection of retinal edema on widefield retinal areas and prediction of ME recurrence in eyes with BRVO.

New Interface for Faster Proteoform Analysis: Immunoprecipitation Coupled with SampleStream-Mass Spectrometry.

Different proteoform products of the same gene can exhibit differing associations with health and disease, and their patterns of modifications may offer more precise markers of phenotypic differences between individuals. However, currently employed protein-biomarker discovery and quantification tools, such as bottom-up proteomics and ELISAs, are mostly proteoform-unaware. Moreover, the current throughput for proteoform-level analyses by liquid chromatography mass spectrometry (LCMS) for quantitative top-down proteomics is incompatible with population-level biomarker surveys requiring robust, faster proteoform analysis. To this end, we developed immunoprecipitation coupled to SampleStream mass spectrometry (IP-SampleStream-MS) as a high-throughput, automated technique for the targeted quantification of proteoforms. We applied IP-SampleStream-MS to serum samples of 25 individuals to assess the proteoform abundances of apolipoproteins A-I (ApoA-I) and C-III (ApoC-III). The results for ApoA-I were compared to those of LCMS for these individuals, with IP-SampleStream-MS showing a >7-fold higher throughput with >50% better analytical variation. Proteoform abundances measured by IP-SampleStream-MS correlated strongly to LCMS-based values (R2 = 0.6-0.9) and produced convergent proteoform-to-phenotype associations, namely, the abundance of canonical ApoA-I was associated with lower HDL-C (R = 0.5) and glycated ApoA-I with higher fasting glucose (R = 0.6). We also observed proteoform-to-phenotype associations for ApoC-III, 22 glycoproteoforms of which were characterized in this study. The abundance of ApoC-III modified by a single N-acetyl hexosamine (HexNAc) was associated with indices of obesity, such as BMI, weight, and waist circumference (R ∼ 0.7). These data show IP-SampleStream-MS to be a robust, scalable workflow for high-throughput associations of proteoforms to phenotypes.

A Wide-Field Optical Coherence Tomography Normative Database Considering the Fovea-Disc Relationship for Glaucoma Detection.

Purpose: One purpose of this study was to collect wide-field swept-source optical coherence tomography (SS-OCT) data from healthy eyes and build a wide-filed normative database. Another purpose was to compare the glaucoma diagnostic ability of new parameters based on this normative database to the parameters that are currently in use, such as the peripapillary retinal nerve fiber layer (RNFL), macular ganglion cell-inner plexiform layer, and ganglion cell complex (GCC) thickness. Methods: This study had 220 healthy eyes and 292 eyes with early-stage glaucoma (EG) and moderate-stage glaucoma (MG) enrolled. Using the wide-field SS-OCT images (12 × 9 mm) of healthy eyes, a wide-field normative database was constructed by transforming and combining the individual images into a uniform template using the fovea and optic disc centers as fixed landmarks. Adjustment for the disc size was conducted. With this normative database, new parameters based on the ratio of the fovea-disc distance (FDD) consisting of the fovea-disc relationship were evaluated. The glaucoma diagnostic ability was assessed based on the area under the receiver operating characteristic curve (AUC). Results: Among the new peripapillary parameters, the RNFL of the circumference of the circle with diameter 0.8 FDD showed the highest AUC value for EG and MG, but the value was not significantly superior to that of the initial RNFL (AUC = 0.940 vs. 0.937, P = 0.631). Among the macular parameters, the GCC of the area of the circle of 1.5 FDD showed the highest AUC value for EG and MG, and the value was significantly superior to that of initial GCC (AUC = 0.929 vs. 0.919, P = 0.033). However, there was no significant difference between the initial and adjusted GCC thickness in patients included in the EG or MG groups separately. Conclusions: A wide-field normative database was built to consider the relationship between the fovea and the optic disc. Considering this aspect, we found that the GCC analysis using a broader area presented a significantly greater glaucoma diagnostic performance for EG and MG in the macula than the initial parameter for the GCC. Translational Relevance: Based on this wide-field normative database, the clinical use of a wide-field deviation map may help diagnose the patients with EG and MG in the future.

Rate of central corneal thickness changes in primary angle closure eyes: long-term follow-up results.

BACKGROUND: Central corneal thickness (CCT) and its association with intraocular pressure, which is a pivotal parameter in glaucoma management, has previously been reported. In this study, we intended to investigate the long-term change of CCT in terms of rate in eyes with primary angle-closure (PAC). Additionally, we aimed to analyze events that could affect CCT. METHODS: In this retrospective study, 26 patients with PAC who had a follow-up period of more than 5 years were analyzed. The rate of CCT changes from baseline was evaluated from the serial CCT measurements over the average follow-up period. The pattern of CCT change rate according to modes of treatment and history of angle-closure attack was analyzed using the repeated linear mixed model analysis. RESULTS: A total of 52 eyes were enrolled. The CCT reduction rate of the entire study population was - 0.72 ± 0.22 µm/yr (P = 0.001) with statistical significance. The CCT thinning rate of the laser peripheral iridotomy (PI) group was - 0.53 ± 0.25 µm/yr (P = 0.034) and that of the surgical trabeculectomy group was - 1.32 ± 0.43 µm/yr (P = 0.002), and it was not statistically significant (P = 0.112). The rate of CCT thinning in patients with a history of acute angle-closure attack was - 0.81 ± 0.31 µm/yr (P = 0.009) and that in patients without an attack was - 0.63 ± 0.30 µm/yr (P = 0.001), and it was not statistically significant (P = 0.680). Baseline CCT appeared to be the only significant factor affecting the rate of CCT changes (P < 0.001). CONCLUSIONS: We found a significant reduction in CCT over a long observation period in PAC eyes. We also found that the rates of CCT reduction were not affected by different treatment modalities or acute angle-closure attacks. The analysis of long-term CCT changes in conjunction with baseline CCT would also be helpful in the clinical evaluation of the PAC patients.

Optic disc melanocytoma with normal tension glaucoma and angle closure glaucoma: Two case reports.

RATIONALE: Optic disc melanocytoma is an ophthalmic tumor that arises from melanocytes, and is a variant of the melanocytic nevus. Here we report 2 cases of optic disc melanocytoma in Asian patient: one associated with normal tension glaucoma (NTG), and the other associated with angle closure glaucoma (ACG). PATIENT CONCERNS: Case 1 is a 57-year-old Asian female presented to our department for a general ophthalmic examination. Incidentally, brownish pigmented lesion was found on dilated fundus examination of her right eye. The fundus examination and optical coherence tomography (OCT) examination revealed a mass within optic disc, and superotemporal retinal nerve fiber layer (RNFL) thinning. The Humphrey visual field test showed corresponding visual field defect. Fluorescein angiography showed no leakage around the lesion. Case 2 is a 78-year-old Asian woman presented with complaints of acute bilateral ocular pain. The initial examination revealed shallow anterior chamber. Under the impression of intermittent angle closure attack, prophylactic laser peripheral iridotomy were performed. On dilated fundus examination, black pigmented lesion was found at superior sector of optic disc. Further examination revealed bilateral superotemporal, inferotemporal RNFL thinning on OCT, and spatially corresponding visual field defects. DIAGNOSES: Clinical diagnosis of NTG was made for case 1 patient. Although it was a little distant from typical glaucomatous changes, nevertheless she had RNFL defect compatible with visual field defects. Considering her normal IOP and angle structures, we believe NTG was a probable diagnosis for the patient. In case 2, we made diagnosis of ACG presenting as intermittent angle closure attack because of her presenting symptoms, narrowing of anterior chamber and angle structures found on gonioscopic and slit lamp examinations. INTERVENTIONS: In Case 1, we prescribe 0.005% latanoprost ophthalmic solution. In Case 2, at first prophylactic laser peripheral iridotomy was performed. Then, topical eyedrops administration was started, and the patient was examined periodically. OUTCOMES: In Case 1, at 6 months' follow-up, OCT and visual field test showed no progression. In Case 2, to this date, the optic disc melanocytoma remains stable for over a 6-year-follow-up period. LESSONS: The fact that NTG and ACG can coexist in patients with melanocytoma of optic disc should be recognized, and the possibility of such should appropriately be evaluated.

Novel Interface for High-Throughput Analysis of Biotherapeutics by Electrospray Mass Spectrometry.

With the rapid rise of therapeutic antibodies and antibody-drug conjugates, significant investments have been made in developing workflows that utilize mass spectrometry to detect these intact molecules, the large fragments generated by their selective digestion, and the peptides generated by traditional proteomics workflows. The resultant data is used to gain insight into a wide range of parameters, including primary sequence, disulfide bonding, glycosylation patterns, biotransformation, and more. However, many of the technologies utilized to couple these workflows to mass spectrometers have significant limitations that force nonoptimal modifications to upstream sample preparation steps, limit the throughput of high-volume workflows, and prevent the harmonization of diverse experiments onto a single hardware platform. Here, we describe a new analytical platform that enables direct and high-throughput coupling to electrospray ionization mass spectrometry. The SampleStream platform is compatible with both native and denaturing electrospray, operates with a throughput of up to 15 s/sample, provides extensive concentration of dilute samples, and affords similar sensitivity to comparable liquid chromatographic methods.

Traumatic optic neuropathy-associated progressive thinning of the retinal nerve fiber layer and ganglion cell complex: two case reports.

BACKGROUND: Traumatic optic neuropathy (TON) is a form of optic nerve injury that occurs secondary to trauma and is etiologically associated with acute axonal loss with severe vision loss. Here, we reported longitudinal changes in the peripapillary retinal nerve fiber layer (RNFL) and macular ganglion cell complex (GCC) using wide-field swept source optical coherence tomography (SS-OCT) in two cases of TON and identified the source of the damage. CASE PRESENTATION: (Case 1) A 65-year-old man was admitted to the hospital due to an injury in the right eye (OD) and was subsequently diagnosed with indirect TON. He was then treated with high-doses of intravenous steroids. Wide-field SS-OCT was performed at the baseline and after 1 day, 2 days, 1 week, 1 month, and 4 months. The wide-field deviation map detected thinning earlier in the macular GCC than in the peripapillary RNFL. (Case 2) A 63-year-old man was admitted to the hospital with a fractured left maxilla-zygomatic complex attributed to blunt-force trauma to the head and loss of vision in his left eye (OS). He was diagnosed with indirect TON and treated with high-doses of intravenous steroids. Wide-field SS-OCT was performed at the baseline and after 1 week, 2 weeks, 2 months 5 months, and 7 months. The wide-field deviation map detected thinning earlier in the peripapillary RNFL than in the macular GCC. CONCLUSIONS: Wide-field SS-OCT facilitated the identification of various sequential progression patterns in patients with TON. Furthermore, the area in which the structural damage was first detected was seen differently in the peripapillary and macular deviation maps for each case. Thus, wide-field imaging, which includes the macular and peripapillary areas, are useful in monitoring TON.

Multidimensional Top-Down Proteomics of Brain-Region-Specific Mouse Brain Proteoforms Responsive to Cocaine and Estradiol.

Cocaine addiction afflicts nearly 1 million adults in the United States, and to date, there are no known treatments approved for this psychiatric condition. Women are particularly vulnerable to developing a cocaine use disorder and suffer from more serious cardiac consequences than men when using cocaine. Estrogen is one biological factor contributing to the increased risk for females to develop problematic cocaine use. Animal studies have demonstrated that estrogen (17ß-estradiol or E2) enhances the rewarding properties of cocaine. Although E2 affects the dopamine system, the molecular and cellular mechanisms of E2-enhanced cocaine reward have not been characterized. In this study, quantitative top-down proteomics was used to measure intact proteins in specific regions of the female mouse brain after mice were trained for cocaine-conditioned place preference, a behavioral test of cocaine reward. Several proteoform changes occurred in the ventral tegmental area after combined cocaine and E2 treatments, with the most numerous proteoform alterations on myelin basic protein, indicating possible changes in white matter structure. There were also changes in histone H4, protein phosphatase inhibitors, cholecystokinin, and calmodulin proteoforms. These observations provide insight into estrogen signaling in the brain and may guide new approaches to treating women with cocaine use disorder.

MALDI-TOF MS-based total serum protein fingerprinting for liver cancer diagnosis.

Serum is one of the most commonly used samples in many studies to identify protein biomarkers to diagnose cancer. Although conventional enzyme-linked immunosorbent assay (ELISA) or liquid chromatography-mass spectrometry (LC-MS)-based methods have been applied as clinical tools for diagnosing cancer, there have been troublesome problems, such as inferior multiplexing capabilities, high development costs and long turnaround times, which are inappropriate for high-throughput analytical platforms. Here, we developed a simple and robust cancer diagnostic method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based total serum protein fingerprinting. First, serum samples were simply diluted with distilled water and subsequently spotted onto a MALDI plate without prior chromatographic purification or separation. The sample preparation method was enough to collect reproducible total serum protein fingerprints and would be highly advantageous for high-throughput assay. Each of the integrated main spectrum profiles (MSPs), which are representative of liver cancer patients (n = 40) or healthy controls (n = 80), was automatically generated by the MALDI Biotyper 3 software. The reliability of the integrated MSPs was successfully evaluated in comparison with a blind test set (n = 31), which consisted of 13 liver cancer patients and 18 healthy controls. Additionally, our partial least squares discriminant analysis (PLS-DA) demonstrated a statistically significant difference in MALDI-TOF MS-based total serum protein fingerprints between liver cancer patients and healthy controls. Taken together, this work suggests that this method may be an effective high-throughput platform technology for various cancer diagnoses and disease evaluations.

Discovery of glycocholic acid and taurochenodeoxycholic acid as phenotypic biomarkers in cholangiocarcinoma.

Although several biomarkers can be used to distinguish cholangiocarcinoma (CCA) from healthy controls, differentiating the disease from benign biliary disease (BBD) or pancreatic cancer (PC) is a challenge. CCA biomarkers are associated with low specificity or have not been validated in relation to the biological effects of CCA. In this study, we quantitatively analyzed 15 biliary bile acids in CCA (n = 30), BBD (n = 57) and PC (n = 17) patients and discovered glycocholic acid (GCA) and taurochenodeoxycholic acid (TCDCA) as specific CCA biomarkers. Firstly, we showed that the average concentration of total biliary bile acids in CCA patients was quantitatively less than in other patient groups. In addition, the average composition ratio of primary bile acids and conjugated bile acids in CCA patients was the highest in all patient groups. The average composition ratio of GCA (35.6%) in CCA patients was significantly higher than in other patient groups. Conversely, the average composition ratio of TCDCA (13.8%) in CCA patients was significantly lower in all patient groups. To verify the biological effects of GCA and TCDCA, we analyzed the gene expression of bile acid receptors associated with the development of CCA in a CCA cell line. The gene expression of transmembrane G protein coupled receptor (TGR5) and sphingosine 1-phosphate receptor 2 (S1PR2) in CCA cells treated with GCA was 8.6-fold and 3.4-fold higher compared with control (untreated with bile acids), respectively. Gene expression of TGR5 and S1PR2 in TCDCA-treated cells was not significantly different from the control. Taken together, our study identified GCA and TCDCA as phenotype-specific biomarkers for CCA.

Top-Down Proteomics Enables Comparative Analysis of Brain Proteoforms Between Mouse Strains.

Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains.

An On-Line Platform for the Analysis of Permethylated Oligosaccharides Using Capillary Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry.

Permethylation is one of the most practical modifications for mass spectrometric analysis of carbohydrates. In this study, we showed a facile platform for the on-line permethylation and analysis of acidic N-glycans using capillary liquid chromatography-electrospray ionization-tandem mass spectrometry to elucidate the N-glycan structures derived from glycoprotein samples. Two capillaries, individually packed with NaOH and C18 powder, were coupled to electrospray ion trap mass spectrometer. Prior to mass spectrometry, oligosaccharides were permethylated in NaOH-packed capillary by methyl iodide, and the permethylated glycans were consequently extracted from residual reagents, such as NaOH and methyl iodide, in C18-capillary, instead of using conventional chloroform extraction. After permethylation and purification, the permethylated glycans were released from C18 capillary by acetonitrile, and sprayed into mass spectrometer. Using this on-line strategy, a picomolar range of sialylated N-glycans from bovine fetuin were analyzed by tandem mass spectrometry.

Mass spectrometry-based N-linked glycomic profiling as a means for tracking pancreatic cancer metastasis.

The aberrant glycosylation profile on the surface of cancer cells has been recognized for its potential diagnostic value towards assessing tumor progression. In this study, we initially investigate N-glycan profiles on the surface of normal (HPDE) and cancerous (Capan-1, Panc-1, and MIA PaCa-2) pancreatic cell lines, which are from different sites of pancreatic tumor. The enzymatically deglycosylated total N-glycans are permethylated via a quantitative solid-phase method and then analyzed by using MALDI-TOF MS and MALDI-QIT-TOF MS. We demonstrate that the level of high-mannose type glycans is higher among Capan-1 cells-pancreatic cancer cells that have metastasized to the liver-than that observed among Panc-1 and MIA PaCa-2 cells-pancreatic cancer cells from the pancreas duct head and tail regions, respectively. Furthermore, the relative abundance of highly-branched sialyted N-glycans is significantly up-regulated on Panc-1 and MIA PaCa-2 pancreatic cancer cells compared to that of normal HPDE pancreas cells. Taken together, these results indicate that specific N-glycosylation profile changes in pancreatic cancer cells can be used to not only distinguish between normal and cancerous cells but also provide more information on their location and metastatic potential.

Stable isotopic labeling-based quantitative targeted glycomics (i-QTaG).

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling-based quantitative targeted glycomics (i-QTaG) technique for the comparative and quantitative analysis of total N-glycans using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N-glycans using a model glycoprotein (bovine fetuin). Moreover, the i-QTaG using MALDI-TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of (13) C6 /(12) C6 -2-aminobenzoic acid-labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N-glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N-glycan peaks from i-QTaG method showed a good linearity (R(2) > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2-AA labeled N-glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up-regulation of the Lewis antigen (~82%) in sera from prostate cancer patients. In this proof-of-concept study, we demonstrated that the i-QTaG method, which enables to achieve a reliable comparative quantitation of total N-glycans via MALDI-TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics.

Comparative N-linked glycan analysis of wild-type and α1,3-galactosyltransferase gene knock-out pig fibroblasts using mass spectrometry approaches.

Carbohydrate antigens expressed on pig cells are considered to be major barriers in pig-to-human xenotransplantation. Even after α1,3-galactosyltransferase gene knock-out (GalT-KO) pigs are generated, potential non-Gal antigens are still existed. However, to the best of our knowledge there is no extensive study analyzing N-glycans expressed on the GalT-KO pig tissues or cells. Here, we identified and quantified totally 47 N-glycans from wild-type (WT) and GalT-KO pig fibroblasts using mass spectrometry. First, our results confirmed the absence of galactose-alpha-1,3-galactose (α-Gal) residue in the GalT-KO pig cells. Interestingly, we showed that the level of overall fucosylated N-glycans from GalT-KO pig fibroblasts is much higher than from WT pig fibroblasts. Moreover, the relative quantity of the N-glycolylneuraminic acid (NeuGc) antigen is slightly higher in the GalT-KO pigs. Thus, this study will contribute to a better understanding of cellular glycan alterations on GalT-KO pigs for successful xenotransplantation.