Glycoblotting of Egg White Reveals Diverse N-Glycan Expression in

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Glycoblotting of Egg White Reveals Diverse N-glycan Expression in Quail Species Jurgen T. Sanes, Hiroshi Hinou, Yuan Chuan Lee, and Shin-Ichiro Nishimura J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04782 • Publication Date (Web): 11 Dec 2018 Downloaded from http://pubs.acs.org on December 18, 2018

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Journal of Agricultural and Food Chemistry

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Glycoblotting of Egg White Reveals Diverse N-glycan

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Expression in Quail Species

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Jurgen T. Sanes a, Hiroshi Hinou a, Yuan Chuan Lee b, and Shin-Ichiro Nishimura a*

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a

Faculty of Advanced Life Science and Graduate School of Life Science, Hokkaido University,

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N21, W11, Kita-ku, Sapporo 001-0021, Japan. b Biology

Department, Johns Hopkins University, 3400 North Charles Street, Baltimore,

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Maryland 21218, United States

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KEYWORDS: N-Glycans; Glycan Diversity; Inter-species Glycan Comparison; Glycoblotting;

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Egg White; Galliformes; Quail

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ABSTRACT

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The glycan part of glycoproteins is known to be involved in the structure and modulatory functions

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of glycoproteins, serving as ligands for cell to cell interactions, and as specific ligands for cell to

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microbe interactions. It is believed that intraspecies and interspecies variations in glycosylation

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exist. As an approach to better understand glycan diversity, egg whites (EW) from four different

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quail species are studied by the well-established glycoblotting procedure, a glycan enrichment and

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analysis method. N-glycans were classified and the profiles were established for quail egg white

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samples which showed 21 relevant glycan peaks; 18 peaks were expressed significantly, and 10

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glycan peaks are found to be abundant in certain species. The result establishes glycan profiles for

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Blue-scaled, Bobwhite, Japanese, and Mountain quail egg whites and shows unique difference

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among glycan expressions, particularly, high-mannose in Japanese quail and tetra-antennary

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glycan structure for other quail species.

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INTRODUCTION

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Glycans in glycoproteins are formed by post-translational modification of peptides. Intra-species

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and inter-species glycan variation in glycoproteins is known to exist, but less information about

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glycan structural diversity is available 1. We chose to study EWs, because they are a nutrient

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source for the embryo, and most importantly, it serves as a barrier to harmful microbes that invade

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the developing embryo 2. Thus, it is a very ideal material for examining the inter-species diversity

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of glycans 3-5.

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A comprehensive profiling of N-glycans of the EWs from different avian species was

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conducted previously 6 which showcased that members of Galloanserae can be classified into 2

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major groups and 5 submajor clusters based on MALDI-TOF/MS analysis. It was rationalized that

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glycan expression patterns are influenced by features such as body size, and the influence of

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lifestyle and diet of the birds. The comprehensive glycan analysis was achieved very efficiently

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using the Glycoblotting/SweetBlot technology developed in this laboratory 7. The above-

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mentioned work compares between two avian orders, e.g. Galliformes and Anseriformes, and not

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between within the same order of birds 6. The current work was conducted to compare glycan

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expression within one avian order.

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In this paper, the N-glycan expression from EW of four quail species, Blue-Scaled Quail

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(BSQ, Callipepla squamata), Northern Bobwhite Quail (BWQ, Colinus virginianus), Japanese

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Quail (JQ, Coturnix japonica), and Mountain Quail (MQ, Oreortyx pictus)

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These quail species belong to the order Galliformes of ground-feeding birds. The glycan analyses

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were conducted by the glycoblotting enrichment procedure for glycans. Glycoblotting glycan

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enrichment makes use of the stability of hemiacetals under acidic medium to form hydrazone

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bonds with a hydrazide functionalized polymer that would differentiate the glycans from other

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were analyzed.

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biomolecules. The enriched glycans could be easily washed, derivatized, and labeled for mass

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spectrometry analysis 10. At the time of writing, this is the first paper for N-glycan inter-species

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comparison among quails in the order Galliformes.

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MATERIALS AND METHODS

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The procedure for digestion and glycoblotting was based on the published article

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modifications and was performed as follows:

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Enzymatic Release of N-Glycans from Quail Egg White

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with few

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Japanese quail eggs were bought from the supermarket and the egg white separated from

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the yolk. The BSQEW, BWQEW, and MQEW were taken from an in-house egg white collection.

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Egg whites from 10 eggs provided ~50 mL of sample which was homogenized and then dried. A

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1.0 mg lyophilized quail EW (BSQ, BWQ, JQ, and MQ) was weighed and placed in Eppendorf

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tubes. Three trials were prepared for each quail type. The egg white powder was dissolved in 20

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µL 200 mM ammonium bicarbonate. Addition of a 26 µL 100 µM disialyloctasaccharide (Tokyo

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Chemical Industry Co., LTD.) internal standard followed. A 54 µL mixture containing 0.06% 1-

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propanesulfonic acid, 2-hydroxyl-3-myristamido (PHM) with 12 mM dithiothreitol (DTT) in 105

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mM NH4HCO3 was added and incubated at 60°C for 90 min. After incubation, a 10 µL freshly

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prepared 123 mM iodoacetamide was added and incubated in the dark for 1 h at room temperature.

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Digestion with 10 µL of 40 U/ µL sequence grade trypsin (Sigma Aldrich) in 1mM HCl was

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performed and incubated overnight at 37°C. The trypsin enzyme was heat inactivated in a heat

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block at 90°C for 10 min. The trypsin-digested solution was cooled at room temperature before

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adding 2 units of PNGase F (Roche) and incubated overnight at 37°C, followed by addition of a

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10 µL of 0.5 units/µL Proteinase K at 37°C and incubation for 3 hours. The Proteinase-K (Roche)


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was heat inactivated at 90°C for 10 min. The enzyme digested samples were then placed in a

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SpeedVac to dry.

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Glycoblotting Methodology for Enrichment of N-Glycans

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Five hundred µL BlotGlycoH bead suspension (10 mg/mL) was added to each of the 96-

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well multiScreen Solvinert filter plate. The 96-well filter plate was then attached to a vacuum

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manifold and the water removed. The dried digest containing released N-glycans was reconstituted

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with 20 µL Milli Q water and 180 µL of 2% acetic acid/acetonitrile. The 96-well plate was

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incubated at 80°C for 45 min or until dry. Washing with 200 µL of 2 M guanidine-HCl in

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NH4HCO3, H2O, and 1% triethylamine in methanol followed. Each solvent washing was performed

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twice and vacuumed after every solvent addition. The unreacted hydrazide functional beads were

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then capped by addition of freshly prepared 100 µL 10 % acetic anhydride in methanol and

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incubated at RT for 30 min. The capping solvent was removed in vacuum. Washing with 200 µL

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of 10 mM HCl, methanol, dioxane was then conducted. Washing with each solvent was done twice

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and vacuumed after each solvent addition. Addition of 100 µL 100 mM 3-methyl-1-p-tolyltriazene

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(MTT) in dioxane was conducted and the 96-well plate incubated at 60°C until dry. The 96-well

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plate was washed twice with 200 µL of dioxane, water, methanol, and water, with vacuuming

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between washings. A 20 µL of 50 mM O-benzyloxyamine hydrochloride (BOA) and 180 µL of 2

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% acetic acid/acetonitrile were added to the wells. The 96-well plate was then incubated at 80°C

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until the wells are dry. Elution of the glycans was then performed by addition of 100 µL of MilliQ

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H2O. Elution was conducted twice. The eluted labeled-glycans were pooled and dried in

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SpeedVac.

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MALDI-TOF MS and MALDI-TOF/TOF Analysis


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The dried labeled-glycans were reconstituted with 20 µL of ultrapure H2O. The mass

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spectral analysis was with an Autoflex III (Bruker Daltonics) in reflector, positive ion mode,

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typically totaling 200x10 shots with acceleration voltage, reflector voltage, and pulsed ion

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extraction settings of 25.3 kV, 26.4 kV, and 100 ns, respectively. The matrix recommended by the

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Bruker Dalton company was prepared by dissolving a mixture of 20 mg/mL 2,5-dihydroxybenzoic

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acid (DHB) and 1 mM NaCl in 30% acetonitrile containing 0.1% trifluoroacetic acid in water. A

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1 µL of the prepared DHB matrix was spotted on the Anchorchip Target Plate MTP 384 Target

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Plate (polished steel TF, Bruker), dried, followed by spotting with 1 µL of the reconstituted

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solution containing the labeled glycans. The experimental masses were obtained using the

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FlexAnalysis 3.0 software (Bruker Daltonics) and the structures annotated using the GlycoMod

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Database (https://web.expasy.org/glycomod/)11,12 and the Functional Glycomics Gateway

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(http://www.functionalglycomics.org/glycomics/molecule/jsp/carbohydrate/searchByCompositio

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n.jsp)13 websites. In MALDI-TOF/TOF Mode, precursor ions were accelerated to 8 kV and the

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fragments further accelerated to 20.1 kV in positive ion mode. MS/MS analysis was conducted

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using Ultraflex III (Bruker Daltonics) to further confirm the identity of important N-glycan

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structures.

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Glycotyping of N-glycans in Quail Egg Whites and Statistical Analysis

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The area of the curve was taken and normalized with the internal standard. From the results

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obtained as total amount of glycans in pmols, the N-glycans could be classified into hybrid,

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complex, high-mannose, and paucimannose structures. The hybrid and complex glycans were

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classified into bisecting and non-bisecting structures. Furthermore, hybrid and complex glycans

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were classified based on antennary types. Estimated mole percentage of each glycan type was

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tabulated to further distinguish the difference of the glycan structures expressed in quail EWs and


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identify which of the glycan types were expressed differently. Distinct, abundant, and trace N-

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glycan structures were identified. Single-way Analysis of Variance (ANOVA) and Tukey’s

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multiple comparison post-test were used for statistical analysis.

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RESULTS AND DISCUSSION

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N-Glycans in Blue-Scaled Quail Egg White (BSQEW)

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Figure 1 shows all major N-glycans for a single trial (see also Figure S1) and the structural

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compositions is displayed in Table 1. Nineteen mass/charge peaks corresponding to N-glycans

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were found in the analysis of BSQEW shown in Table 2. A description on how to calculate the

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theoretical m/z is presented in the supporting information. The peak at 2175 m/z pertains to the

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disialyloctasaccharide internal standard (2600 pmol) to which the relevant glycan m/z values were

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normalized with. Highest and lowest glycan amounts were seen for 1647 and 1972 m/z values,

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respectively. Statistical significant difference was found among ions under 99.9% confidence level

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using one-way analysis of variance as test statistic.

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Based from the total N-glycan amount of 232±22 nmols, glycotyping reveals the glycans

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in BSQEW were estimated to be

Glycoblotting of Egg White Reveals Diverse N-Glycan Expression in

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