1 Title: Creating a Mass Spectral Reference Library for

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Creating a Mass Spectral Reference Library for Oligosaccharides in Human Milk Connie Africano Remoroza, Tytus D. Mak, Maria Lorna De Leoz, Yuri A Mirokhin, and Stephen E. Stein Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01176 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 5, 2018

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Analytical Chemistry

Title: Creating a Mass Spectral Reference Library for Oligosaccharides in Human Milk Short-title: Development of a material based MS library of oligosaccharides

Authors: Connie A. Remoroza Mass Spectrometry Data Center, Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD, 20899-8362, USA [email protected]

Tytus D. Mak Mass Spectrometry Data Center, Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD, 20899-8362, USA [email protected]

Maria Lorna A. De Leoz Mass Spectrometry Data Center, Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD, 20899-8362, USA [email protected]

Yuri A. Mirokhin Mass Spectrometry Data Center, Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD, 20899-8362, USA [email protected]

Stephen E. Stein Mass Spectrometry Data Center, Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, MD, 20899-8362, USA [email protected]

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Creating a Mass Spectral Reference Library for Oligosaccharides in Human Milk

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Connie A. Remoroza, Tytus D. Mak, Maria Lorna A. De Leoz, Yuri A. Mirokhin, Stephen E.

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Stein

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Mass Spectrometry Data Center, Biomolecular Measurement Division, National Institute of Standards

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and Technology, Gaithersburg, MD, 20899-8362, USA

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*[email protected]

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Abstract

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We report the development and availability of a mass spectral reference library for

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oligosaccharides in human milk. This represents a new variety of spectral library that includes

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consensus spectra of compounds annotated through various data analysis methods – a concept

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that can be extended to other varieties of biological fluids. Oligosaccharides from the NIST

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Standard Reference Material (SRM) 1953, composed of human milk pooled from 100

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breastfeeding mothers, were identified and characterized using Hydrophilic Interaction Liquid

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Chromatography Electrospray Ionization Tandem Mass Spectrometry (HILIC-ESI-MS/MS) and

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the NIST 17 Tandem MS Library. Consensus reference spectra were generated, incorporated into

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a searchable library and matched using the newly developed hybrid search algorithm to elucidate

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unknown oligosaccharides. The NIST hybrid search program facilitates the structural assignment

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of complex oligosaccharides especially when reference standards are not commercially available.

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High accuracy mass measurement for precursor and product ions, as well as the relatively high

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MS/MS signal intensities of various oligosaccharide precursors with Fourier transform ion trap

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(FT-IT) and higher energy dissociation (HCD) fragmentation techniques, enabled the assignment

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of multiple free and underivatized fucosyllacto- and sialyllacto-oligosaccharide spectra. Neutral

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and sialylated isomeric oligosaccharides have distinct retention times, allowing the identification

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of 74 oligosaccharides in the reference material. This collection of newly characterized spectra

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based on a searchable, reference MS library of annotated oligosaccharides, can be applied to

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analyze similar compounds in other types of milk or any biological fluid containing milk

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oligosaccharides.

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Keywords: HILIC-ESI-MS/MS, Human milk, SRM 1953, oligosaccharides, Tandem Mass

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Spectral Library, Fourier Transform-IT, Hybrid search

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Introduction

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Human milk is the gold standard for healthy human infant feeding. Human milk contains

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unique bioactive oligosaccharides that play a significant role in brain development and increased

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immunity to infection in infants1-2. Milk oligosaccharides are typically composed of three to ten

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monosaccharide units, consisting of glucose (Glc), galactose (Gal), N-acetyl-glucosamine

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(GlcNAc), fucose (Fuc) and sialic acid (Neu5Ac). The core group present at the reducing end of

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milk oligosaccharides is either lactose (Galβ1–4Glc) or N-acetyl-lactosamine (Galβ1–

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4GlcNAc)3. The most common oligosaccharides in human milk have a lactose unit (Galβ 1−

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4Glc) at the reducing end.

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Because milk oligosaccharides are highly polar, appropriate separation and identification

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techniques are required to characterize unknown compounds. Enzyme digestion by

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exoglycosidases combined with size exclusion chromatography and Capillary electrophoresis

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(CE)4 or porous graphitized carbon (PGC) separation techniques have been used frequently5.

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Several studies have shown that Hydrophilic Interaction Liquid Chromatography coupled to

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mass spectrometry and/or fluorescence detection (HILIC-ESI-MS/FLD) enable sufficient

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separation for the characterization of neutral and acidic oligosaccharides from plant materials6-7

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and mammalian milk8. The elucidation of unknown oligosaccharides by HILIC-MS/MS alone

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can be challenging especially because reference standards for many of these oligosaccharides are

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not commercially available.

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One objective of this work is to facilitate analysis in the identification of these

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oligosaccharides through the development of a library of tandem mass spectra. Mass spectral

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(MS) libraries enable the tentative identification of unknown compounds in complex matrices by

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matching known fragmentation patterns of electrospray derived ions present in tandem MS

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libraries9. Recently, this method was enhanced by matching the unknown and MS library spectra

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with different parent ions based upon consistently mass shifted peaks. This strategy is termed the

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Hybrid Library Search10 and simplifies the recognition of unknown compounds in the sample

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based on their similarity to known and well-characterized reference spectra.

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Another objective is to develop methods for creating libraries that include recurring

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spectra of components not commercially available, but identifiable to a meaningful extent using

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current data analysis methods. Such ‘material-oriented’ libraries are needed to deal with complex

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biological samples analyzed by mass spectrometry.

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This study reports the creation of a MS library of oligosaccharides from the NIST

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Standard Reference Material® on human milk (SRM 1953), which is significant because there is

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little published characterization of the highly polar and complex composition of oligosaccharides

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in SRM 1953. Additionally, we describe the process of the structural assignment for isomeric

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oligosaccharides using the NIST 17 Tandem MS Library11 and hybrid search method, a process

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of general applicability.

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Materials and Methods

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Standard Reference Material (SRM) 1953 was obtained from the National Institute of

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Standards and Technology (Gaithersburg, MD). SRM 1953 is a human milk pool from one

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hundred breastfeeding mothers (https://www-s.nist.gov/srm1953). The human milk sample was

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stored in a sterile container and kept frozen (−80°C) until use. Water used in the sample

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preparation was LC-MS grade. All other chemicals used were of analytical grade.

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Extraction and purification of human milk oligosaccharides

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Oligosaccharides from SRM1953 (2 mL) were extracted and purified by solid phase

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extraction (SPE) followed by drying as previously described12. Briefly, 2 mL of SRM 1953 was

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centrifuged at 14000 × g, 4˚C for 30 mins. The liquid layer was transferred by pipet and mixed

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with four volumes of 2:1 v/v of chloroform-methanol solvent and centrifuged at 14000 × g, 4˚C

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for 30 mins. Proteins were precipitated overnight at 4˚C by adding two volumes of ethanol into

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the mixture, then centrifuged at 14000 × g, 4˚C for 30 mins. The decanted liquid was evaporated

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to dryness prior solid phase extraction and HILIC-ESI-MS analyses (Supporting Information

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S2).

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UHPLC-HILIC-MS/MS Analysis of Oligosaccharides

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An Ultimate 3000 UHPLC system (Thermo Scientific) coupled to an Orbitrap mass

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spectrometer (Thermo ScientificTM Orbitrap FusionTM LumosTM) was used for the analysis of the

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samples. The chromatographic separation was performed on ACQUITY Glycoprotein BEH

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Amide column, 300Å (1.7 µm, 2.1 mm × 150 mm, Waters Corporation, Milford, MA, U.S.A.).

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The acquisition time was 65 min, and the mobile phase had a flow rate of 400 µL/min, pH 4.5

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and with a column oven temperature of 35˚C. The injection volume was 10 µL. The composition

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of the two mobile phases was 10 mmol/L ammonium formate with 0.1% (v/v) formic acid (A)

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and 99.9% (v/v) ACN with 0.1% (v/v) formic acid (B). The elution program was performed as

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follows: 1.5 min isocratic 95 % (v/v) B; 8.5 min linear gradient from 95% (v/v) to 80% (v/v) B;

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50 min linear gradient from 80% (v/v) to 50% (v/v) of B followed by 5 min of column washing

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with a linear gradient from 50% (v/v) to 2% (v/v) B including column re-equilibration with 95%

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(v/v) B. During the column washing the flow rate was set at 250 µL/min. The electrospray MS

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detection was performed in positive and negative detection mode both for neutral and acidic

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oligosaccharides with the ion source voltage set to +/- 3.5 kV; the capillary temperature 250˚C;

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sheath gas 15 (arbitrary units); auxiliary gas 10 (arbitrary units). Spectra were acquired using

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both ion trap and beam-type collision cell fragmentation, both with spectrum measurement at

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high mass accuracy in orbitrap mass analyzer. The former is referred as Fourier transform ion

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trap (FT-IT), in which all spectra were acquired at the ‘Normalized Collision Energy’ setting

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(NCE) of 35% and Q value of 0.25. The latter is called ‘HCD’ (higher energy dissociation’) by

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the instrument maker, although fragmentation patterns are equivalent to those of most triple

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quadrupole and QTOF instruments at comparable collision energies13. These spectra were

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acquired at NCE values of 10, 15, 20, 25, 30, 40 and 50. Each sample was analyzed in triplicate.

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Unidentified Spectra of Oligosaccharides for Mass Spectral Matching

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The acquired FT-IT and HCD MS2 spectra from the raw HILIC-MS/MS data were sorted

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and clustered into consensus spectra using NIST algorithms14 to create a library of unknown

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consensus spectra. A consensus spectrum is a weighted average of the similar spectra having the

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same precursor ion. Each spectrum must have a minimum match factor (MF) score of 999 based

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on similarity in peak relative intensities and fragment masses (Supporting Information S2).

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Methods for Identification and Annotation of Unknown Spectra

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The following describes the systematic analysis of neutral and acidic oligosaccharides in

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SRM 1953. Identification of oligosaccharides in the unknown MS library of SRM 1953 was

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done manually, based on the literature, and using results of searches against the NIST Tandem

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spectral library. Spectra were first examined individually then corresponding entries were

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matched in the unknown MS library. Nine considerations in making these identifications are

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described below. The first three of these used NIST MS Search 2.3 software15 while the last six

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were done manually (Supporting Information S2).

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1. Library MS Search Results

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Spectra from the unknown MS library were searched against the NIST 17 Tandem MS

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Library using the NIST MS Search 2.3 software15 using Simple and MS/MS hybrid search

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methods. Search parameters such as precursor m/z and product ion masses were set to error

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tolerances of 10 × 10-6 mg/Kg and 50 × 10-6 mg/Kg, respectively. The search software generates

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the possible oligosaccharide structures based on the similarity of peak intensity and masses

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between the consensus spectrum and the library reference oligosaccharide spectrum.

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2. Hybrid Search Peak Alignment

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The hybrid search method matches query spectra with library spectra that differ by discrete

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chemical groups. The basic principle of the search is that when two precursor ions differ only in

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a single modification that does not greatly affect the fragmentation mechanism, each product ion

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peak in one spectrum of one precursor corresponds to a peak created by exactly the same

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fragmentation in the other precursor spectrum10. This is done by shifting library peaks by the

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difference in the query and library mass (DeltaMass). For example, reduction of carbohydrates

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with a reducing group modifies the core unit lactose to lactitol, thereby adding two H atoms (m/z

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2.004) to the precursor ion m/z. This method is incorporated into the freely available NIST MS

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Search 2.3 software15. The software is intended for the mass spectral matching of unknown and

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library tandem mass spectra (high accuracy), qualitative characterization and illustration of fully

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annotated MS2 spectra of oligosaccharides in human milk.

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3. Fragment Annotation

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The corresponding B/Y and C/Z type ions were used to annotate and distinguish isomeric

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structures as previously described16 and by using SimGlycan17. Utilizing these fragmentation

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rules, fragment annotation was comprehensively conducted for each spectrum as described

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(Supporting Information S1). Initially, all possible fragments were acquired in Glypy 0.11.318 for

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the spectrum when considering all single- and double-cleavages that could occur with the

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associated glycan structure. Theoretical m/z values were then combinatorically generated from

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these neutral fragment masses when considering all common adduct types, water losses and

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gains, and isotopic shifts. Finally, annotations were assigned to a peak if the theoretical m/z value

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matched the experimental m/z value to within 10 × 10-6 mg/Kg. This annotation provides

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information on the chemical composition, branching site and sequence type present in the

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oligosaccharide.

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Specific illustrations of these ideas are discussed in the following sections.

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Results and Discussion

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Elution Profile of Milk Oligosaccharides. Elution of different oligosaccharides in the

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sample is caused by HILIC using different commercially available milk oligosaccharides

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(Supplementary Table S1). Base peak chromatograms of neutral and acidic fractions are

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displayed in Figure 1.

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Figure 1A displays the elution profile of fucosyllactose (2’FL), lacto-N-tetraose (LNT),

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lacto-N-difucohexaose (LNDFH) and monofucosyllactose-N-hexaose (MFLNH), known to be

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the most abundant neutral oligosaccharides in human milk. The milk oligosaccharides showed an

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excellent separation on a HILIC column related to their size. The elution of 2-FL prior to

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Neu5Ac-lactose (3-SL) illustrates the selectivity of HILIC. Sialylated-oligosaccharides display

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increased polarity relative to fucosylated oligosaccharides because they contain an additional

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carboxylate ion (COO-). The clear distinction in the elution pattern of fucosylated and/or

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sialylated oligosaccharides confirmed the HILIC separation, which is based both on size and

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polarity. Neutral oligosaccharides, FL, LNFP and F-LNH isomers (Fig. 1A) and sialylated

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oligosaccharides, SL, LST and S-LNFP isomers (Fig. 1B) were distinguished.

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Figure 1. Base peak HILIC-MS elution profile of free oligosaccharides in SRM 1953 human

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milk in positive detection mode. A. Neutral oligosaccharides. B. Acidic oligosaccharides. Refer

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to Table 2 for the description and annotation of peaks. Annotation number: A4121 means 4

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Hexose; 1 Fucose; 2 GlcNAc; 1 Neu5Ac.

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MS/MS Analysis

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Structural assignment of isomeric oligosaccharides is challenging due to the

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heterogeneity and complexity of monosaccharide composition and linkages. The following

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sections described the annotation of unknown experimental HCD and FT-IT MS2 spectra in the

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sample. The chemical composition constraints and glycosidic bonds and cross-ring

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fragmentation patterns rules were applied in the annotation of similar precursor ions (isomers),

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singly and doubly charged precursor ions of neutral and sialylated oligosaccharides. This method

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demonstrates the recognition of different branching patterns and linkages in the structures of

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fucosylated and sialylated oligosaccharides.

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Neutral Milk Oligosaccharides. Monofucosylated lacto-N-hexaose isomers (MFLNH

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III and MFpLNH IV) isomers eluted at different retention times with precursor ions m/z 1219.4

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[M+H]+ have product ions that are found useful to distinguish branched from linear structure

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(Supplementary Figure S2.1). This technique was then used to predict the possible chemical

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structures of three trifucosyl iso-lacto-N-octaose isomers eluting at longer retention time (37 to

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39) min (Fig.1A).

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It is known that singly charged state ions in FT-IT spectra do not allow the trapping of

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fragments with m/z values lower than one-third of the precursor mass19. Figure 2 illustrates the

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MS2 spectra of singly [M-H]- (m/z 1874.67) and doubly [M-2H]-2 (m/z 936.83) charged for

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oligosaccharide eluted at 38.56 min. Fragment ions C, Z and A type ions are dominant. The

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unknown spectra for N5330 isomer has the peak signal at m/z 1037.3632, evidence of the

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internal fucose residue at the β1-6 branch and consistent with previously reported TFiLNO b20.

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Furthermore, product ions of [M-2H]-2 such as m/z 364.1233, m/z 544.1863 and m/z 672.2325

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indicate the Gal(β1-4)Fuc(α1-3)GlcNAc sequence at the terminal β1-3 branch with two cross-

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2,4

3,5

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ring-glycosidic linkages

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internal ring cleavage of GlcNAc at the terminal β1-3 branch. The information provided by the

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low mass ions from a doubly charge spectra

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trifucosyl octasaccharides20 TFiLNOa, TFiLNOb (Supplementary Figure S2.2), and the proposed

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new structure of N5330 oligosaccharide.

X2a/Z3 (m/z 815.2878) and

X2a/Y3 (m/z 965.3423) illustrating the

is important in the structural assignment of

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Figure 2. Negative-ion FT-IT MS2 fragmentation pattern of trifucosyl iso-lacto-N-octaose.

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Annotation of deprotonated singly [M-H]- and doubly [M-2H]-2 charged states molecular ions

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enabled to distinguish isomeric structures. Annotation number: N5330 denotes N: Neutral, 5

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Hexose; 3 Fucose; 3 GlcNAc; 0 Neu5Ac. C3/Y5 means glycosidic-glycosidic linkage;

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means cross-ring - glycosidic linkage.

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2,4

X2a/Z3

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Sialylated Milk Oligosaccharides. The HILIC elution profile of sialylated

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oligosaccharides is shown in Figure 1B. The 3-SL having a terminal Neu5Ac(α2-3) linked to a

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lactose unit elutes before 6-SL with a Neu5Ac(α2-6) linkage. It was observed that LSTa having

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a terminal Neu5Ac(α2-3) linked to a lactose unit elutes before LSTb with Neu5Ac(α2-

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6)GlcNAc and LSTc with a Neu5Ac(α2-6) linkage, respectively.

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Moreover, unknown precursor ions m/z 1162.436 of oligosaccharides were identified as

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sialyl-lacto-N-fucopentaose (S-LNFP) isomers eluted at 26.5 min to 28.8 min (Figures 3A &

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3B). S-LNFP I and S-LNFP II were previously identified in human milk21. Note that fragment

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ions of ammoniated precursor are protonated due to loss of ammonia22. As expected, S-LNFP

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isomer exhibit prominent B and Y type ions in positive mode detection. Diagnostic ions such as

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m/z 495.1797 (S-LNFP I) and m/z 454.1570 (S-LNFP II) characterize the linkages Neu5Ac(α2-

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6) and Neu5Ac(α2-3), respectively. The m/z 495.1797 ion is evidence that the Neu5Ac residue

281

links to GlcNAc residue as previously observed23. Product ions of m/z 454.1570 and m/z

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495.1797 are not present in A3111b as observed with the product ions of LSTc indicating that

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the Neu5Ac residue is α2-6 linked to a terminal galactose. These observations suggest that S-

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LNFP I, S-LNFP II (Table 2) elute from HILIC prior to the proposed isomeric structure A3111b.

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The latter oligosaccharide was reported to be the conjugate glycan of glycoprotein or glycolipid

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group belonging to the sialyltransferase gene family24, but has not been reported previously as

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free oligosaccharide in human milk.

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Figure 3. Identification and annotation of sialylated lacto-N-pentaose isomers with precursor

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ions of m/z 1162.436. (A) S-LNFP I and previously unreported (B) S-LNFP (A3111b). B3/Y3a

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means glycosidic-glycosidic linkage.

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MS Library Aided Identification of Milk Oligosaccharides

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MS library searches could produce high scores when library and the unknown spectra

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have similar or different precursor m/z values (DeltaMass). For mass spectral matching, HILIC-

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MS2 data of milk oligosaccharides in SRM 1953 were processed and clustered into high mass

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accuracy HCD/FT-IT MS2 of unknown consensus spectra using a nearest-neighbor clustering

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algorithm14 with the following constraints: first, two spectra cannot belong to the same cluster if

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they have different charges, NCE, or library ID. Second, differences in precursor m/z values and

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retention time have a threshold of 20 × 10-6 mg/Kg and ± 0.3 min, respectively.

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Unknown spectra were then matched by direct and hybrid search against the NIST 17

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Tandem MS Library using an automated search software NIST Mass Spectral Search Program

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(version v2.3). The program uses a modified vector dot product to calculate a match factor 9-10, 25-

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26

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direct search matched oligosaccharides in the NIST 17 library spectra with values ranging from

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811 to 965 with a median of 909. This strategy is complementary to the previously reported

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chromatography-retention time8, 27 based experiments.

that ranges from 0 (no peaks in common) to 999 (identical spectra). As shown in Table I, the

310 311

Direct Identification. Oligosaccharides that matched compounds in the NIST 17 library

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were tentatively identified using the conventional direct search, where both the precursor m/z,

313

charge and spectrum must match. A good consensus spectrum match typically had a match factor

314

(MF) score > 800. Reverse match factors (RMF) treat peaks not in the library as possible

315

contaminants and yield high scores. Since different isomers may have indistinguishable spectra,

316

it is essential to use other factors to assign isomeric structures. Table 1 shows fifteen milk

317

oligosaccharides with various precursor ions and normalized collision energies that matched

318

NIST 17 library entries with MF ranging from 850 to 992 while the reverse match factor (RMF)

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varies from 871 to 995. Neutral and sialylated oligosaccharides present in the sample such as

320

lacto-N-tetraose (LNT), lacto-N-fucopentaose (LNFP) and sialyllactose (SL) produced MF most

321

often above 900.

322 323 324 325 326

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Table 1. MS/MS Direct Search of the consensus MS2 spectra in milk SRM 1953 against NIST

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17 Tandem MS Library for glycans. Name

RT

Theoretical m/z

Experimental m/z

Neutral Oligosaccharides 2’Fucosyllactose, 2’FL 13.17 511.1633 511.1624 Lex-X Trisaccharide, Le-X 13.46 552.1893 552.1899 Le-A Trisaccharide, Le-A 25.11 512.1974 512.1980 Lacto-N-tetraose, LNT 18.79 730.2376 730.2380 Lacto-N-neotetraose, LNnT 19.18 730.2376 730.2399 Lacto-N-fucotetraose I, LNFP I 21.77 446.6384 446.6376 Lacto-N-fucotetraose III, LNFP III 22.75 876.2955 876.2956 Difucolacto-N-hexaose c, DFLNHc 30.21 1387.4856 1387.4862 Difucoparalacto-N-hexaose II, DFpLNH 32.43 1387.4856 1387.4856 II Trifucoparalacto-N-hexaose, TFpLNH 34.92 1533.5435 1533.5435 Acidic Oligosaccharides 3’Sialyllactose, 3’SL 17.01 656.2009 656.2033 6’Sialyllactose, 6’SL 18.20 656.2009 656.1980 23.70 657.2349 657.2360 3’-α-Sialyl-N-acetyllactosamine, 3’SLN 25.47 657.2349 657.2354 6’-α-Sialyl-N-acetyllactosamine, 6’SLN Sialyllacto-N-tetraose b, LSTb 24.55 1021.3330 1021.3399 *FT-ITMS = 35% a Normalized Collision Energy (NCE) = 10 eV to 50 eV b Match Factor (MF) Score c Reverse Match Factor (RMF) Score (non-matching peaks in query spectra are ignored)

329

Hybrid

Search

Identification.

MS

b

c

Precursor Type

Collision Energy a (NCE)

MF Score

[M+Na]+ [M+Na]+ [M +H-H2O]+ [M+Na]+ [M+Na]+ [M+H+K]2+ [M+Na]+ [M+Na]+ [M+Na]+

25 * 20 30 30 15 40 * *

965 811 987 909 945 933 942 850 927

988 914 993 909 978 980 870 882 957

[M+Na]+

*

835

891

20 20 15 15 40

899 900 900 992 857

994 919 919 995 871

requires

an

+

[M+Na] [M+Na]+ [M+H-H2O]+ [M+Na]+ [M+Na]+

reference

library

RMF Score

in-depth

330

characterization of available data, especially when the experimentally acquired spectra produce

331

match factor scores below 800, indicating lack of identity with available MS libraries. The

332

hybrid search method can assist in the identification of several varieties of oligosaccharides. The

333

m/z difference between the consensus spectrum and mass library spectrum (DeltaMass) was

334

previously described for hybrid search identification of peptides10 and fentanyl-related

335

compounds26. We now described for the first time the extension of this method in the

336

identification of reduced oligosaccharides or oligosaccharides differ with a single or multiple

337

sugar units.

338

One example is its ability to link reduced to non-reduced spectra, by shifting peaks

339

containing the reduced group by two Da. The reduction is often used in carbohydrate analyses to

340

simplify oligosaccharide chromatographic analysis27. This is illustrated in Figure 4A where one

16


Page 17 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


341

unknown spectrum (m/z 878.312) was searched against the NIST 17 MS library, finding the non-

342

reduced glycan LNFP III. As expected, the calculated mass difference is due to the DeltaMass

343

between the m/z values of the Y-type fragment ions of the non-reduced versus reduced

344

oligosaccharides. With the direct search, the simple match factor (sMF) score is 309 (relative to a

345

maximum of 999); however, after shifting the Y-type fragment ions (gray line) by two Da (pink

346

line) produced a hybrid match factor (hMF) of 826 (Fig. 4B).

347

Another example is the ability of the hybrid search to link glycans differing by a single

348

sugar unit to confirm the direct MS search of LNT. This is illustrated in Figures 4C, 4D and

349

Supplementary Fig. S2.9, where the unknown spectrum (m/z 730.2378) matches with the library

350

spectrum of 3α,4β,3α galactotetraose with a DeltaMass of 41.027 Da. This is consistent with the

351

unknown compound containing a GlcNAc sugar unit instead of the Gal residue in the library

352

compound. This is demonstrated in Fig. 1D, where fragment ions m/z 509.1470, m/z 527.1575

353

and m/z 671.1996 are shifted by 41.027 Da to m/z 388.1214 (B2), m/z 406.1317 (C2), m/z

354

550.1741 (B3), m/z 568.1842 (Y3) and m/z 712.2263. Note that LNT is one of the most abundant

355

neutral and non-fucolactosylated milk oligosaccharides in the sample, illustrating how the hybrid

356

search strategy aids the identification of unknown spectra. This strategy may be useful in the

357

identification of permethylated and other derivatized oligosaccharides.

358

17



359 360

Figure 4. Hybrid MS Library Search identifications. Precursor m/z values of adduct ion

361

[M+Na]+ (A) m/z 878.3116 (reduced); (B) LNFP III (m/z 876.2963); (C) m/z 730.2378; (D)

362

3α,4β,3α-Galactotetraose (m/z 689.2111). The head-to-tail plot shows the spectral matching of

363

product ions of the unknown (red) against the known ions (blue) in NIST Tandem MS Library

364

2017. Shifted peak (gray line), inserted/predicted peak (pink line). Original match factor score

365

(sMF) and hybrid match factor (hMF). DeltaMass is the difference of precursor m/z values

366

between the unknown consensus spectrum and the NIST 17 library spectrum.

367 368

MS Library of Annotated Oligosaccharides in NIST Human Milk Reference Material

369

Raw HILIC MS/MS data of 196 runs were processed, clustered to create consensus

370

spectral files, and searched using the hybrid search and Tandem MS Library11 as described

371

above to create a reference material-based library.

372

Table 2 displays the list of 74 oligosaccharides that were identified and elucidated in

373

human milk sample, of which 45 are neutral and 29 are sialylated oligosaccharides. The MS

374

library of identified and annotated oligosaccharides has different adduct ions and normalized

375

collision energies using HCD and FT-IT fragmentation techniques. Among the precursor and

376

product ions, positive adduct ions of [M+NH4]+ and [M+H]+ were abundant. Precursor ions [M-

18


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Page 19 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


377

H]-, and [M-H+H2CO2]- are the common adduct ions observed in negative detection mode.

378

Annotation of the negative HCD/FT-IT MS2 spectra allows the distinction of various fucosyl

379

(α1-2, α1-3/α1-4) glycosidic linkages and cross ring cleavages (A-type ions) present in the

380

oligosaccharide structure.

381

The hybrid search technique enabled identification of reduced known oligosaccharides and 12

382

previously unreported free oligosaccharides in human milk. The high mass accuracy and high

383

signal resolution of the HCD/FT-IT spectra confirmed most of the oligosaccharides using both

384

positive and negative detection. Extensive analysis of the FT-IT spectra enabled resolution and

385

annotation of about 30% of the precursor ions in the higher mass region (< m/z 2000) using the

386

dual charge state fragmentation strategy.

387

So far, the remaining partially identified oligosaccharides at longer retention time (11-12

388

sugar units) require additional analysis for identification because of the ambiguity in structural

389

assignment of terminal fucose, galactose, and sialic acid linkages (Supporting Fig. 2S.12).

390 391 392 393 394 395 396 397 398 399

19



Page 20 of 28

400 401 402 403 404 405 406 407 408

Table 2. Annotation of peaks in Figure 2 derived from SRM 1953 human milk sample by HILIC-MS/MS using HCD and FT-IT fragmentation techniques. Name

Proposed Structureǂ

RT (min)

Precursor Type +

Neutral Oligosaccharides 2’-FL

12.91 13.99

a, b, c, d b

12.26

a, c

21

3’-FL

Collision Energy # NCE

Name

Precursor Type

f, g, h, i g

+ 20-50, *

Acidic Oligosaccharides 3’-SL

25, *

6’-SL

*

3’-S-3-FL

21

H-Tri

RT (min)

Proposed Structureǂ

15.82

21

-

19.96

a, b, c a, b, c b, c

23.71

l

10-50, *

25.47

l

10-40, *

23.92

a, b

24.73

a, b, c

25.23

a, b, c

f

20-25, *

26.10

a, b

f

*

26.10

c

26.55

a, b

f

10-25, *

f

*

17.18

21

Collision Energy # NCE

10-40, * 15-50, * f, g

10-50, *

28

11

Le-X Tri

13.80

a, c

10, 40, *

3’-SLN

22.95

l

20

6’-SLN

15.61

b, c

10-50, *

LSTa

16.04

b, c

10-50, *

LSTb

18.75

b, c

f, g, I, j

10-50, *

LSTc

19.16

a, c

f, g

10-50, *

S-LNFP I

19.29

a, c

10, *

F-LSTc

29

Le-A Tri

8

23

30

LDFT

g, i

f

10-50, *

21 21

3’GalL

31

10-50, *

21

LNT 21

LNnT

21

21

21

Le B tetra

10-20, *

32

30

LNFP I

21.77

e

f, g

10-30, *

S-LNFP II 21

21

LNFP V

22.09

c

f, g

10-40, *

A3111a

28.30

a, b

22.71

a, b, c

f, g

10-50, *

A3111b

28.45

c

22.99

a, b, c

f, g

10-50, *

DSLNT

29.59

a, b, e

21

LNFP III

10-40, *

21

LNFP II 21

21

20


f

10-20, *

Page 21 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


N4010a

23.46

a, b, c

f

10-30, *

S-LNnH II

30.00

a, b

f

10-25, *

31.40

e

f, g

*

f

10-40, *

32

N4010b

24.00

a, b

f

10, *

S-LNH

LNDFH I

26.10

a, b, c

g

10-30, *

A4121a

31.48

a

26.60

c

g, h

*

A4121b

32.30

a, b

26.10

a, b

f, g

10-30, *

FS-LNH

33.33

f, g

20, *

DSLNH

32

21

LNDFH II

10-20, *

21

LNH

f

20, *

21 21

LNnH

26.65

33.40

a, b

*

21 21

IFLNH I

27.85

a

*

FS-LNH I

f

*

32

32

MFLNH I

33.72

28.11

b

f

10-25, *

A4121c

33.86

a

*

28.59

a, b, e

f, g, j

10-30, *

MSDFLNH

34.73

c

f, g

10-25, *

29.54

a, b

f

10-15, *

MSDFLNnH

34.95

c

f, g

15, 30, *

29.71

a, b

f

10-20, *

A4221a

35.03

a, b

g

20, *

31.20

b, e

f, g

10-20, *

A4221b

35.30

a, b

g

20, *

32.09

b

g

10, 15

DS-FLNH II

34.65

j

*

32.29

a, b, c

g

10-30, *

A4122a

35.42

a, b

32.53

a, b, c

f, g

15-30, *

DS-FLNH I

35.77

e

32.65

c

34.07

a, b, c

21

MFLNH III 21

32

MFpLNH IV

23

32

IFLNH III 32

DFLNH a

21

DFLNH b 21

DFLNH c

*

32

DFpLNH II 21

21

DFpLNnH

*

21

TFLNH

k

20-40, *

21

21


j

20-25, *


TFLNnH

34.77

a, b

g

20, 25, *

TFpLNH

34.98

a, b

g

*

J

15-30, *

Page 22 of 28

13

5130c

33.60 27

FLNO

34.32

a, b, c

f, g

10-30, *

34.66

a, b

g, j

*

34.97

a, b

f, k

*

35.95

a

f, k

*

36.20

a, e

k

*

35.63

e

36.69

a, b, e

f, k

*

37.05

a, b, e

f, k

*

37.26

f, j

*

38.56

f, j

*

39.03

f, j

*

32

FLNnO 32

N5230a

32

DFLNO II 32

DFLNnO II 32

LND

*

33

DFLNO I 32

DFLNnO I 32

TFiLNO a 20

N5330

TFiLNO b 20

409 410 411

Annotation code A3111 denotes N=Neutral, A=Acidic, 3 Hexoses, 1 Fucose, 1 GlcNAc, 1 Neu5Ac Positive mode: a [M+H]+ b[M+NH4]+ c[M+Na]+ d[2M+Na]+ e[M+2NH4]2+ / [M+H+K]2+ Neutral loss l[M-H2O-H]+ Negative mode: f [M-H]- g[M+H2CO2 -H]- h[M+HC2F3O2 -H]- i[2M-H]j [M-2H]2- k[M+2H2CO2 -2H]2*IT-FTMS = 35% # Normalized Collision Energy (NCE) = 10, 15, 20, 25, 30, 40, 50 eV ǂ Reference

Linkages α 1-6/α 1-2 β 1-4 β1-3/β1-6 α 2-3/α 2-6 6 4 3

2

Searching unknown spectra using the MS library of annotated milk oligosaccharides

412

The MS library of oligosaccharides in this study derived from human milk SRM 1953 is

413

available online34 along with search software and can be readily applied to other bovine milk

414

samples and biological fluids (Supplementary Figures S2.6 & S2.7). The MS library consists of

415

469 positive and negative ion spectra having 45 neutral and 29 acidic oligosaccharides. All

22


Page 23 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


416

fragments ions of MS2 spectra are comprehensively annotated. Figure 5 illustrates the library

417

search of unknown spectrum against the mass spectral data base of milk oligosaccharides. The

418

head to tail comparison between unknown spectrum and LNFP I show the similarity of fragment

419

ions in terms of peak intensity and their m/z values as interpreted by the MF score of 938.

420 421

Figure 5. Overview of NIST MS Search interface illustrating the data information and

422

comparison of the MS2 spectra between the unknown (top) and the annotated peaks of LNFP I

423

(bottom). The head-to-tail comparison of the unknown spectrum (cluster 018619) and LNFP I

424

(middle). Match factor (MF) score = 938, Reverse MF score = 978.

425 426

Conclusions

427

Human milk reference material SRM 1953 containing neutral and acidic oligosaccharides

428

was analyzed on HILIC coupled to electrospray ionization with an Orbitrap-based mass

429

spectrometer using HCD and FT-IT fragmentation techniques. Consensus library spectra of MS2

430

ions were generated from the raw HILIC-MS data. The NIST Tandem MS Library with its new

431

hybrid search algorithm facilitated the identification of unknown spectra of both non-reduced

432

and reduced oligosaccharides. This strategy enabled spectral matching between the consensus

433

and library spectra despite shifts in m/z values. Moreover, the high mass accuracy both for

23



434

precursor and product ions was sufficient to enable the assignment of 74 oligosaccharides

435

including novel glycan structures. A clear distinction could be made in the elution pattern of

436

isomeric fucosylated and sialylated oligosaccharides using previously established elution orders

437

related to size and polarity. The reduction of oligosaccharides by sodium borohydride increased

438

product chromatographic retention times and peak quality resulting to an addition of two

439

hydrogen atoms to the precursor ions. The hybrid search can reliably find reduced

440

oligosaccharides in comparison to non-reduced analogs, and vice versa, enabling an increase in

441

match factors. The SRM 1953 milk oligosaccharide MS library has been demonstrated to aid in

442

identifying oligosaccharides in other biological or milk samples.

443

Supporting information.

444

Supporting Information S1.

445 446

Milk SRM 1953 Glycan MS Library https://chemdata.nist.gov/srmd-1953-glycan/spectra Supporting Information S2.

447

Detailed methods on sample preparation and processing of spectra

448

Structural assignment of neutral and acidic oligosaccharides

449

Supplementary Tables

450

Table S1. List of commercially available oligosaccharides in the study

451

Table S2. Diagnostic ions used for the identification and annotation of the consensus

452

spectrum in the MS library of unidentified spectra.

453

Acknowledgements

454

We thank Dr. Sanford Markey for detailed discussion. We also thank Drs. Dmitrii

24


Page 24 of 28

Page 25 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


455

Tchekhovskoi, Arun Moorthy, Meghan Burke and Yuxue Liang for fruitful discussions and

456

technical support on NIST MS Search software, hybrid search method and LC-MS instrument

457

settings. Finally, we thank Joshua Klein for his contribution to the fragment ion annotation using

458

glypy Python package.

459

Disclaimer

460

The commercial instruments and materials are used for the experimental part the study.

461

Such identification does not intend recommendation or endorsement by the National Institute of

462

Standards and Technology, nor does it intend that the materials or instruments used are

463

necessarily the best available for the purpose. The authors declare no competing financial

464

interest.

465

References

466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485

1. Claps, S.; Di Napoli, M. A.; Sepe, L.; Caputo, A. R.; Rufrano, D.; Di Trana, A.; Annicchiarico, G.; Fedele, V., Sialyloligosaccharides content in colostrum and milk of two goat breeds. Small Ruminant Research 2014, 121 (1), 116-119. 2. Donovan, S. G., Glenn; Newburg, David, Prebiotics in Infant Nutrition. In Mead Johnson Nutrition, United States of America, 2009. 3. Manz, C.; Pagel, K., Glycan analysis by ion mobility-mass spectrometry and gas-phase spectroscopy. Current Opinion in Chemical Biology 2018, 42, 16-24. 4. Difilippo, E.; Willems, H. A. M.; Vendrig, J. C.; Fink-Gremmels, J.; Gruppen, H.; Schols, H. A., Comparison of Milk Oligosaccharides Pattern in Colostrum of Different Horse Breeds. Journal of Agricultural and Food Chemistry 2015, 63 (19), 4805-4814. 5. Zhou, S.; Huang, Y.; Dong, X.; Peng, W.; Veillon, L.; Kitagawa, D. A. S.; Aquino, A. J. A.; Mechref, Y., Isomeric Separation of Permethylated Glycans by Porous Graphitic Carbon (PGC)-LC-MS/MS at High Temperatures. Analytical Chemistry 2017, 89 (12), 6590-6597. 6. Remoroza, C.; Cord-Landwehr, S.; Leijdekkers, A. G. M.; Moerschbacher, B. M.; Schols, H. A.; Gruppen, H., Combined HILIC-ELSD/ESI-MSn enables the separation, identification and quantification of sugar beet pectin derived oligomers. Carbohydrate Polymers 2012, 90 (1), 41-48. 7. Leijdekkers, A. G.; Huang, J.-H.; Bakx, E. J.; Gruppen, H.; Schols, H. A., Identification of novel isomeric pectic oligosaccharides using hydrophilic interaction chromatography coupled to traveling-wave ion mobility mass spectrometry. Carbohydrate research 2015, 404, 1-8.

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1 Title: Creating a Mass Spectral Reference Library for

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