molecules

Article
Comparison of Different HILIC Stationary Phases in
the Separation of Hemopexin and Immunoglobulin G
Glycopeptides and Their Isomers
Katarina Molnarova and Petr Kozlík *
Department of Analytical Chemistry, Faculty of Science, Charles University,
Hlavova 8, 128-43 Prague, Czech Republic; katarina.molnarova@natur.cuni.cz
* Correspondence: petr.kozlik@natur.cuni.cz

Academic Editors: Patrick M. Martin and Sandrine Bouquillon




Received: 21 September 2020; Accepted: 10 October 2020; Published: 13 October 2020

Abstract: Protein glycosylation analysis is challenging due to the structural variety of complex
conjugates. However, chromatographically separating glycans attached to tryptic peptides enables
their site-specific characterization. For this purpose, we have shown the importance of selecting a
suitable hydrophilic interaction liquid chromatography (HILIC) stationary phase in the separation of
glycopeptides and their isomers. Three different HILIC stationary phases, i.e., HALO® penta-HILIC,
Glycan ethylene bridged hybrid (BEH) Amide, and ZIC-HILIC, were compared in the separation
of complex N-glycopeptides of hemopexin and Immunoglobulin G glycoproteins. The retention
time increased with the polarity of the glycans attached to the same peptide backbone in all HILIC
columns tested in this study, except for the ZIC-HILIC column when adding sialic acid to the glycan
moiety, which caused electrostatic repulsion with the negatively charged sulfobetaine functional
group, thereby decreasing retention. The HALO® penta-HILIC column provided the best separation
results, and the ZIC-HILIC column the worst. Moreover, we showed the potential of these HILIC
columns for the isomeric separation of fucosylated and sialylated glycoforms. Therefore, HILIC is a
useful tool for the comprehensive characterization of glycoproteins and their isomers.

Keywords: glycoproteomics; glycopeptides; separation of glycopeptide isomers; glycopeptide

separation; hydrophilic interaction liquid chromatography

1. Introduction
Glycosylation is one of the most important and extensive post-translation protein modifications [1].
Protein glycosylation includes a complex series of enzymatic steps that result in the formation of
protein-bound oligosaccharides with various biological functions [1–3]. Alterations in glycosylation
patterns affect many biological processes, such as protein folding, intracellular sorting, secretion,
and host-microbial recognition [4,5]. In addition, abnormal protein glycosylation has been used as a
diagnostic tool for many cancers, including breast, prostate, and ovarian cancer [6–9]. Yet, despite
recent bioanalytical advances in glycomics, glycoprotein analysis remains a challenge.
The most commonly used method for glycoprotein analysis is reversed-phase liquid
chromatography (RP-LC) with tandem mass spectrometry (MS/MS). However, RP-LC is unable
to adequately resolve glycopeptides into their glycoforms [10–12]. An alternative chromatographic
mode to RP-LC in glycopeptide analysis is to use porous graphitized carbon (PGC). PGC has
been successfully applied in the isomeric separation of different N-glycopeptides [13,14]. Another
chromatographic option for glycopeptide separation is hydrophilic interaction liquid chromatography
(HILIC) [15]. HILIC is a powerful LC mode for the separation of polar analytes insufficiently retained
in RP-LC [10,16]. Moreover, HILIC is also attractive in glycopeptides enrichment [17,18], and HILIC

Molecules 2020, 25, 4655; doi:10.3390/molecules25204655 www.mdpi.com/journal/molecules

Molecules 2020, 25, 4655 2 of 13

has been applied in the separation of the isomeric glycoforms differing only in linkage position
and/or branching [15,19–21].
Changes in glycopeptide isomeric structures can also be used as potential biomarkers [9,22].
As such, Huang et al. have used HILIC to separate sialylated N-glycan isomers of fetuin differing
only in α2–3 and α2–6 linkages [19]. Kozlik et al. separated core- and outer-arm-linked fucose
into bi-antennary and tri-antennary glycoforms and the antennary position of sialic acid linked via
α2–6 linkage of the hemopexin glycopeptides by HILIC [16].
In recent years, various commercial HILIC phases have been described with surface chemistries
far more diverse than RP and which can provide substantially different selectivities. Among the
most commonly and successfully used HILIC columns in glycopeptide separations, amide [23,24],
ZIC-HILIC [25,26], and penta-HILIC [10,27] stand out for their resolution and versatility. For example,
Gilar et al. successfully applied an amide column to the glyco-profiling of a therapeutic monoclonal
antibody and proteins with several N-linked and O-linked glycosylation sites [28]. Although
ZIC-HILIC columns are used for glycopeptide enrichment rather than for their separation [29–31],
Hernandez-Hernandez et al. has used a ZIC-HILIC column to separate sialylated trisaccharide isomers
of bovine caseinomacropeptide according to the sialic acid attachment site [32]. The Penta-HILIC
column showed good separation potential for the analysis of hemopexin glycopeptides [16] and for
the analysis of bovine fetuin glycopeptides and human IgG glycopeptides [19].
In this study, we investigated and compared the separation potential of three commercially
available HILIC columns, i.e., HALO® penta-HILIC, Glycan BEH Amide, and a zwitterionic type
ZIC-HILIC column. The penta-HILIC uses proprietary bonding chemistry that includes five hydroxyl
groups on the bonded ligand. The ZIC-HILIC column contains a zwitterionic sulfoalkylbetaine
stationary phase covalently attached to porous silica. The amide column is based on the ethylene
bridged hybrid (BEH) particle technology with trifunctionally-bonded amide phase. For comparison
purposes, we studied glycopeptides prepared by trypsin digestion of well-characterized glycoproteins,
i.e., human hemopexin and Immunoglobulin G. The findings of this study may therefore help to
optimize the HILIC method for glycopeptide analysis.

2. Results and Discussion

In this study, we compared the separation potential of three different HILIC stationary phases in the
analysis of complex N-glycopeptides of hemopexin and IgG glycoproteins. For this purpose, we selected
the most intense tryptic glycopeptides of the aforementioned glycoproteins. All studied glycopeptides
are outlined in Table 1. In this article, we use the following abbreviations for N-glycans: A2G2 for a
bi-antennary glycan with two terminal galactoses, A3G3 for a tri-antennary glycan terminated with
three galactoses, S for sialic acid, and F for fucose. For the chromatograms shown in the figures, we
exported extracted-ion chromatograms (EIC) to OriginPro 8.5.0 (OriginLab Corporation, Northampton,
MA, USA), normalized them to specific glycoform, and overlaid them. First, we optimized the gradient
program to enhance, as much as possible, the resolution of the studied glycopeptides. For HALO®
penta-HILIC and ACQUITY UPLC Glycan BEH Amide, we used the same gradient program (see
Instrumentation and experimental conditions). However, in the ZIC-HILIC column, a slower gradient
program was applied to obtain satisfactory peak shapes and adequate retention and resolution.
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A2G2
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187 A2G2
A2G2
A2G2 A2G2F1
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A2G2S1
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A2G2S2
A2G2S2 A3G3S1
A3G3S1
A3G3S1
SWPAVGN
SWPAVGNSWPAVGN
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SWPAVGN
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453
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A2G2S2 A3G3S1
A3G3S1
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A3G3S1
453
A2G2 A2G2F1
A2G2F1 A2G2S1 A2G2S2 A3G3S1
ALPQPQN
ALPQPQN
ALPQPQN 453 VTSLLGCTH
VTSLLGCTH
453453
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VTSLLGCTH
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EEQYN
EEQYN IgG180
180STYR
STYR A2G2 A2G2
A2G2
A2G2 A2G2
A2G2 A2G2 A2G2F1
A2G2 A2G2F1
A2G2F1
A2G2F1 A2G2F1 A2G2S1
A2G2F1
A2G2F1
A2G2F1 A2G2S1
A2G2S1
A2G2S1 A2G2S1 A2G2S2
A2G2S1
A2G2S1
A2G2S1
A2G2S1 A2G2S2
A2G2S2
A2G2S2A2G2S2
A2G2S2
A2G2S2
A2G2S2
IgG
IgG
IgG A2G2 A2G2F1 A2G2S2
(IgG1)
(IgG1) 180
EEQYN
EEQYN
EEQYNIgG 180
IgG
IgG IgG
IgG
180
STYR
180
IgG STYR
STYR
IgG
IgG A2F1
A2F1 A2G1F
A2G1F G1A3F1
G1A3F1 A2G2F1
A2G2F1
EEQYN 180 IgG
STYR (IgG1)
EEQYN
EEQYN
EEQYN (IgG1)
(IgG1)
(IgG1) 180 180
180STYR
180STYR
STYR
A2F1
A2F1A2F1
A2F1 A2G1F
A2G1F
A2G1F G1A3F1
G1A3F1
G1A3F1
G1A3F1 A2G2F1
A2G2F1
A2G2F1
EEQYN
EEQYN
EEQYN
EEQYN (IgG1)
(IgG1)
(IgG1)
180 180180
STYR STYR
180 STYR
STYR
180
EEQYN
EEQYN
EEQFN
EEQFN EEQYN
EEQYN
EEQYN 180
176
176
180
180
STFR
STFR
180
STYR
STYR
180 STYR
STYR
STYR A2F1
A2F1
A2F1 A2G1F
A2G1F
A2G1F G1A3F1
G1A3F1
G1A3F1 A2G2F1
A2G2F1
A2G2F1
EEQFN (IgG1)
(IgG1) (IgG1)
(IgG1)
176
(IgG1) (IgG1)
(IgG1)
(IgG1)
STFR
(IgG1) (IgG2)
(IgG2)
(IgG2) A2F1 A2F1
A2F1
A2F1 A2F1 A2G1F
A2F1 A2G1F
A2G1F
A2G1F A2G1F G1A3F1
A2G1F G1A3F1
G1A3F1
G1A3F1 G1A3F1 A2G2F1
G1A3F1 A2G2F1
A2G2F1
A2G2F1A2G2F1
A2G2F1
EEQFN
EEQFN
EEQFN 176176 176
176
STFR STFR
STFR A2F1
A2G1F
A2G1F A2F1
A2F1 A2G1F
A2G1F
A2G1F
A2G2F1
A2G2F1
A2G2F1 G1A3F1
G1A3F1
G1A3F1 A2G2F1
A2G2F1
A2G2F1
EEQFN
EEQFN
EEQFN
A2G2 (IgG2)
(IgG2)
(IgG2)
=
176 176
176STFR
STFR
176 STFR glycan with two terminal galactoses; A3G3 = tri-antennary glycan terminated with three
bi-antennary
A2G2 =176 bi-antennary A2G1FA2G1F
A2G1F A2G2F1
A2G2F1
A2G2F1
EEQFN
A2G2
EEQFN
EEQFN (IgG2)
(IgG2)
galactoses;
EEQFN (IgG2)
176STFR
=176SSTFR
176 STFR
STFR
176 sialic acid; glycan
bi-antennary
=176 Fglycan
= fucose. with
withtwo twoterminal terminal galactoses;
galactoses;A3G3 A3G3==tri-antennary
tri-antennaryglycanglycanterminated
terminated
EEQFN EEQFN
EEQFN
EEQFN
EEQFN 176 176
STFR
176 176
STFRSTFR
STFR
STFR A2G1F
A2G1F
A2G1F A2G2F1
A2G2F1
A2G2F1
with
withthree threegalactoses;
galactoses;SS==sialic sialicacid;
acid;FF==fucose. fucose.
A2G2A2G2
A2G2 (IgG2)
(IgG2)=(IgG2)
(IgG2)
(IgG2)=(IgG2)
(IgG2) bi-antennary
=bi-antennary
bi-antennary
(IgG2)
(IgG2) glycan
glycan
glycan with
with
with two two
two terminal
terminal
terminal galactoses;
galactoses;
galactoses; A3G3A3G3
A3G3 = tri-antennary
tri-antennary
= =tri-antennary glycan
glycan
glycan terminated
terminated
terminated
2.1. with Separation of Glycopeptides A2G1F A2G1F
by A2G1F
A2G1F
A2G1F A2G1F
A2G1F
A2G1F
HILIC
A2G1F A2G2F1
A2G2F1A2G2F1
A2G2F1 A2G2F1
A2G2F1
A2G2F1
A2G2F1
A2G2F1
A2G2
A2G2
A2G2
with
withthree = =
three= bi-antennary
bi-antennary
bi-antennary
threegalactoses;galactoses; glycan
glycan
glycan
galactoses;S S= =sialicS = with
with
with
sialic
sialicacid; two
acid; two
two F =
acid;F F= fucose. terminal
terminal
terminal
fucose.
= fucose. galactoses;
galactoses;
galactoses; A3G3A3G3
A3G3 == = tri-antennary
tri-antennary
tri-antennary glycan
glycan
glycan terminated
terminated
terminated
2.1.
2.1.Separation
SeparationofofGlycopeptides Glycopeptidesby byHILIC
HILIC
with
with
with
A2G2
A2G2 =three
three
=three galactoses;
galactoses;
galactoses;
===bi-antennary
bi-antennary Sglycan
S===glycan
sialic
Sglycan sialic
sialic acid;
acid;
withacid; Fterminal
two FF=terminal
=fucose.
=terminal
fucose.
fucose. galactoses; A3G3 ===tri-antennary
tri-antennary glycan terminated
A2G2A2G2
As A2G2 A2G2
A2G2
previously
A2G2
A2G2 =bi-antennary
bi-antennary =bi-antennary
bi-antennary
==bi-antennary
bi-antennarydescribed,
bi-antennary glycan
glycan glycan glycan
glycan
glycan with
inwith
with two
HILIC,
with two
with
with two
with
with two two
two twoterminal
glycopeptide
two terminal
terminal
terminal galactoses;
galactoses;
galactoses;
terminal
terminal A3G3
galactoses;
galactoses;
retention
galactoses;
galactoses;
galactoses; A3G3
A3G3 A3G3
A3G3A3G3
A3G3 =mainly
=isA3G3 =tri-antennary
tri-antennary
==tri-antennary
driven
tri-antennary glycan
glycan
glycan
=tri-antennary
tri-antennary
by
tri-antennary glycan
the
glycan
glycan terminated
terminated
terminated
glycan terminated
terminated
glycan moiety
terminated
glycan terminated
terminated
2.1.2.1.
2.1. Separation
Separation
Separation
As
As
with
with
with previously
previously
three
three
three of
ofof Glycopeptides
Glycopeptides
Glycopeptides
galactoses;described,
galactoses;
galactoses; described,
S S= =
=S sialic
=the sialicby
by
sialicbyHILIC
HILIC
in
in HILIC
HILIC,
acid;HILIC,
acid; F F = = glycopeptide
fucose.glycopeptide
fucose. retention
retention is
is mainly
mainly driven
driven by
by the
the glycan
glycan
of the with three
with with
with
with three
glycopeptide
with galactoses;
three
three
three
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for
for hemopexin
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column
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not
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and
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column
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the SWPAVGN
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187
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penta-HILIC
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glycoforms,
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as
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and
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inin the
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187 CSSALR
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wereco-eluted
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inchromatographicGlycan
chromatographic
chromatographic was BEH
BEH
observed amide
amide
resolution
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studied
CSSALR
and and
and and
and hemopexin
hemopexin
peptide.
non-fucosylated
non-fucosylated
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and
and and
and non-fucosylated
non-fucosylated
non-fucosylated
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non-fucosylated Inglycopeptides
glycopeptides
addition,
glycoforms,
glycoforms,
glycoforms,
glycoforms, Table
glycoforms,
glycoforms,
glycoforms,
glycoforms,
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as was
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as
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(Supplementary
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summarizes
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resolution)
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all
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all VTSLLGCTH
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hemopexin peptide
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and A2G2F1
glycopeptidesA2G2F1 of of
was
wasboth
both peptides;
observedpeptides;
observed inin A2G2S2
A2G2S2
thethe HALO
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and® ®A3G3S1 ®®A3G3S1
penta-HILIC
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SWPAVGN
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and
187
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glycopeptides
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CSSALR
CSSALR
CSSALR
187
187 retention
CSSALR
CSSALR
187
187 187
187 CSSALR
CSSALR
CSSALR
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were
peptide.
peptide. were
were
times
peptide.
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peptide. The co-eluted
co-eluted
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of TheThe
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the
glycopeptides Glycan
Glycan
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chromatographic
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chromatographic BEH
in
chromatographic
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BEH
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amide
studied
resolution
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resolution the
column,
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resolution HILIC
resolution
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(at
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almost
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A2G2S1
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baseline
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of
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column.
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resolution)
resolution)
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resolution)
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ofof
resolution) ofof453453
all
ofofall
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glycopeptides
453
all
allglycopeptides
VTSLLGCTH
VTSLLGCTH
453
all VTSLLGCTH
allstudied
allstudied
studied
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of all
studied studied
studied
studied were
were
were
hemopexin
hemopexin
hemopexin peptide
peptide
hemopexin peptide
hemopexin
hemopexin
hemopexin
hemopexin co-eluted
co-eluted
co-eluted
and and
and A2G2F1inin
glycopeptides
glycopeptides
glycopeptides inthe
A2G2F1
A2G2F1
glycopeptides
glycopeptides
glycopeptides
glycopeptides
glycopeptides theGlycan
the Glycan
ofGlycan
of
was ofboth
was
was both
both
was
waswas BEH
BEH
BEH
observed
was
was observed
observed
observed
observed amide
amide
peptides;
peptides;
peptides; amide
observed
observed
observed inin incolumn,
column,
in
the
in column,
A2G2S2
A2G2S2
A2G2S2
inthe
the the
in
the
in HALO
the and
HALO
HALO
the
the namely:
HALO
HALO namely:
namely:
and
and
HALO ® A3G3S1
HALO
HALO A3G3S1
®®A3G3S1
® ®A2G2S1
®A2G2S1
A2G2S1
®penta-HILIC
®penta-HILIC
®®penta-HILIC
penta-HILIC
penta-HILIC ofof of
the
penta-HILIC
penta-HILIC
penta-HILIC ofof
the
the of
resolution) all studied hemopexin glycopeptides was observed in the HALO penta-HILIC
the
the
the
column.
column.
column.
column.
column.ALPQPQN
ALPQPQN
ALPQPQN Several
Several
column.
column.
column. Several
Several
Several
453 453
453
453 VTSLLGCTH
VTSLLGCTH
VTSLLGCTH
glycopeptides
glycopeptides
glycopeptides
glycopeptides
Several
Several
Several glycopeptides
glycopeptides
glycopeptides
glycopeptides were were
were peptide
peptide
peptide
were
werewere
were co-eluted
co-eluted
co-eluted
were and
co-eluted and
and
co-eluted
co-eluted
co-eluted inin
co-eluted A2G2F1
A2G2F1
A2G2F1
inin
inthe
the the
inthe
in
the
in ofof
Glycan
Glycan
the the
the of
Glycan
Glycan both
both
both
BEH
Glycan
Glycan
Glycan
Glycan BEH
BEH BEH
BEH peptides;
peptides;
peptides;
BEH
BEH amide
amide
amide
amide
BEH amide
amide amide A2G2S2
A2G2S2
A2G2S2
column,
column,
column,
column,
amide column,
column,
column, and and
and
namely:
namely:
namely:
namely:
column, namely:
namely: A3G3S1
A3G3S1
A3G3S1
namely: A2G2S1
A2G2S1
A2G2S1
A2G2S1
namely: A2G2S1 ofof
A2G2S1 of
A2G2S1 the
ofof
A2G2S1 the
theof
of
ofof of of
of
column. Several glycopeptides were co-eluted in the Glycan BEH amide column, namely: A2G2S1
the
the
the
the the ALPQPQN
the
the
the ALPQPQN
ALPQPQN
ALPQPQN
the ALPQPQN
ALPQPQN
ALPQPQN
ALPQPQN
ALPQPQN
453 453
453 453
453VTSLLGCTH
VTSLLGCTH
VTSLLGCTH
VTSLLGCTH
453 453
453 VTSLLGCTH
VTSLLGCTH
VTSLLGCTH
453
453 VTSLLGCTH
VTSLLGCTH peptide
peptide
peptide
peptide peptide
peptide
peptide
peptide andand
peptide and
and and and
and andA2G2F1
A2G2F1
A2G2F1
A2G2F1
andA2G2F1 A2G2F1
A2G2F1
A2G2F1
A2G2F1 ofof
of both of
ofofboth
ofboth
bothof
both
of both
both peptides;
both
both peptides;
peptides;
peptides; peptides;
peptides;
peptides;
peptides;
peptides; A2G2S2
A2G2S2
A2G2S2
A2G2S2 A2G2S2 A2G2S2
A2G2S2
A2G2S2
A2G2S2 and
and
andand and A3G3S1
andand A3G3S1
and
and A3G3S1
A3G3S1
A3G3S1 A3G3S1
A3G3S1
A3G3S1
A3G3S1 of
of theofof the
the
of
ofof the
of
the
of the
the the
the
 Molecules 2020, 25, x FOR PEER REVIEW 4 of 13

SWPAVGN
Molecules 187CSSALR
2020, 25, 4655peptide. In addition, Table S1 (Supplementary Materials) summarizes
4 ofthe
13
intact masses and retention times of these glycopeptides in the studied HILIC columns.

Figure1.1.Separation
Figure Separationofofglycopeptides
glycopeptidesofofhemopexin
hemopexinininHALO
HALO penta-hydrophilic
®® penta- hydrophilicinteraction
interactionliquid
liquid
chromatography (HILIC) (A), Glycan ethylene bridged hybrid (BEH) amide (B), and ZIC-HILIC
chromatography (HILIC) (A), Glycan ethylene bridged hybrid (BEH) amide (B), and ZIC-HILIC (C) (C)
columns. PEP1 refers to SWPAVGN 187CSSALR, and PEP2 to ALPQPQN453
187
columns. PEP1 refers to SWPAVGN CSSALR, and PEP2 to ALPQPQN VTSLLGCTH.
453VTSLLGCTH.

Asmentioned
As mentionedabove,above, wewe chose
chose twotwo occupied
occupied peptides
peptides of theofIgG
the glycoprotein,
IgG glycoprotein,whichwhich only
only differ
differ
by twoby two amino
amino acids acids
in theinpeptide
the peptide sequence
sequence (IgG1:
(IgG1: EEQYN 180
EEQYNSTYR 180 STYRand and IgG2:EEQFN
IgG2: 176
EEQFN STFR).
176STFR).
Figure2 2shows
Figure shows that
that thethe A2G1F1
A2G1F1 glycoform
glycoform of IgG2
of the the IgG2
peptide peptide
had thehad the lowest
lowest retention
retention in each in each
HILIC
HILIC because
column column tyrosine
because(Y)tyrosine (Y) is more
is more hydrophilic hydrophilic
than phenylalanine than(F);phenylalanine (F); thus,with
thus, the glycopeptides the
glycopeptides
this backbone elutewith earlier
this backbone
[19]. Aselute earlier
shown [19]. As
in Figure shown
2A,B, thisinglycoform
Figures 2A and B,
eluted in this
twoglycoform
peaks in
eluted
the HALO ® penta-HILIC
in two peaks in theand HALO ® penta-HILIC and Glycan BEH amide column, most likely due to a
Glycan BEH amide column, most likely due to a partial separation of
partial
the separation
isobaric of theresulting
glycoforms isobaric glycoforms resulting from
from the attachment of thethe attachment
galactose of the
to each ofgalactose to each of
the two antennae
theand
(α3 twoα6),
antennae (α3 anddescribed
as previously α6), as previously
[19]. In thedescribed
ZIC-HILIC [19].column,
In the ZIC-HILIC
this isomeric column, this isomeric
glycoforms eluted
glycoforms eluted as one peak (see Figure 2C). Adding one galactose to
as one peak (see Figure 2C). Adding one galactose to the A2F1 or the A2G1F1 glycoform increased the the A2F1 or the A2G1F1
glycoform
retention increased
time the retention
by approximately timeinby
1 min HALO® penta-HILIC
theapproximately 1 min incolumn.
the HALO ® penta-HILIC column.
However, the addition of
However,
one the addition of
N-acetylhexosamine toone N-acetylhexosamine
the center galactose moleculeto theofcenter galactose
the A2G1F1 molecule of
glycopeptide the A2G1F1
increased the
glycopeptide
retention time byincreased
only 0.4the minretention time by only
because galactose 0.4= min
(log P −2.6)because
is more galactose
polar than(log P = −2.6) is more
N-acetylhexosamine
polar
(log P =than
−1.7)N-acetylhexosamine (log P = −1.7) (https://www.ncbi.nlm.nih.gov/pccompound).
(https://www.ncbi.nlm.nih.gov/pccompound). A similar trend was observed in theA
similarBEH
Glycan trend was observed
amide column, albeitin thewith
Glycan BEH
smaller amide
shifts column, albeit
in retention withFigure
times (see smaller shifts
2B). in retention
Similarly to the
times (seeofFigure
separation 2B). Similarly
hemopexin to thetheseparation
glycopeptides, ZIC-HILICofcolumn hemopexinprovidedglycopeptides, the ZIC-HILIC
the highest retention times
column
and provided
the poorest the highest
resolution of the retention
studied IgG times and the poorest
glycopeptides, as shown resolution
in Figureof2C.the Westudied
observed IgG
a
glycopeptides, as shown in Figure 2C. We observed a total co-elution
total co-elution of the A2F1 and A2G1F1 glycoforms of the IgG1 peptide. Moreover, we did not detectof the A2F1 and A2G1F1
glycoforms
the of the IgG1
G1A3F1 glycoform (forpeptide.
no plausibleMoreover,
reason).we did not detect the G1A3F1 glycoform (for no
plausible reason).
Molecules 2020, 25, 4655 5 of 13
Molecules 2020, 25, x FOR PEER REVIEW 5 of 13

Figure
Figure 2. Separation
2. Separation of glycopeptides
of glycopeptides of IgG
of IgG in HALO
in HALO ® penta-HILIC
® penta-HILIC (A), Glycan BEH amide (B), and
(A), Glycan BEH amide (B), and
ZIC-HILIC
ZIC-HILIC (C)(C) columns.
columns. PEP1
PEP1 refers
refers to EEQYN
to EEQYN 180 180
STYRSTYR
andand PEP2
PEP2 to to EEQFN
EEQFN 176176 STFR.
STFR.

2.1.1. Separation
2.1.1. of of
Separation Fucosylated Glycopeptides
Fucosylated Glycopeptides
Enhanced
Enhanced fucosylation
fucosylation hashasbeen
been proposed
proposed asasa biomarker
a biomarker forfor
monitoring
monitoring liver disease
liver disease [35–37].
[35–37].
Accordingly, LC separation of fucosylated glycoform
Accordingly, LC separation of fucosylated glycoform isomers can improve isomers can improve analytical workflow
workflow in
in disease
diseaseprogression.
progression.As As ® penta-HILIC column in
we we havehaveshown shown previously,
previously, the HALO
the HALO ® penta-HILIC column in nanoscale
nanoscale
was ablewas able to separate
to separate mono-fucosylated
mono-fucosylated glycopeptides
glycopeptides of hemopexin,
of hemopexin, which only which onlyin
differed differed
the core-
in or
theouter
core- arm-linked
or outer arm-linked fucose positions
fucose positions [16]. We [16]. We separated
separated fucosylated fucosylated
glycopeptide glycopeptide
isomers of
isomers of hemopexin
hemopexin glycoproteins
glycoproteins during the during the comparative
comparative study of study of different
different HILIC HILIC stationary
stationary phases.
phases. Figure 3A,D shows two well-separated peaks for hemopexin SWPAVGN 187 CSSALR and
Figures 3A and D shows two well-separated peaks for hemopexin SWPAVGN CSSALR 187 and
ALPQPQN 453 ®
ALPQPQN VTSLLGCTH
453VTSLLGCTHinin the HALO
the HALO penta-HILIC
® penta-HILICcolumn column at at
chromatographic
chromatographic resolutions
resolutions of of
1.99 and
1.99 and1.36, respectively.
1.36, respectively. The The assignment
assignment of of
core or or
core outer arm
outer armfucosylation
fucosylation to to
chromatographic
chromatographic
peaks was confirmed by exoglycosidase digestion in our previous study
peaks was confirmed by exoglycosidase digestion in our previous study [16]. Our assumption [16]. Our assumption waswas
correct based m/z1058.45 187
correct basedononthe thefragmentation
fragmentation spectra spectra of A2G2F1
A2G2F1 with withm/z 1058.45(SWPAVGN
(SWPAVGN CSSALR)
187CSSALR) and
and 1168.84 (ALPQPQN 453 VTSLLGCTH). In core fucosylation, a fragment of m/z 1753.80 or m/z
1168.84 (ALPQPQN VTSLLGCTH). In core fucosylation, a fragment of m/z 1753.80 or m/z 2085.10
453

2085.10 was identified

was identified (Figure(Figure S1 (Supplementary
S1 (Supplementary Materials)).
Materials)). ThisThis fragment
fragment waswas notnot detected
detected ininthe
thefragmentation
fragmentationspectra spectraofofthe theouter
outerarm-fucosylated
arm-fucosylatedglycopeptide.
glycopeptide.Although
Althoughthe theseparation
separationininthe
SWPAVGN187187
theSWPAVGN CSSALRsequence
CSSALR sequenceofofthe the A2G2F1
A2G2F1 glycoform was was better
betterininthe
theGlycan
Glycan BEHBEH Amide
Amide
column
column = 1.72),
(R (R = 1.72),wewe were
were unable
unable to to
separate
separate thethe isobaric glycoforms
isobaric glycoforms in in
thethe
other
other hemopexin
hemopexin
sequence
sequence (Figure
(Figure3E). Despite
3E). Despite optimizing
optimizing thethe
gradient
gradient program
programforfor
thethe
ZIC-HILIC
ZIC-HILIC column,
column, thethe
core-
core-
and outer arm-fucosylated glycoforms were not separated, either in the SWPAVGN 187 CSSALR or in
and outer arm-fucosylated glycoforms were not separated, either in the SWPAVGN 187

in the
the ALPQPQN 453
ALPQPQN453VTSLLGCTH VTSLLGCTHsequences sequences (Figure 3C,F). Isomeric glycoforms eluted
(Figure 3C,F). Isomeric glycoforms eluted in ain a single
single broad
broad peak. Moreover, only the HALO ® penta-HILIC column separated the fucosylated bi-antennary
peak. Moreover, only the HALO penta-HILIC column separated the fucosylated bi-antennary
®

isomeric
isomeric glycoforms
glycoforms of of
different
different peptide
peptide backbones.
backbones.
Molecules 2020,25,
Molecules2020, 25,4655
x FOR PEER REVIEW 6 6ofof1313

Figure 3. Normalized EIC chromatograms of A2G2F1 glycoforms of SWPAVGN187 CSSALR (A–C) and
Figure 3. 453
Normalized EIC chromatograms of A2G2F1 glycoforms of SWPAVGN187CSSALR 187 (A–C)
ALPQPQN VTSLLGCTH (D–F) in different HILIC columns. PEP1 refers to SWPAVGN CSSALR,
and ALPQPQN453VTSLLGCTH (D–F) in different HILIC columns. PEP1 refers to
and PEP2 to ALPQPQN453 VTSLLGCTH.
SWPAVGN CSSALR, and PEP2 to ALPQPQN VTSLLGCTH.
187 453

2.1.2. Separation of Sialylated Glycopeptides

2.1.2. Separation of Sialylated Glycopeptides
As shown in Figure 4, the EIC chromatograms of mono-sialylated glycoforms of hemopexin have
multipleAs isobaric
shown in Figure
peaks. 4, the
Based onEIC
our chromatograms
previous study [16], of mono-sialylated
mono-sialylatedglycoforms
hemopexinofglycoforms
hemopexin
contain isomers that differ in the localization of sialic acid on different arms (α3 antenna or α6hemopexin
have multiple isobaric peaks. Based on our previous study [16], mono-sialylated antenna).
Inglycoforms
each column, containwe isomers
obtained that differ inresolutions,
different the localization of sialic
achieving theacid onresolution
best different arms
in the(α3HALO ®
antenna
or α6 antenna).
penta-HILIC In each
column, column,
wherein thewetwoobtained
isobaric different
glycoforms resolutions, achieving
were separated withthe best resolution
a resolution of 3.09in
the HALO ® penta-HILIC
187 column, wherein the two isobaric
for the SWPAVGN CSSALR and of 2.24 for the ALPQPQN VTSLLGCTH sequence. Neither 453 glycoforms were separated with
thea
resolution
Glycan BEH of 3.09column
amide for thenor SWPAVGN
the ZIC-HILIC187 CSSALR
columnand wasofable
2.24
to for the ALPQPQN
baseline separate theseVTSLLGCTH
453
glycoforms
sequence. Neither the Glycan BEH amide column nor the ZIC-HILIC
of both peptides (Figure 4B–F). The Glycan BEH amide column provided a resolution of 1.02 column was able to baseline
for the
SWPAVGN CSSALR and of 0.82 for the ALPQPQN VTSLLGCTH peptide. The ZIC-HILICprovided
separate these
187 glycoforms of both peptides (Figure 4B–F).
453 The Glycan BEH amide column column
a resolution
provided of 1.02
the worst for the SWPAVGN
resolutions. 187CSSALR and of 0.82 for the ALPQPQN
The resolution was 0.74 for the SWPAVGN187 CSSALR 453VTSLLGCTH
peptide and
peptide. The ZIC-HILIC 453 column
0.43 for the ALPQPQN VTSLLGCTH peptide. provided the worst resolutions. The resolution was 0.74 for the
SWPAVGN 187
The separationCSSALR peptide
of the A3G3S1 andglycoform
0.43 for theofALPQPQN
the SWPAVGN 453 VTSLLGCTH
187 peptide in the HALO®
CSSALRpeptide.
The separation
penta-HILIC column of is the
shownA3G3S1 glycoform
in Figure of the SWPAVGN
S2A (Supplementary 187 CSSALRdisplaying
Materials), peptide inthree
the HALO
peaks ®
penta-HILIC
with resolutioncolumn
R1,2 = 1.28is shown
and R2,3in =Figure
0.80. In S2Athe(Supplementary
Glycan BEH amide Materials),
column,displaying three were
only two peaks peaks
with resolution
separated (FigureRS2B 1,2 = (Supplementary
1.28 and R2,3 = 0.80. In the Glycan
Materials)), BEH amide
in contrast to onlycolumn,
one peakonly twoZIC-HILIC
in the peaks were
separated (Figure S2B (Supplementary Materials)), in contrast to only one
column (Figure S2C (Supplementary Materials)), most likely as a result of partial isomer separation peak in the ZIC-HILIC
column (Figure S2C (Supplementary Materials)), most likely as a
of the α2–6 sialic acid (either the unbranched α6-antenna or the branched α3-antenna withresult of partial isomer separation
β2- or
of the α2–6 sialic
β4-branching). acidpoint,
At this (either
wetheareunbranched
still unable toα6-antenna or thepeak
elucidate which branched α3-antenna
corresponds to whichwithisomer.
β2- or
β4-branching). At this point, we are still unable to elucidate which peak corresponds to which
isomer.
Molecules 2020,
Molecules 25, x4655
2020, 25, FOR PEER REVIEW 77 of
of 13
13

A) HALO® penta-HILIC B) Glycan BEH amide C) ZIC-HILIC

column column column

100
100 100

Normalized intensity (%)

Normalized intensity (%)

Normalized intensity (%)

80
80 PEP1 80 PEP1 PEP1

60
60 60

40
40 40

20
20 20

0
0 0 39.5 40.0 40.5 41.0 41.5 42.0 42.5 43.0 43.5
23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5 27.0 29.5 30.0 30.5 31.0 31.5 32.0 32.5
Time (min) Time (min) Time (min)

D) HALO® penta-HILIC E) Glycan BEH amide F) ZIC-HILIC

column column column
100 100 100
Normalized intensity (%)

Normalized intensity (%)

Normalized intensity (%)

PEP2 PEP2 PEP2
80 80 80

60 60 60

40 40 40

20 20 20

0 0 0
25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5 29.0 29.5 30.0 30.5 31.0 31.5 32.0 32.5 39.5 40.0 40.5 41.0 41.5 42.0 42.5 43.0 43.5
Time (min) Time (min) Time (min)

Figure 4. Normalized EIC chromatograms of A2G2S1 glycoforms of SWPAVGN187 CSSALR (A–C) and
Figure 4. Normalized EIC chromatograms of A2G2S1 glycoforms of SWPAVGN187CSSALR (A–C)
ALPQPQN453 VTSLLGCTH (D–F) in different HILIC columns. PEP1 refers to SWPAVGN187 CSSALR,
and ALPQPQN453VTSLLGCTH (D–F) in different HILIC columns. PEP1 refers to
and PEP2 to ALPQPQN453 VTSLLGCTH.
SWPAVGN187CSSALR, and PEP2 to ALPQPQN453VTSLLGCTH.
2.2. Column Temperature Effect on the Separation of Glycopeptides Isomers
2.2. Column Temperature Effect on the Separation of Glycopeptides Isomers
In line with Mechref et al., who studied the column temperature effect on the isomeric separation
In line with Mechref
of N-glycopeptides by PGCet[13],
al., we
who studied
tested threethe columncolumn
different temperature effect on
temperatures, i.e.,the
40,isomeric
50, and
separation
◦ of N-glycopeptides
60 C. The retention by PGC [13],
times and resolutions westudied
of all tested three different
isomeric column temperatures,
glycopeptides separated in the i.e., 40,
three
50, and 60 °C. The retention times and resolutions of all studied isomeric
HILIC columns are shown in Table 2. In most cases, the retention time decreased with an increase glycopeptides separated in
the three HILIC columns are shown in Table 2. In most cases, the retention time decreased with an
in temperature.
increase
HALO ® penta-HILIC column: For hemopexin with the glycan composition of A2G2F1,
in temperature.
the resolution decreased with an increase in temperature for the SWPAVGN of CSSALR
HALO ® penta-HILIC column: For hemopexin with the glycan composition 187 A2G2F1, and the
resolution decreased
ALPQPQN 453 VTSLLGCTH with sequences
an increase in 2).
(Table temperature
In turn, for for the the mono-sialylated
SWPAVGN CSSALR 187
glycoforms and
ALPQPQN 453VTSLLGCTH sequences (Table 2). In turn, for the mono-sialylated glycoforms of
of hemopexin, the resolution increased with temperature, albeit only for the peptide sequence
hemopexin,453the
ALPQPQN resolution For
VTSLLGCTH. increased
the other with temperature,
hemopexin albeit only
glycopeptide, for the peptide
the resolution decreasedsequence
at 50 ◦ C
ALPQPQN 453
column temperature VTSLLGCTH. For theatother
but increased 60 C.hemopexin
◦ The column glycopeptide,
temperature the alsoresolution
affected the decreased
separation at of
50 the
°C
column temperature but increased at 60 °C. The column 187temperature
A3G3S1 glycoform of hemopexin (sequence SWPAVGN CSSALR). At 40 C, we were able to partly also affected
◦ the separation of
the A3G3S1
separate glycoform
the three ofglycoforms
isobaric hemopexinof(sequence SWPAVGN
A3G3S1. However, at 50CSSALR).
187 ◦ At 40
C, the second °C, started
peak we were able to
co-eluting
partlythe
with separate the three
third peak, isobaric
and the secondglycoforms
and the thirdof A3G3S1.
peaks fully However,
co-eluted at at
5060°C,◦ C,
theassecond
shownpeak started
in Figure S3
co-eluting with the third peak, and the second and the third peaks fully
(Supplementary Materials). In the separation of the A2G1F glycoform of IgG2 (EEQFN STFR), the co-eluted at 60 °C,
176 as shown
in Figure slightly
resolution S3 (Supplementary
improved when Materials).
increasingIn thethe separation
column temperatureof the A2G1F
to 50 ◦ C, butglycoform
worsened of at 60IgG2◦ C.
(EEQFN STFR), the resolution slightly improved when increasing the column temperature to 50
176

°C, but worsened at 60 °C.

Glycan BEH Amide column: The resolution of fucosylated bi-antennary hemopexin with the
sequence SWPAVGN187CSSALR improved by increasing the column temperature to 50 °C, but
worsened at 60 °C. We observed the same tendency for the A2G2S1 glycoform with the sequence
ALPQPQN453VTSLLGCTH. Conversely, the resolution slightly increased at 50 °C and 60 °C for
mono-sialylated bi-antennary hemopexin with the SWPAVGN187CSSALR sequence. In the
separation of mono-sialylated tri-antennary glycan (A3G3S1) of hemopexin with the
SWPAVGN187CSSALR sequence, the resolution worsened with the increase in column temperature.
Molecules 2020, 25, 4655 8 of 13

Table 2. Chromatographic parameters: tR , retention time; R, resolution of the HILIC separation of glycopeptide isomers of hemopexin and IgG. ND means not
detected analyte.

HALO® Penta-HILIC Glycan BEH Amide ZIC-HILIC

Column Temperature
40 ◦ C 50 ◦ C 60 ◦ C 40 ◦ C 50 ◦ C 60 ◦ C 40 ◦ C 50 ◦ C 60 ◦ C
Hemopexin
tR R tR R tR R tR R tR R tR R tR R tR R tR R
SWPAVGN187 CSSALR
A2G2F1 (core) 17.8 17.5 17.1 30.6 30.5 30.3
1.99 1.85 1.81 1.71 2.34 1.82 43.5 − 43.1 − 43.0 −
A2G2F1 (outer arm) 18.2 17.8 17.5 30.9 30.8 30.6
A2G2S1 (isomer 1) 24.7 24.4 24.1 30.8 30.7 30.6 41.3 41.0 41.1
3.09 2.89 3.33 1.02 1.05 1.12 0.74 0.86 0.89
A2G2S1 (isomer 2) 25.2 24.9 24.7 30.9 30.9 30.7 41.6 41.3 41.4
A3G3S1 (isomer 1) 25.9 25.7 25.4 31.3 31.3 31.1
1.28 0.39 1.33 1.23 0.74 0.73 41.8 − 41.7 ND
A3G3S1 (isomer 2) 26.2 26.0 25.8 31.5 31.4 31.2
1.80 0.37
A3G3S1 (isomer 3) 26.4 26.1 ND ND ND ND ND ND ND
ALPQPQN453 VTSLLGCTH
A2G2F1 (core) 19.7 19.3 18.9
1.36 1.09 1.07 30.6 − 30.5 − 30.2 − 42.7 − ND ND
A2G2F1 (outer arm) 19.9 19.6 19.2
A2G2S1 (isomer 1) 26.9 26.7 26.3 30.5 30.4 30.3 40.8 40.6 40.5
2.24 2.72 3.11 0.82 1.01 0.99 0.43 0.50 0.61
A2G2S1 (isomer 2) 27.3 27.1 26.8 30.6 30.6 30.4 40.9 40.7 40.7
IgG2
EEQFN176 STFR
A2G1F (isomer 1) 19.5 19.4 0.80 19.2 30.8 30.7 30.5 1.02 43.9 − 43.7 − 43.5 −
0.74 0.73 0.90 0.97
A2G1F (isomer 2) 19.7 19.6 19.4 30.9 30.8 30.6
Molecules 2020, 25, 4655 9 of 13

Glycan BEH Amide column: The resolution of fucosylated bi-antennary hemopexin with the
sequence SWPAVGN187 CSSALR improved by increasing the column temperature to 50 ◦ C, but
worsened at 60 ◦ C. We observed the same tendency for the A2G2S1 glycoform with the sequence
ALPQPQN453 VTSLLGCTH. Conversely, the resolution slightly increased at 50 ◦ C and 60 ◦ C for
mono-sialylated bi-antennary hemopexin with the SWPAVGN187 CSSALR sequence. In the separation
of mono-sialylated tri-antennary glycan (A3G3S1) of hemopexin with the SWPAVGN187 CSSALR
sequence, the resolution worsened with the increase in column temperature. The resolution of the
A2G1F isomer of IgG2 with the EEQFN176 STFR sequence improved with the increase in temperature.
SeQuant ZIC-HILIC column: In the isomeric separation of the A2G2S1 glycoform of the
SWPAVGN187 CSSALR peptide, increasing the temperature improved the chromatographic resolution
(see Figure S4 (Supplementary Materials)). We also observed a slightly better resolution for the A2G2S1
glycoform of the ALPQPQN453 VTSLLGCTH peptide, as shown in Figure S4 (Supplementary Materials).
However, the increased temperature did not affect the separation of the outer arm, the core fucosylation
of the studied hemopexin glycopeptides, or the A2G1F glycoform of IgG2.
The increased temperature helped in the isomeric separation of some glycoforms but not others.
The separation mechanism of HILIC is very complex, and we cannot reach a general conclusion of the
column temperature effect on isomeric separation in HILIC at this point. More detailed studies are
necessary to improve our understanding of the impact of column temperature on the LC separation of
isomeric glycopeptides by HILIC.

3. Materials and Methods

3.1. Chemicals
Acetonitrile (LC-MS grade), formic acid (LC-MS grade), iodoacetamid (purity ≥ 99%), dithiotreitol
(purity ≥ 99%), and SOLu-Trypsin were purchased from Sigma-Aldrich (St.Louis, MO, USA), as well
as the IgG standard from human serum (UniProtKB ID, IgG1, P01857; IgG2, P01859). The hemopexin
standard from human plasma (P02790) was purchased from Athens Research and Technology, Inc.
(Athens, GA, USA). Water (LC-MS grade), ammonium hydrogen carbonate (LC-MS grade), and
acetic acid (LC-MS grade) were supplied by Merck (Darmstadt, Germany). α2–3,6,8,9 neuraminidase
(P0722S), and GlycoBuffer 1 was purchased from New England BioLabs (Ipswich, MA, USA).

3.2. Sample Preparation

The glycopeptide standards were prepared by trypsin digestion of 30 µL of hemopexin (10 µg µL−1 )
and 100 µL of IgG (2 µg µL−1 ) according to a previously described protocol [38]. After trypsin digestion,
the samples were desalted using solid-phase extraction (SPE) on a Sep-Pak Vac C18 cartridge (Waters,
Milford). The following desalting procedure was used: Condition step: 2 × 1 mL of 100% acetonitrile
and 1 mL of 2% acetic acid; loading step; washing step: 0.5 mL of 2% acetic acid; elution step: 0.5 mL
of 70% acetonitrile with 2% acetic acid. 100 µL of hemopexin digest was mixed with 100 µL of IgG
digest. In total, 100 µL of the mixture was subjected to desialylation by adding 14 µL of GlycoBuffer 1
and 14 µL of neuraminidase overnight at 37 ◦ C. The desialylated sample was desalted, as mentioned
above, and eluted in 0.3 mL 70% acetonitrile with 2% acetic acid. Then, 75 µL of the glycoprotein
tryptic digest mixture was mixed with 75 µL of the desialylated sample. The mixture was evaporated
and reconstituted in 65% acetonitrile with 0.1% formic acid, analyzing 1 µL by HILIC.

3.3. Instrumentation and Experimental Conditions

The measurements were performed on an Agilent 1290 Infinity II LC System with a binary pump
(Agilent Technologies, Inc., Waldbronn, Germany) coupled with maXis™ Q-TOF mass spectrometer
(Bruker Daltonics, Bremen, Germany). The glycopeptide separation was tested in three different HILIC
columns: HALO® penta-HILIC (2.1 × 150 mm; 2.7 µm; Advanced Materials Technology, Wilmington,
Delaware, USA), ACQUITY UPLC Glycan BEH Amide Column (2.1 × 150 mm; 1.7 µm; Waters
Molecules 2020, 25, 4655 10 of 13

Corporation, Milford, MA, USA) and SeQuant ZIC-HILIC (2.1 × 150 mm; 3.5 µm; Merck, Darmstadt,
Germany). The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in
acetonitrile (B). The gradient program was optimized to enhance, as much as possible, the resolution
of all glycopeptides analyzed in this study. In the case of HALO® penta-HILIC and ACQUITY UPLC
Glycan BEH Amide, the following gradient ((min)/% B) was used: 0/80, 10/80, 25/65, 35/40, 40/40,
43/80, 55/80. For the SeQuant ZIC-HILIC column, a shallower gradient program was used: 0/80, 10/80,
35/65, 45/40, 55/40, 58/40, 70/80. The flow rate was 0.3 mL min−1 for every column, and the column
temperature was maintained at 40 ◦ C (if not stated otherwise).

3.4. Mass Spectrometry Settings

The glycopeptides were analyzed in a data-dependent acquisition mode. The Hystar 3.2 and
otofControl 4.0 software (Bruker Daltonics, Bremen, Germany) were used for data acquisition. The
acquired MS data were analyzed using Bruker DataAnalysis 4.4. The experimental conditions for the
source were set as follows: End plate offset 500 V, capillary voltage 3500 V, dry gas 8 L min−1 with 250 ◦ C.
For mass spectrometer calibration, an ESI-L-low concentration tune mix (Agilent Technologies, Santa
Clara, California) was used in a mid-positive calibration mode. The MS spectra were acquired at a mass
range of m/z 100–3000 at a spectra rate of 2 Hz with precursor selection in the range of 600–3000 m/z,
setting the preferred charges states between +2 and +4. After MS acquisition, stepping-energy the five
most intense precursors were subjected to collision-induced dissociation (CID), setting the collision
energy to 70 eV and collecting mixed energy spectra at 100% collision energy for 50% of the cycle time
and 50% collision energy for other 50% of the cycle time, with the product-ion spectra rate ranging
from 4 to 16 Hz, depending on precursor intensity. In this operation mode, ion counts from each
subdivision were summed together. Glycopeptides were identified manually from the precursor
masses and CID spectra. The fragmentation spectra of all the studied glycopeptides are shown in
Figure S1 (Supplementary Materials).

4. Conclusions
In this study, we compared three different HILIC stationary phases, i.e., HALO® penta-HILIC,
Glycan BEH Amide, and ZIC-HILIC in the separation of complex N-glycopeptides of hemopexin
and IgG glycoproteins. We showed the effect of the composition of the glycans on the retention and
separation of glycopeptides. Adding a monosaccharide unit to a glycan moiety enhances glycopeptide
retention. Sialic acid in a glycan moiety significantly prolonged the retention times of hemopexin
glycopeptides in the HALO® penta-HILIC column. In contrast, in the Glycan BEH Amide column,
this effect was unremarkable. In the ZIC-HILIC column, we observed electrostatic repulsion between
the negatively charged sulfobetaine functional group and the negatively charged sialic groups of the
glycans, thereby decreasing retention. The HALO® penta-HILIC column provided the best separation
of the studied glycopeptides and the ZIC-HILIC column the worst. We also demonstrated the potential
of the HILIC columns tested in this study for the isomeric separation of fucosylated and sialylated
glycoforms. The HALO® penta-HILIC column provided good separation of core- and outer arm-linked
fucose of the A2G2F1 glycoform of both hemopexin peptides. The Glycan BEH Amide column was able
to separate only the A2G2F1 isomeric glycoforms of the SWPAVGN187 CSSALR peptide, whereas the
ZIC-HILIC column did not separate any A2G2F1 isomers. The HALO® penta-HILIC column showed
the best separation of sialylated glycoforms, followed by the Glycan BEH Amide column, whereas
the ZIC-HILIC column provided a very poor chromatographic resolution. Choosing the right HILIC
stationary phase is a crucial step when optimizing the HILIC method. Therefore, the findings of this
study may help to develop the HILIC method for glycopeptide analysis because the HILIC technique
is a potentially useful tool for the comprehensive characterization of glycoproteins and their isomers.

Supplementary Materials: The following are available online, Fragmentation spectra of the A2G2F1 glycoform of
the SWPAVGN187 CSSALR and ALPQPQN453 VTSLLGCTH peptide in the HALO® penta-HILIC column (Figure
S1 (Supplementary Materials)). Separation of the A3G3S1 glycoform of the SWPAVGN187 CSSALR peptide of
Molecules 2020, 25, 4655 11 of 13

hemopexin in different HILIC columns (Figure S2 (Supplementary Materials)) and column temperature effect
on the separation of this glycoform in the HALO® penta-HILIC column (Figure S3 (Supplementary Materials)).
Separation of the A2G2S1 glycoform of hemopexin in both peptide sequences at different column temperatures in
the ZIC-HILIC column (Figure S4 (Supplementary Materials)).
Author Contributions: K.M.: Conceptualization, Methodology, Investigation, Writing—original draft P.K.:
Supervision, Writing—review and editing, Resources, Funding acquisition. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by the Czech Science Foundation, Grant No 19-18005Y. The work was
supported in part by the Charles University project SVV260560.
Acknowledgments: The authors also thank Carlos V. Melo for editing the manuscript.
Conflicts of Interest: The authors have declared no conflict of interest.

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Sample Availability: Samples of the compounds (hemopexin and IgG) are available from the authors.

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