Supplemental Table 1. Technological developments in glycoproteomics.
Sample
Glycoprotein
HeLa cells
N-linked
Standard
proteins and
human serum
N-linked
Cell surface
proteins of
Chang liver and
HepG2 cells
N-linked
Normal and
cancerous
frozen prostate
tissues
N-linked
Sample Prep
Enrichment/Release/
Labeling
Cell culture, oxidation
of membrane proteins,
cell lysis; membrane
glycoprotein capture on
hydrazide beads, onbead tryptic digestion;
same for cytosolic
proteins; PNGase
F/H218O release,
Tryptic digestion of
proteins, followed by
oxidation; glycopeptide
capture on hydrazide
beads; CH2O/CD2O
labeling on the bead or
in solution; PNGase F
release.
Cell surface
glycoprotein oxidation,
cell lysis and
glycoprotein capture on
hydrazide beads; tryptic
digestion; PNGase F
release of
glycopeptides;
light/heavy dimethyl
peptide labeling.
Microdissection
followed by cell lysis,
tryptic digestion,
periodate oxidation and
conjugation to
hydrazine resins;
CE/LC-MS Platform
Findings
Ref
N-linked glycoproteins
Peptide analysis
NanoLC-ESI/MS/MS
(Orbitrap MS); Validation:
NXT/S consensus & search
for non-enzymatic
spontaneous N deamidation.
A total of 268 N-glycosylation sites in 106
glycoproteins in the membrane and cytosolic fractions,
were identified; roles in cell-cell recognition, binding
migration and signaling; dozens of new glycoproteins
and sites.
10
Peptide analysis
NanoLC-ESI/MS/MS
(Triple TOF 5600);
heavy/light dimethyl labeled
based quantitation.
Hydrazide bead capture with heavy/light dimethyl
labeling has enabled better enrichment recovery over
solution based labeling (up to 330% improvement) and
improved sensitivity (from 10 µg standard proteins);
117 glycosites quantified in serum.
11
Peptide analysis
SCX prefractionation of
nonglycosylated peptides;
nanoLC-ESI/MS/MS (LTQOrbitrap XL); transmembrane
domains predicted by the
topology algorithm TMHMM;
Validation: NXS/T consensus;
stable isotope dimethyl based
MS quantitation.
Peptide analysis
NanoLC-ESI/MS/MS (Triple
TOF 5600 for generation of
data independent SWATH-MS
maps (32 x 26 m/z windows);
hotgun analysis with Orbitrap
XL-MS/MS for checking
Glycoprotein identification accomplished by glyco and
non-glycopeptides; non-glycopeptides used for
quantitation; glycosite occupancy was assessed with
dimethyl labelled glyco and non-glycopeptides; 341 cell
surface glycoproteins identified (82 % specificity); 33
glycoproteins changed expression level between the two
cell lines.
12
Over 1400 N-glycosites per sample identified; 220
proteins associated with various cancer aggressiveness
and metastatic processes showed significant quantitative
changes.
13
1
PNGase F release of
glycopeptides.
Secretome of
human
hepatocellular
carcinoma cells
N-linked
HeLa, mouse
liver cells
Core
fucosylate
d N-linked
HeLa cells
N-linked
Wheat flour
albumin
N-linked
Standard
proteins and
mouse liver
N-linked
Yeast
N-linked
FASP tryptic digestion
of secreted proteins;
hydrazide bead or ZICHILIC enrichment;
PNGase/H218O release.
Tryptic digestion of
protein extracts; HILIC
followed by lentil lectin
enrichment;
endoglycosidase F3
release.
Tryptic digestion of
proteins; enrichment
with reactor with
monolithic C12
hydrophobic &
monolithic HILIC
hydrophilic media;
PNGase F release.
Tryptic digestion of
wheat albumin proteins;
ZIC-HILIC/cotton wool
enrichment; PNGAse A
H218O release.
Tryptic digestion of
protein samples; amino
phenyl boronic acid
functionalized
detonation
nanodiamonds
enrichment I
glycopeptides;
PNGase F/H218O
release.
Tryptic digestion of
protein extracts;
sample quality in CID mode
and with the triple TOF for
generating the spectral
libraries.
Peptide analysis
NanoLC-ESI/MS/MS (Q
Exactive Orbitrap); labelfree/area-based MS
quantitation.
Peptide analysis
NanoLC-ESI/MS/MS (Q
Exactive Orbitrap);
Validation: NXT/S consensus
1213 unique N-glycosites from 611 N-glycoproteins
identified; differential regulation of glycoproteins
involved in metastasis.
14
The use of the stepped fragmentation function with
glycan diagnostic ion capability enabled the
identification of 1364 and 856 glycopeptides in HeLa
and mouse liver, respectively.
15
Peptide analysis
MALDI-TOF/TOF.
Protein purification/desalting, tryptic digestion,
enrichment and deglycosylation performed in one
monolithic glycoproteomic reactor; 486 N-glycosylation
sites identified in 104 HeLa cells; 2.5 fmol detection
limit for protein standards.
16
Peptide analysis
MALDI-MS (Ultraflex
MALDI-TOF/TOF MS);
NanoLC-ESI/MS/MS (Q
Exactive Orbitrap).
Peptide analysis
MALDI-MS; NanoLCESI/MS/MS (Orbitrap);
Validation: NXS/T consensus.
A total of 78 N-glycosylation sites in 67 albumin
proteins were assigned. Several of the identified
glycoproteins show sequence similarity to known food
allergens.
17
50-fold improvement in sensitivity when compared to
other enrichment methods
(detection from 5 x10-8 - 5 x10-10 M concentration
solutions).
18
Peptide analysis
A cost effective and generic method that enabled the
identification of 816 N-glycosylation sites in 332
19
2
E. coli proteins
N-linked
Human blood
serum
N-linked
Standard
proteins and
disease-free and
breast cancer
sera
N-linked
Human plasma
N-linked
glycopeptide capture on
boronic acid
functionalized magnetic
beads; elution with
acidic solutions
(HCOOH & TFA);
ConA/WGA
enrichment for
comparison;
PNGAse F/H218O
release.
Lectin beads; chemical
cleavage of glycosidic
bonds (TFMS); first
Glc-NAc attached to
Asn was not cleaved
(amide bond).
Serum depletion from
highly abundant
proteins, followed by
tryptic digestion;
WGA/ConA/RCA120 &
PNGase F; or,
WGA/ConA/RCA120 &
ZIC-HILIC & PNGase
F release; or,
ZIC-HILIC & PNGase
F release.
Protein enrichment
using tandem lectin
affinity on monolithic
columns with surface
immobilized ConA,
WGA and RCA-1;
tryptic digestion of the
eluate; PNGase F
release.
Glycoprotein
enrichment on serial
affinity
High pH RPLC
prefractionation; nanoLCESI/MS/MS (LTQ-Orbitrap
Elite); Validation: NXS/T
consensus.
glycoproteins (194 membrane proteins); enrichment
specificity with boronic acid was higher than with
lectins and more sensitive.
Peptide analysis
Peptides containing the first
Glc-NAc were analyzed by
LC-MS/MS (Magic C18AQ;
LTQ Orbitrap Elite)
46% more N-glycosylation sites identified with
chemical deglycosylation than with Endo H enzymatic
digestion.
20
Peptide analysis
IEF or high-pH RPLC
fractionation; nanoLCESI/MS/MS (LTQ-FT);
Validation: NXS/T consensus;
check for spontaneous
deamidation.
The combined enrichment strategy resulted in a 14-32 %
increase in the number of detected N-glycosites; sample
fractionation by high-pH or IEF further resulted in 3.1
and 1.8-fold, respectively, increase in the identified
glycosites; a total of 615 N-glycosites from 312
glycoproteins were mapped.
21
Peptide analysis
NanoLC-ESI/MS/MS (LTQOrbitrap).
The impact of the order of the three lectin columns was
investigated; the sequence WGA/ConA/RCA-1 captured
the largest number of glycoproteins; 113 glycoproteins
identified, and a panel of 23 non-redundant differentially
expressed glycoprotein cancer marker candidates
uncovered.
22
Peptide analysis
NanoLC-ESI/MS/MS
(LTQ-Orbitrap).
Patterns of protein capture from the two series of
affinity columns were investigated; independent
occurrence of different affinity-targetable glycan
23
3
Urine and
plasma
N-linked
Complex
biantennary
glycans and
chicken
ovalbumin
Ovalbumin and
human serum
N-linked
glycans
Wild type and
mutant CHO
cell lines
N-linked
N-glycans
chromatography with
immobilized lectin
and antibody selectors
LEL/HPA/antiLe*Ab/anti-sLe*Ab, or
anti-sLe*Ab/antiLe*Ab/HPA/LEL;
protein elution and
digestion from the
affinity columns;
PNGase F release.
10-30 kDa MW cutoff
filter for glycoprotein
capture; PNGase
F/H218O release of
glycans; glycan
permethylation; onfilter proteolytic
digestion of
deglycosylated proteins.
Anion-doped liquid
matrix (G3CA doped
with BF4- and NO3-) for
negative ion mode
MALDI-MS.
Glycoprotein capture on
carbon-functionalized
ordered graphene/
mesoporous silica
composites with short
mesoporous channels
and high surface area;
PNGase F release.
Filter-Aided N-Glycan
Separation (FANGS);
permethylation.
features, and multiple targetable glycan features were
co-resident in the same glycoprotein were recognized.
Glycan and peptide analysis
MALDI-MS (MDS Sciex
4800); IEF peptide
prefractionation; nanoLCESI/MS/MS (LTQ-Orbitrap
XL); Validation: NXS/T
consensus; 18O incorporated
deamidation.
The GlycoFilter technology allows for simple and
comprehensive characterization of glycans and peptides;
865 and 295 glycosites were identified in urine and
plasma samples, respectively.
24
Glycan analysis
MALDI-QIT-TOF-MS
(AXIMA Resonance) in
negative ion mode.
Subfemtomole detection limits with the new matrix
composition in negative ion mode; sensitivity
improvement for negative ion MS2 that facilitates
identification of glycan structures.
25
Glycan analysis
MALDI-TOF.
25 N-linked glycans from ovalbumin, and 48 N-linked
glycans from 400 nL healthy pristine human serum were
detected.
26
Glycan analysis
MALDI-TOF-MS (Ultraflex
III TOF/TOF); MALDI-FTICR-MS (9T Solarix FT);
DHB matrix.
Differences in N-glycan profiles were due to
degradation or mislocalization of glycosylation enzymes
in the mutant CHO cells;
The method is applicable for N-glycan analysis in low
amounts of samples (e.g. ~106 cells).
27
4
CHO cells
N-linked
Mouse brain
and human
kidney tissue
N-linked
HIV gp120
N-linked
Standard or
recombinant
proteins
N-linked
Human serum
N-linked
Poly-lysine coated
plates were used for the
adhesion and isolation
of plasma membranes;
tryptic peptides were
enriched with HILIC
SPE and N-linked
glycans released with
PNGase F and
permethylated; DHB
matrix.
On-tissue digestion
with PNGase F (and
sialidase).
The protein was
conjugated to
Aminolink resin and the
N-glycans were
released with PNGase F
and purified with
Carbograph columns.
N-linked glycans were
released from
glycoproteins with
hydrazine, PNGase F,
or Endo H.
PNGase F release of
glycans, followed by
sodium borohydride
reduction and
enrichment on
C8/graphitized carbon
cartridges.
Glycan analysis
MALDI-TOF-MS (MALDI
MSP 96) in positive ion mode;
Validation: 2AA labeled Nlinked glycans were analyzed
by NP-HPLC with
fluorescence detection.
The adhesion-based isolation method of the plasma
membrane prevents contamination of the samples with
high-mannose species originating from other cell
compartments.
28
Glycan analysis
MALDI-IMS (7T Solarix 70
dual source FTICR with
SmartBeam II 1000Hz laser);
Validation: off-tissue analysis
of N-linked glycans by
MALDI-MS (DHB matrix)
and with HPLC.
Glycan analysis
MALDI-MS Resonance,
Nano-LC-ESI/MS/MS (TSQ
Quantum, Orbitrap Velos Pro
with HCD).
First implementation of imaging mass spectrometry for
spatial profiling of N-glycans on tissue samples;
potential applications in clinical diagnostics; an
estimated 30 N-glycans were detected on-tissue in
mouse brain.
29
Isobaric aldehyde reactive tags (iART), 114 and 115 Da,
have been developed for glycan quantitation by tandem
MS; iART can be expanded to six-plex tags for
concurrent analysis of six samples.
30
Glycan analysis
ESI-Synapt G2 travelling
wave ion mobility MS; CID
performed after mobility
separation.
Glycan analysis
LC-MS (QTOF).
Structure determination of N-glycans was achieved by
travelling wave ion mobility and negative ion CID; ion
mobility enabled better differentiation of fragment ions
from glycan molecular ions produced in the ion source;
potential for separating isomers needs improvement.
A subambient pressure ionization with nanoESI (SPIN)
interface was developed; the SPIN interface enabled the
identification of higher charge state ions and of ~25 %
more glycans than a regular heated capillary interface.
31
5
32
Saccharomyces
cerevisiae
proteins
N-linked
Human EGFR
Trypsin digested
glycopeptides were
enriched using ClickTE HILIC material
(synthesized by linking
cysteine to silica
through thiolene click
chemistry); before LCMS/MS analysis, the
glycopeptides were
deglycosylated in
parallel with either
PNGase F or Endo H
enzymes.
Tryptic digestion of
EGFR.
Bovine fetuin
N-linked
Tryptic digestion of the
glycoprotein, followed
by TMT labeling.
Human liver
N-linked
Click maltose-HILIC
for enrichment of
tryptic N-linked
glycopeptides;
Glycopeptide analysis
Nano LC-MS/MS (LTQOrbitrap); Validation: a)
identified the know Nglycosylation sites in
horseradish peroxidase (HRP),
b) NXS/T consensus.
135 N-glycosylation sites were detected in 79 proteins
from S. cerevisiae. The method could be used
potentially for studying congenital disorders of
glycosylation (CDG).
33
Glycopeptide analysis
NanoLC-ESI/MS/MS
(Orbitrap Fusion trybrid with
HCD, CID and ETD); CID
spectra processed by SweetHeart.
Glycopeptide analysis
LC-MS/MS (TriVersa
NanoMate infusion with ETD
or ECD, LTQ-Orbitrap XL
ETD); CID for glyco structure
elucidation (preserving intact
peptide backbone);
ETD/MS/MS for glycosite
localization; in-source pseudoMS3 survey scan for
identification of diagnostic
oxonium ions; TMT labeled
glycopeptide quantitation,
confirmed by more precise
HCD of nonglycopeptides
sand glycopeptides.
Glycopeptide and peptide
analysis
2D-LC-ESI/MS/MS
(phosphate monolithic column
A novel data dependent HCD-pd (accurate-massproduct-dependent)-CID/ETD decision tree workflow
was demonstrated for the analysis of glycoproteins; it
efficiently utilizes information from all fragmentation
methods (HCD, CID and ETD) to sequence the peptide
and the glycan.
Five MS events including a survey scan at m/z 204,
high-resolution FT full scan, CID, ETD and HCD were
conducted alternatively for data acquisition at each LC
elution time point; the method enable the identification
of 23 glycoforms from the 5 glycosylated sites on
bovine fetuin; TMT labeling was used to identify and
quantify unpredicted glycopeptides or other
modifications.
34
2210 N-glycosylated proteins and 4783 N-glycosylation
sites with the sequence motif of N-!P-[S/T/C] were
detected. This number is the highest reported so far for
the human liver proteome. (Note: The highlighted motif
36
6
35
Rat brain
membrane
proteins
N-linked
Synthetic
glycopeptides
with 6279 Nglycosites
N-linked
Blood plasma
N-linked,
sialylated
hydrazide bead
enrichment of
glycoproteins or tryptic
glycopeptides.
PNGase F release of Nglycans from extracted
rat brain proteins,
removal of
glycosylamines from
the reducing terminus,
N-glycan reduction
with NaBH4,
purification on PGC
microcolumns; rat brain
proteins were digested
with trypsin,
dephosphorylated, and
the glycopeptides were
enriched on a ZICHILIC cartridge;
PNGase F release of
glycans from
prefractionated
glycopeptides.
SPOT synthesis
technology for peptide
synthesis.
Sample depletion of
highly abundant
proteins, FASP tryptic
digestion; TiO2
enrichment in SA
glycopeptides;
PNGase F/H218O
release.
and C18 (LTQ Orbitrap Velos
and Triple-TOF 5600);
Validation: NXS/T/C
consensus.
Glycopeptide, peptide and
glycan analysis
RPLC fractionation of
enriched N-glycopeptides;
PGC-LC-ESI/MS/MS for the
analysis of N-glycans (XCT
Plus 3D ion trap); nanoLCESI/MS/MS for the analysis of
N-glycopeptides and
deglycosylated peptides
(LTQ-Orbitrap Velos with
CID/HCD and ETD).
Synthetic glycopeptides
NanoLC-ESI/MS/MS (QTrap,
QTof, LTQ-Orbitrap XL).
is the consensus sequence for N-glycosylation, which
signifies that Pro cannot be the residue near N, but any
other residue can be present at this position. The second
residue C-term from N can be S, T, or C.)
A combined glycomics and glycoproteomics approach
was developed for characterizing N-linked glycosylation
heterogeneity; the GlycoMod tool and CID MS/MS
were used for elucidating the N-glycan structures;
deglycosylyated, deamidated peptides were used to
create a database of formerly glycosylated peptides; Nglycopeptides were identified by searching for a peptide
variable modification from a list of 71 glycan
compositions; 863 intact N-linked glycopeptides from
161 rat brain proteins were identified.
An SRMAtlas resource comprising a library of SRM
assays for 5568 N-glycosites was developed; the
resource enables multiplexed evaluation of clinically
relevant cancer-associated N-glycoproteins; consistent
quantification over 5 orders of magnitudes in 120
plasma samples was performed, demonstrating the
potential for evaluating biomarker candidates.
N-linked-sialylated glycoproteins
Peptide analysis
A novel method that resulted in the identification of 982
High-pH RPLC
glycosylation sites in 413 proteins.
prefractionation; nanoLCESI/MS/MS (Q Exactive
Orbitrap); Validation: NXS/T
consensus.
7
37
38
39
B-cells
N-linked,
sialic acid
and
terminal
Gal or
GalNAc
Fetuin, colonic
adenocarcinom
a cell line,
insect cell line
N-linked
sialylated,
high
mannose
EGFR2 isolated
from cell
lysates and
synthetic
glycans
N-linked
neutral
and
sialylated
Enrichment of
biotinylated
glycoproteins using
streptavidin-coated
beads; derivatization: a)
sialic acids oxidized
periodate; b) terminal
Gal or GalNAc are
oxidized by galactose
oxidase; both oxidation
procedures generate
aldehydes; biotinylation
of the aldehyde groups
is performed with
aminooxy-biotin;
PNGase F digestion
released glycans from
glycoproteins;
deglycosylated proteins
were digested with
trypsin.
Tryptic digestion of
proteins; C18 retention
of complex sialylated
N-glycans and
separation from
smaller, high mannose
structures in the flowthrough; step-wise
elution with various
concentrations of
organic solvent and
acid; permethylation.
N-glycan release with
PNGase F, and labeling
with 3-AQ.
Peptide analysis
(deglycosylated enriched
glycopeptides)
SCX-RPLC-MS/MS (LTQ);
Validation: NXS/T/C
consensus sequence.
The method is useful for direct labeling of living cells
that allows targeted proteomics of glycoprotein
subpopulations on cells and identification of changes in
glycosylation profiles; a total of 175 unique N-linked
glycosylation sites were identified in 108 non-redundant
proteins.
40
Glycan analysis
(MALDI-MS).
Improved glycomic mapping by the separation of
different types of N-glycans.
41
Glycan analysis
(MALDI-QIT/TOF).
3-aminoquinoline (3-AQ)/α-cyano4-hydroxycinnamic
acid was used for glycan labeling for sensitive detection
in negative mode MALDI-MS; detection response was
linear in the 0.5-5000 fmol range, and the method was
applicable to N-linked neutral and sialylated glycans;
the fragmentation pattern of 2-AQ labeled N-glycans
42,
43
8
Protein
standards and
CHO cells
N-linked
neutral
and
sialylated
Glycans from standard
and cell extract proteins
were released with
PNGase F and purified
by SPE.
Glycan analysis
CE-ESI/MS/MS negative
mode (API 4000 triple
quadrupole and TOF-MS).
Haptoglobin
and human
plasma
Trisialylat
ed Nlinked
Glycan analysis
Nano-HILIC-MS/MS (LTQ
Orbitrap XL or Elite);
Validation: 2AB-labled
sialylated glycan standard.
Human plasma
and fibrinogen
N-linked
sialylated
WAX separation of Nlinked glycans;
chemical derivatization
with DMT-MM/MeOH
for charge
neutralization of
sialylated glycans.
HILIC SPE with 96well plates; linkage
specific modifications:
ethyl esterification of
α2,6-linked sialic acids
and lactonization of
α2,3-linked sialic acids.
Morning (AM)
and afternoon
(PM) urine
samples from
the same donor
N-linked
neutral
and
sialylated
PNGase F digestion of
filtered urine protein
samples; derivatization
was performed using
the Dual Reactions for
Analytical Glycomics
(DRAG) methodology:
a) the reducing end of
N-glycans was labeled
with 2-AA or 2(13C6)AA by reductive
amination; b) sialic
acids were neutralized
by methylamidation.
Glycan analysis
MALDI-MS (MSD SCIEX
4800) in positive ion
reflectron mode, DHB matrix;
Validation: human serum IgG
and bovine fetuin.
Glycan analysis
MALDI-TOF-MS and
MALDI-TOF/TOF-MS/MS in
positive reflectron mode
(UltraFlextreme MS with
SmartBeam II laser); DHB
matrix; Validation: HILICUPLC of 2AA labeled Nglycans.
9
exhibited simple and informative tandem mass spectra,
similar to underivatized glycans.
A flow-through microvial interface that enables CE-MS
analysis of neutral and sialylated glycans, without
derivatization, was developed; over 90 glycans were
identified including glycans with multiple sialylation
sites; glycan heterogeneity of highly sialylated Nglycans, promoted by extensive acetylation, was
identified.
Potential for the detection of pancreatic cancer specific
changes in the relative abundance of trisialylated Nglycans in plasma; seven trisialylated N-linked glycans
were quantified with a mean CV of 9.3% in triplicate
samples.
The derivatization allows mass spectrometric linkagespecific identification of N-linked glycans containing
α2,6-linked α2,3-linked sialic acids; the newly
developed derivatization method is performed under
relatively mild conditions and does not require highly
purified glycans, allowing high-throughput sample
preparation in a 96-well plate format; in the human Nglycome, relative quantitation of more than 100 distinct
N-glycan compositions containing sialic acid linkages
was reported.
Based on their m/z values, 34 pairs of N-glycans were
identified during the quantitative evaluation of the
differences between the AM and PM urine samples;
DRAG is suitable for quantitative comparisons of Nglycan profiles in different samples; neutralization of
sialic acids allows direct quantitative analysis of acidic
and neutral glycans within a sample.
44
45
46
47
Recombinant
erythropoietin
O-linked
Protein capture on
PVDF membrane; Oglycans released by
reductive β-elimination.
Human
fibrinogen
O-linked
Kappa casein;
OmpA/MotB
protein from
“superbug” A.
baummannii
CSF
O-linked/
acidic
Bovine type II
collagen α-1
chain (CO2A1)
O-linked
and
hydroxyla
tion
Digestion with trypsin
or proteinase K;
glycopeptide
fractionation with
HILIC chromatography.
Protein digestion with
Glu-C and/or trypsin,
followed by enrichment
of glycopeptides by
ZIC-HILIC.
PNGase F treatment of
CSF samples, periodate
oxidation, hydrazide
capture of O-linked
glycoproteins, on-bead
tryptic digestion;formic
acid hydrolysis and
release of desialylated
O-glycopeptides.
Trypsin and GluCdigested peptides.
CHO cells
O-linked
O-linked
Cells were cultured in
the presence of
GalNAz; secreted and
cytoplasmic GalNAz
O-linked glycoproteins
Glycan analysis
MicroLC-MS/MS of glycans
with PGC microcolumns
(Agilent 1100 LC/ MSD Trap
XCT Plus).
Glycopeptide analysis
Glycopeptides analyzed by 1D
and 2D nanoLC-MSn (n = 1, 2,
and 3) using an ion trap mass
spectrometer.
Glycopeptide analysis
NanoLC-MS with ultraviolet
photodissociation (UVPD) in
negative ion mod (LTQ
Velos).
Peptide and O-glycopeptide
analysis
NanoLC-ESI/MS/MS;
(LTQ-FT with CID and ECD
or Orbitrap Velos/Orbitrap
Elite with HCD/ETD).
Glycopeptide analysis
NanoLC-ESI/MS/MS
LTQ-Orbitrap Velos;
CID/HCD-MS/MS performed
for peptide and glycopeptide
identifications;
CID/HCD/ETD-MS/MS
performed on separate samples
to confirm glycosylation sites.
Peptide analysis
SCX fractionation of peptides
and nanoLC-ESI/MS/MS
(-TOF Premier).
10
The method was capable of distinguishing between
different isobaric glycan isomers; for obtaining detailed
information about glycosylation site heterogeneity, the
method can be performed in conjunction with
glycopeptide analysis using capillary LC-MS/MS.
The protocol requires microgram quantities of protein or
protein mixture and has fmol sensitivity.
Seven novel O-linked glycosylation sites in human
fibrinogen were identified.
48
The automated method was shown to be able to identify
acidic glycopeptides by sequencing both glycans and
peptides in the same run; a novel O-glycosylation site
was identified in OmpA/MotB outer membrane protein
from A. baummannii.
O-glycosylation on Ser/Thr of extracellular proteins was
investigated; CID-MS2/MS3 was used for glycopeptide
identification, and ECD/ETD to pinpoint the correct
glycosylation site; the core-1-like HexHexNAc-Ostructure to one to four Ser/Thr residues was vastly
dominant and enabled automated MASCOT search
protocols; 106 O-glycosylations were characterized; Pro
residues were found preferentially in the n-1, n+1 and/or
n+3 positions, relative to Ser/Thr.
Comprehensive characterization of O-glycosylation and
hydroxylation of CO2A1 protein (sugar moiety
attachments to hydroxy-lysine, i.e., HyK); 23 Lys
residues were observed as unmodified, hydroxylated or
glycosylated with Glc-Gal or Gal moieties; the
modifications on these sites varied qualitatively and
quantitatively; in addition, a total of 128 hydroxy-Pro,
i.e., HyP, with unusual motifs, were observed.
50
N-azido-galactosamine (GalNAz) click chemistry was
used to metabolically label CHO cells; GalNAz was
metabolically incorporated into O-linked mucin glycans
to enable the analysis of secreted proteins across several
53
49
51
52
Genetically
engineered
SimpleCells
O-linked
GalNAc
Commercial
sialoglycopepti
des and human
serum
N-linked
O-linked
Sialylated
Etanercept
N-/Olinked
proteins were purified
by click-chemistry
affinity
chromatography and
digested with trypsin;
lyophilized peptides
were labeled with
iTRAQ reagents.
Cell lysates and
glycoprotein enriched
media were digested
with trypsin or
chymotrypsin; C18
SPE, neuraminidase
treatment and lectin
chromatography.
SPE of glycopeptides/
proteins on aldehyde
beads using reductive
amination; PNGase F
release of N-glycans;
NH4OH release of Oglycans/β-elimination;
p-toluidine
derivatization of sialic
acid groups or removal
with neuraminidase.
PNGase F release of Nglycans and sialidase
treatment of the protein
to remove sialic acid
residues from Oglycans, followed by
tryptic digestion;
reductive β-elimination
days of cell culture; mucin-type O-glycosylation is the
most common modification of secreted proteins;
in comparison to standard methods for secretome
analysis. GalNAz improved the number of secreted
protein IDs and the quality of the data by minimizing
the background resulting from non-secreted proteins.
Glycopeptide analysis
IEF prefractionation of
glycopeptides;
NanoLC-ESI/MS/MS
(LTQ-Orbitrap XL with
CID/HCD and ETD).
Twelve human SimpleCell lines were developed
through a genetic engineering strategy that simplifies the
process of O-glycosylation in cells, the elongation of Oglycans being blocked; only simple truncated and
homogeneous O-glycans are produced, which simplifies
enrichment and detection; proteome-wide discovery of
O-glycosylation (O-GalNAc) sites with ETD-MS led to
the first human O-glycoproteome map with ~3000
glycosites and over 600 glycoproteins identified; In
addition, a support vector machine approach was used to
develop a predictor tool for O-GalNAc glycosylation
(NetOGlyc4.0).
Mixed glycosylation and other PTMs
Glycan analysis
A solid phase chemoenzymatic approach for high(MALDI-TOF).
throughput glycoprotein immobilization for glycan
(GIG) extraction and analysis was developed; 66 Nlinked glycans were detected on a single MALDI spot.
N-/O-glycan and Oglycopeptide analysis
UPLC-HILIC with fluorescent
detection of 2-AB labeled N/O-glycans; UPLC-ESI/MSE
for O-glycopeptide
identification; ETD/MS/MS
for O-glycopeptide site
11
Structure elucidation of N-linked glycans, site
heterogeneity determination for the N-linked glycosite
in the Fc portion of the protein (monosaccharide
sequence and linkage information was confirmed by
exoglycosidase array digestions); O-linked glycan
structure verification and all 13 O-glycosylation sites
determined.
54
55
56
Human urine
Sialylated
O-/Nlinked
Kidney tissue
lysates
P-proteins
and
N-linked
Human serum
from healthy
and diseased
patients with
Barrett’s
esophagus,
diplasia,
esophageal
adenocarcinom
a
N-linked
Human blood
serum, late
stage ovarian
cancer
N-linked
of O-glycans from the
protein in
NH3/(NH4)2CO3
solution; N-/O-glycan
labeling with 2-AB.
assignment (Synapt G2-S
HDMS); Glycobase tools used
for preliminary structure
assignment.
Urine samples were
centrifuged and
desalted by dialysis;
trypsin digested
sialylated glycopeptides
were captured with
hydrazide beads.
Tryptic digestion of
protein extracts; TiO2
capture of p-pepties and
hydrazide bead capture
of N-linked
glycopeptides from the
flow-through; PNGase
F release.
Glycopeptide analysis
LC-MSn with CID and ECD
(LTQ-FTICR).
The combination of CID and ECD allowed for the
identification of O-linked glycosylation sites of the
urinary proteome. In the human urine samples, 53
glycoproteins were detected with 58 N-glycosylation
and 63 O-glycosylation sites, of which 29 O-linked sites
were novel.
57
Peptide analysis
NanoLC-ESI/MS/MS with
LTQ-XL for p-peptides, and
QTOF for deglycosylated
Peptide detection; Validation:
NXS/T consensus; Label-free
quantitation with IDEAL-Q
and CORRA programs.
Microfluidics
Glycan analysis
Microchip CE-fluorescence
detection (in-house built CEchip).
Concurrent identification of 437 glycosites/358
phosphosites and 468 glycosites/369 phosphosites in
normal and diseased kidney tissues, respectively.
58
Microchip CE analysis of N-glycans released from 10
µL of serum, followed by PCA and ANOVA analysis of
peak areas and migration times, enabled the
differentiation of diseased and healthy states; the chip
separation channel was 22 cm long, efficiencies were
700,000 plates, and analysis times <100 s.
59
Glycan analysis
Microchip CE-fluorescence
detection (in-house built CEchip).
Statistical analysis of the electrophoresis results showed
significant N-glycan profiles differences between the
control and both pre- and post-treatment cancer samples,
and subtle differences between the pre- and posttreatment samples.
60
PNGase F release of Nglycans from serum
samples; N-glycans
isolated by C18 SepPak cartridge and
activated carbon
cartridge; a portion of
N-glycans desialylated
by sialidase; APTS
labeling for fluorescent
detection.
PNGase F release of Nglycans; N-glycans
isolated by activated
carbon microspin
columns; APTS
labeling for fluorescent
detection.
12
RNase B
N-linked
PNGase F release of
glycans from RNase B;
APTS labeling of
glycans.
Glycan analysis
Microchip CE-fluorescence
detection (in-house built CEchip).
Human blood
serum, model
glycoproteins
N-linked
PNGase F release of Nglycans; deglycosylated
proteins captured by C8
trap; glycans purified
by PGC trap.
Glycan analysis
Online monolithic platform
incorporating an enzyme
reactor coupled to a C8 trap
and an LC-PGCchip/ESI/MS/MS (Agilent
chip/ion trap MS).
Mouse serum
N-linked
PNGase F digestion of
mouse proteins from
serum; glycan
enrichment with
graphitized carbon SPE.
Glycan analysis
NanoLC-PGC-chip/ESIMS/MS (Agilent chip/QTOFMS).
Cell lines of
breast, lung,
cervical,
ovarian, and
lymphatic
cancer
N-linked
Cell culture, plasma
membrane isolation;
PNGase F release of Nglycans; graphite
carbon SPE of Nglycans.
Glycan analysis
NanoLC-PGC-chip/MS
(Agilent chip/TOFMS).
HDL from
blood plasma
N-/Olinked
HDL isolated from
human blood plasma,
followed by tryptic
digestion; PNGase F
release of N-glycans
from HDL; pronase
digestion of HDL for
generating
glycopeptides; graphite
carbon SPE of Nglycans and
Glycan and glycopeptide
analysis
NanoLC-chip-ESI//MS/MS
with enrichment and analytical
C18 or PGC columns (Agilent
chip/Q-TOFMS).
13
Evaluation of separation efficiencies at higher
temperatures (45 C) than ambient, led to the conclusion
that high field strengths (500-1000 V/cm) are necessary
to compensate for the loss in efficiency due to the
increased diffusivity and axial dispersion at such
temperatures.
Developed a novel online enzymatic monolithic
platform for the release and analysis of N-glycans
(fused silica capillary with PNGase F immobilized on
the monolithic packing); the reactor was coupled to a
porous graphitized carbon (PGC)/LC chip; glycan
release could be achieved in 6 min, detection limits
being 100 fmol of glycoprotein from 0.1 μL human
blood serum.
A PGC-LC chip was used for the isomeric separation of
glycans; novel N-glycan modifications detected;
dehydration, O-acetylation, and lactylation; the method
is suitable for isomer-specific glycan profiling of the
serum N-glycome with potential applications in
diagnostics; automated analysis of the raw MS data
against a theoretical library of mouse serum N-proteome
yielded 133 N-glycan compositions.
Demonstrated for the first time direct analysis of cancer
cell membrane N-glycans by nano-LC/MS; hundreds of
N-glycans were identified per cell line, including
multiple isomers for most compositions; different
patterns of glycosylation for different cell types were
revealed, demonstrating value for MS-based biopsy
diagnostics.
A glycomic approach for analyzing HDL is described,
Most N-glycans from HDL particles were highly
sialylated with one or two neuraminic acids (Neu5Ac),
and all O-glycans were sialylated containing a core 1
structure with two Neu5Acs.
61
62
63
64
65
glycopeptides;
ganglioside extraction.
Chemical release of Oglycans by βelimination; the glycans
were purified and
fractionated by porous
graphite carbon
cartridge.
PNGase F release of Nglycans; deglycosylated
proteins precipitated by
ice-cold ethanol;
glycans purified by
graphitized carbon SPE.
Isolation of whey
proteins; PNGase F
release of N-glycans;
glycan purification by
C8 cartridge and
graphitized carbon SPE.
Tear and saliva
from ocular
rosacea patients
O-linked
Glycan analysis
MALDI-FTICR/MS;
NanoLC-PGC-chipESI//MS/MS (Agilent chip/QTOFMS).
Elucidated the structure of the most abundant glycans
found in tear and saliva, showing a correlation between
these two fluids in terms of glycan profile; sulfated
glycans comprised mucin core 1- and core 2-type
structures.
66
Dried blood
spots, human
standard serum
N-linked
Glycan analysis
NanoLC-PGC-chip-ESI/MS/MS (Agilent
chip/TOFMS).
Described a strategy for the analysis of N-glycosylation
patterns from dried blood spots. About ~150 N-glycan
structures from 44 N-glycan compositions were
monitored.
67
Human and
bovine milk
N-linked
Glycan analysis
NanoLC-PGCchip/ESI/MS/MS (Agilent
chip/Q-TOFMS).
The study represents the first comprehensive N-glycan
profile of bovine milk proteins, and the first MS based
confirmation of NeuGc in milk protein bound glycans; a
total of 38 and 51 N-glycan compositions were observed
in human and bovine milk, respectively; high mannose,
neutral and sialylated complex/hybrid glycans were
detectable in both milk sources.
A glyco-analytical multispecific proteolysis (GlycoAMP) strategy was proposed for glycoproteomic
characterizations; the method involves glycoprotein
digestion with multispecific proteases (or protease
cocktail), chromatographic separation by isomerspecific nano-PGC-LC and tandem MS detection; siteand structure-specific, as well as quantitative
information could be generated with this platform.
A method, in-gel non-specific proteolysis for elucidating
glycoproteins (INPEG) was developed; the method
incorporates in-gel separation of proteins and
proteolysis with non-specific enzymes; extracted
glycopeptides separated on the nano-LC-PGC chip
enabled the characterization of site-specific
glycosylation of proteins from human serum and bovine
milk.
68
Model
glycoproteins
N-linked
Glycoproteins digested
by multispecific
proteases;
glycopeptides enriched
by graphitized carbon
solid-phase extraction.
Glycopeptide analysis
NanoLC-PGCchip/ESI/MS/MS (Agilent
chip/Q-TOFMS).
Standard
proteins, human
serum, bovine
milk
N-/Olinked
Glycoproteins were
separated on a gel, and
small N-/Oglycopeptides (w or w/o
sialylation) were
generated by Pronase E
digestion.
Glycopeptide analysis
NanoLC-PGC-ESI/MS/MS
(Agilent chip-Q-TOFMS).
14
69
70
Glycoprotein
standards,
human serum
N-linked
PNGase F release of Nglycans; N-glycans
reduced by sodium
borohydride, and
cleaned up by C8
cartridge and
graphitized carbon SPE.
Glycan analysis
MALDI/FTICR/MS;
NanoLC-PGC-chip-ESI/MS
(Agilent chip/TOFMS).
An N-glycan library based on the most abundant
proteins in serum, containing over 300 entries with 50
structures completely elucidated and over 60 partially
elucidated structures, was developed.
71
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