Download Lecture 10. Glycoproteomics

Lecture 10. Glycoproteomics: Mass
Spectrometric Methods for Analyzing
Glycoproteins and Glycans
Mass Spectrometry in an “Omics” World ME.330.804
Hui Zhang
D
Department
t
t off P
Pathology,
th l
Cli
Clinical
i l Chemistry
Ch i t Division
Di i i
Johns Hopkins University
hzhang32@jhmi edu
[email protected]
Topics
• Glycoprotein classes
• Glycoprotein enrichments
• Release of p
peptides
p
and
glycosite analysis using MS
• Glycomic
Gl
i analysis
l i using
i MS
Glycoproteins
• Common protein modification: over 1/2 of the
mammalian proteins
• Diverse biological processes such as immune
response, cellular regulation, and cell
signaling
g
g
• Alterations in glycosylation patterns are linked
to diseases
Dube DH, Bertozzi CR. Glycans in cancer and inflammation--potential for therapeutics and
diagnostics Nat Rev Drug Discov.
diagnostics.
Discov 2005 Jun;4(6):477
Jun;4(6):477-88.
88
Zhang, H. & Cotter, R.J. Glycoproteomics: New Technology Developments and Applications
Provide Renewed Interest in Glycoproteins. Clinical Proteomics (2008).
Types
yp of Glycoproteins
y p
• N-glycosylation
N glycosylation
To the Asn side chain of proteins containing the sequon Asn-XSer/Thr (where X is any amino acid except Pro)
• O-GalNAc
O GalNAc glycosylation
Begins with the addition of a N-acetylgalactosamine to the OH of
specific Ser or Thr side chains
• O-GlcNAc modification
N-acetylglucosamine addition to the oxygen of specific Ser or Thr
side chains
• Glycosylphosphatidylinositol (GPI)
• Proteoglycans: Glycosaminoglycans (GAG)
Structure and Names of Common Monosaccharide
Varki, A. e. a., Essentials of Glycobiology. Cold Spring Harbor
Laboratory Press: Cold Spring Harbor, NY, 1999.
Major Classes of N-Glycans
Essentials of Glycobiology
Second Edition
6
O-GalNAc Glycans with Different Core Structures
Tn antigen
GalNAcαSer/Thr
Sialyl-Tn antigen
Siaα2-6GalNAcαSer/Thr
Peter-Katalinic J. Methods Enzymol. 2005;405:139-71.
Complex O-GalNAc Glycans with Different Core Structures
Essentials of Glycobiology
Second Edition
Chapter 9, Figure 2
O-linked Glycosylation
Esko, J.
9
O GlcNAc Modifications
O-GlcNAc
O-GlcNAc transferase
O
OH
OH
O
OH
O
O
+
OH
OH
UDP
O
O
+
OH
O
NH
NH
UDP
UDP-GlcNAc
O-GlcNAcase
– Modification at Ser/Thr residues;
– A ubiquitous and dynamic form of protein modification;
– Present in cytosolic proteins and nuclear proteins;
– Some
S
modification
difi i sites
i overlap
l with
i h phosphorylation
h h l i sites;
i
– Protein interactions, signal transduction, glucose sensing;
Implicated in insulin resistance, stress response, and regulation
Courtesy of
off proteosome’s
t
’ functions.
f ti
Yingming Zhao
Torres CR, Hart GW: J Biol Chem 1984, 259(5):3308-3317.
Wang, Z. & Hart, G.W. Clinical Proteomics 4 (2008).
General Structure of GPI Anchors
Essentials of Glycobiology
Second Edition
Chapter 11, Figure 1
Proteoglycans Consist of a Protein Core and One
or More Covalently Attached Glycosaminoglycan
chains
Essentials of Glycobiology
Second Edition
Glycosaminoglycans Consist of Repeating Disaccharide Units
Essentials of Glycobiology
Second Edition
Chapter 16, Figure 3
Keratan Sulfates Contain a Sulfated Poly-N-acetyllactosamine Chain
Essentials of Glycobiology
Second Edition
Chapter 16, Figure 4
Topics
• Glycoprotein classes
• Glycoprotein enrichments
• Release of p
peptides
p
and
glycosite analysis using MS
• Glycomic
Gl
i analysis
l i using
i MS
Glycoprotein Characterization
• Glycoprotein Identification
• Glycosylation type: glycan classes and
their conjugation sites
• Glycan structures
• Glycans on each glycosylation site
• Glycosylation occupancy
• Quantitation
16
November 28, 2012
Characterization of Glycoproteins and
Glycans Using MS
Glycoproteins
Proteolysis
Peptides +Glycopeptides
Analyzer
Separation
Enrichment
Glycopeptides
Detector
R l
Release
glycans
l
Formerly glycosylated peptides + Glycans
Ion trap
Fragmentation
Ionization
Formerly
glycosylated
peptides
Glycans
Sample preparation procedure
Sample plate
MALDI-MS-MSn
Isolation of Glycopeptides
1. D
1
Detection
t ti with
ith specific
ifi llectins
ti or antibodies
tib di
2. Chemical reactions with constituent
monosaccharides-A
monosaccharides
A general method labeling glycans
on proteins involves periodate oxidation followed by
Schiff base formation with amine- or hydrazide-based
probes.
probes
3. Metabolic labeling with chemically reactive
monosaccharides
4. Label in vitro using a purified using a purified
glycosyltransferase
5. LC-based enrichments
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18
1 Lectins
1.
• Carbohydrate-binding
y
gp
proteins
• More than 2,000 lectins
• Many lectins became commercially available
• Multiple lectins with distinct binding specificities
are used in combination or in series
•
•
•
Sharon N, Lis H. History of lectins: from hemagglutinins to biological recognition
molecules. Glycobiology. 2004 Nov;14(11):53R-62R.
http://proline physics iisc ernet in/cgi-bin/cancerdb/input cgi
http://proline.physics.iisc.ernet.in/cgi-bin/cancerdb/input.cgi
http://nscdb.bic.physics.iisc.ernet.in
Lectin Affinity Chromatography
Acronym
Metal Ions Specificity
Elution
Binding
Con A
Ca2+
Mn2+
a-Man>a-Glc 0.1-0.5 M a- N-linked
MeMan
SNA
-
Siaα2-6Gal
or GalNAc
G lNA
0.1-0.5
L t
Lactose
UEA
-
Α-L-Fuc
0.1-0.5 M L- Fuc
Fuc
α2-6-linked
Si
Sia
Geng, M.; Zhang, X.; Bina, M.; Regnier, F., Proteomics of glycoproteins based on affinity selection
of glycopeptides from tryptic digests
digests. J Chromatogr B Biomed Sci Appl 2001, 752,
752 (2),
(2) 293-306
293 306.
Xiong, L.; Andrews, D.; Regnier, F., Comparative proteomics of glycoproteins based on lectin
selection and isotope coding. J Proteome Res 2003, 2, (6), 618-25.
Lectin Affinity
y Capture,
p
, Isotope-coded
p
Tagging
gg g and
Mass Spectrometry to Identify N-linked Glycoproteins
Kaji H, et al. Nat Biotechnol 2003, 21(6):667-672.
Other Affinity Reagents
• Antibodies against glycans: Monoclonal antibodies
against Lewis X antigen. (Baeckström D et al. J Biol
Chem. 1991; 266: 21537-21547)
• Glycoprotein receptors: Mannose-6-phosphate
(M6P) receptors for M6P-motifs containing
glycoproteins
l
t i (Sl
(Sleatt DE ett all P
Proteomics
t
i 2005
2005; 5
5:
1520–1532)
2.Identification and Quantification of N-linked Glycoproteins
Using Hydrazide Chemistry and Mass Spectrometry
Proteolysis
Oxidation
Coupling
Zhang H, Li XJ, Martin DB, Aebersold R Nat
Biotechnol 2003, 21(6):660-666.
Wash
Isotope labeling
Release
Hydrazide beads
y
Glycans
Oxidized glycans
Succinic anhydride
3. Metabolic Incorporation of the N3-GlcNAc into Proteins
C2H5 O
O
C2H5 O
Extracellular
C2H5
OC2H5
O
HN
O
I t
Intracellular
ll l
HO
OH
GlcNAc
Kinase
N N N
OH
O
HN
OH
O
O
N N N
Phospho-NAcetylglucosamine
Mutase
Courtesy of
Yingming Zhao
UDP-GlcNAc
pyrophosphorylase
O-GlcNAc transferase
OH
OH
O
OH
O
O
OH
OH
UDP
O
O
O
OH
NH
+
N3
N3
UDP-N3-GlcNAc
NH
O-GlcNAcase
+
UDP
Chemoselective Conjugation Between N3 and a Phosphine
O
+
Ph2 P
S
Probe
Phosphine
Staudinger reaction
O
Probes:
 Biotin
 Flourescent dyes
 Solid beads
N
H
N3: small, inert, uncharged, non-polar, air-stable, and abiotic.Courtesy of
Science (2000), 287, 2007-2010.
Sprung, R.; Nandi, A.; Chen, Y.; Kim, S. C.; Barma, D.; Falck, J. R.; Zhao, Y.,
Tagging-via-substrate strategy for probing O-GlcNAc modified proteins. J
Proteome Res 2005, 4, (3), 950-7.
Nandi A, Sprung R, Barma DK, Zhao Y, Kim SC, Falck JR, Zhao Y:Global
identification of O-GlcNAc-modified proteins. Anal Chem 2006, 78(2):452-458.
Yingming Zhao
Id ntifi ti n of
Identification
f O
O-N
N3-GlcNAc-modified
Gl NA m difi d Proteins
P t in
OH
O-N3-GlcNAc
O
Conjugation
Affinity purified by
Affinity-purified
streptavidin-beads
HO
OH
O
HN
O
Biotin
2% SDS
8 M Urea
Digestion
HPLC/MS/MS for
protein identification
Courtesy of
Yingming Zhao
4. Carbohydrate-tags Via
Chemo-enzymatic Labeling
• Utilizes a genetically engineered
galactosyltransferase
• Incorporate
I
t ketone
k t
analogs
l
off galactose
l t
t cellular
to
ll l
O-glycosylated proteins
• Incorporate
p
a biotin label through
g coupling
p g with
aminoxy-biotin
•
•
Khidekel N, Ficarro SB, Peters EC, Hsieh-Wilson LC. Exploring the O-GlcNAc proteome: direct
proteins from the brain. Proc Natl Acad Sci U S A 2004;; 101:
identification of O-GlcNAc-modified p
13132-13137.
Khidekel N, Ficarro SB, Clark PM, Bryan MC, Swaney DL, Rexach JE, Sun YE, Coon JJ, Peters EC,
Hsieh-Wilson LC. Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative
proteomics. Nat Chem Biol. 2007; 3: 339-348.
Chemoenzymatic
y
Strategy
gy for Identifying
y g O-GlcNAcglycosylated Proteins from Cellular Lysates
Application of the Strategy Toward Crystallin
a
b
c
d
e
(a) MS analysis revealed the
tagged O-GlcNAc peptide.
(b) MS2 spectrum of the
precursor ion revealed the
signature loss of the ketone
ketonebiotin moiety.
(c) MS3 analysis revealed the
loss of the GlcNAc moiety.
moiety
(d) MS4 analysis generated
additional y and b ions that
were used to sequence the
peptide.
(e) Summary of the y and b
fragment ions.
5 LC
5.
LC-based
based Enrichment
•
•
•
•
•
Hydrophilic interaction LC (HILIC): the hydrophilic nature of
glycopeptides (Wada Y, Tajiri M,Yoshida S. Anal Chem 2004; 76:
6560-6565)
Size exclusion chromatography: masses of N-glycans are
larger than 1200 Da; thus, most N-glycopeptides could be
enriched by size-exclusion chromatography (Alvarez-Manilla G et
al. J Proteome Res 2006;; 5: 701-708))
Boronic acid: Boronic acid forms boronic diesters through
reaction of geminal diols (Sparbier K et al. J Chromatogr B 2006;
840: 29–36)
Strong cation exchanger (SCX): Glycopeptides with a terminal
sialic acid can be enriched by LC on an SCX column
(Lewandrowski U et al. Mol Cell Proteomics 2007; 6: 1933–1941)
Titanium dioxide: Sialic acid binds TiO2 (Larsen MR et al
al. Mol
Cell Proteomics 2007; 6: 1778–1787)
Topics
• Glycoprotein class
• Glycoprotein enrichments
• Release of p
peptides
p
and
glycosite analysis using MS
• Glycomic
Gl
i analysis
l i using
i MS
Release of N-Linked Glycans Using PNGase F
CH2OH
K/R
/
O
O
HO
HO
C
H2
N C
H
• PNGase conversion of Asn to Asp
N
• Mass shift confirms that the peptide was
glycosylated and localizes site of N-linked
S/T
HO
glycosylation
K/R
p
and gglycans
y
with 18O water
• Labelingg ppeptides
Cleavage converts
Asn to Asp
on the glycosylation site
CH2OH
K/R
O
O
NH2
HO
HO
HO C
C
H2
D
S/T
HO
K/R
Reducing glycan and 18O labeling
32
Mass Spectra
p
of Glyco
y and Nonglycopeptides After Releasing N-glycans
Enzymatic Release N-Glycans
Sigma
Enzymatic Release of O-Glycans
Sigma
Chemical Release of O-Glycans
Release of GPI
• Successful cleavage by GPI-specific
GPI specific phospholipases
can be assessed by subsequently analyzing samples
by MS, because removal of the GPI anchor causes a
shift
hift iin molecular
l
l mass. Thi
This iis a common di
diagnostic
ti
method for identifying the presence of a GPI anchor
on a protein of interest
• Another method is to treat the GPI-anchored protein
with nitrous acid, which cleaves the unsubstituted
glucosamine residue that links the glycan to the
phosphatidylinositol (PI).
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37
Enzymatic
y
Release of GPI Anchors
Cleavage sites of phospholipases. Phospholipase C cut just
before the phosphate attached to the R3 moiety.
moiety
Vikipedia
38
Release of Proteoglycans
gy
• Proteoglycans typically contain more glycan than protein.
They may be separated by agarose gel electrophoresis
and by ion-exchange chromatography, which separates on
the basis of the charge conferred by sulfate groups.
• Treatment of proteoglycans with GAG lyases will produce a
shift in mass to remove most of the glycan portion.
• Antibodies that recognize the remaining structures (“stubs”)
may be used in western analysis. The lyases cleave a 4,5
unsaturated uronic acid at the no reducing end
end. Anti-“stub”
Anti stub
antibodies recognize the sulfation of the penultimate Nacetylglucosamine or N-acetylgalactosamine residue.
November 28, 2012
39
Treating Glycoproteins with
Proteases
N-Glycans and O-glycans can be
obtained
bt i d nonselectively
l ti l by
b d
degradation
d ti
of the protein by proteases to generate
glycopeptides.
l
tid
November 28, 2012
40
Chemical Methods to Release Glycans
• Hydrazinolysis: A chemical method that uses
hydrazine to cleave amide bonds (e
(e.g.,
g the
glycosylamine linkage between a sugar residue
and asparagine
p g
or the acetamide bond in Nacetylhexosamines) to release both N-glycans and
O-glycans or, under controlled conditions, cleaves
only
l the
h N
N-glycans.
l
• Anhydrous hydrogen fluoride treatment: cleaves all
th lilinkages
the
k
off glycans
l
while
hil lleaving
i peptide
tid b
bonds
d
and glycopeptide linkage linkages of amino sugars
intact
November 28, 2012
41
Topics
• Glycoprotein classes
• Glycoprotein enrichments
• Release of p
peptides
p
and
glycosite analysis using MS
• Glycomic
Gl
i analysis
l i using
i MS
Control of Glycoconjugate Biosynthesis
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43
Control of Glycan
y
Structures
•
•
•
•
•
•
Expression and activities of enzyme
Nucleotide sugar availability
Kinetics of transports
Glycoprotein expression
Availability of glycosylation sites
Glycans are mixtures of variants (glycoforms)
on a core structure
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44
M
Mass
S
Spectrometric
i A
Analysis
l i of
f Glycans
Gl
• Sample preparation/purification
• Separation
• Ionization
November 28, 2012
45
Sample Preparation Strategies for Glycans
Joseph
p Zaia Mass Spectrometry
p
y and the Emerging
g g Field of
Glycomics. Chemistry & Biology (2008) 15, 881–892.
November 28, 2012
46
S p
Separation
ti n of
f Glycans
Gl
n f
for MS An
Analysis
l i
• Reversed phase LC/MS: reductive
amination is applied
pp
to increase the
hydrophobicity
• Graphitized carbon chromatographychromatography
MS: separate structural isomers
• Hydrophilic interaction chromatography
(HILIC)
• Lectin
L ti affinity
ffi it
November 28, 2012
47
Glycomics
y
Using
g Mass Spectrometry
p
y
• Putative structures are assigned to each molecular
q g
glycan
y
composition
p
ion based on the usuallyy unique
for a given mass.
• Prior knowledge of N- and O-glycan biosynthesis.
• Assignments can be confirmed in a second
experiment employing ESI-MS/MS instrumentation by
selecting
g each molecular ion for collisional activation
and recording its fragment ion spectrum.
• Additional information can be provided by MS
experiments
i
t on chemical
h i l and
d enzymatic
ti di
digests,
t th
the
choice of which is guided by the sequence
information provided by mass mapping and MS/MS
experiments.
November 28, 2012
48
Glycan Fragmentation Ions
Domon and
Costello, 1988
Joseph Zaia Mass Spectrometry and the Emerging Field of Glycomics. Chemistry & Biology
(2008) 15, 881–892.
November 28, 2012
49
Data from a Glycomics Study of N-glycans from Mouse Kidney
Essentials of Glycobiology
Second Edition
Chapter 47, Figure 6
Linkage Analysis
• The p
principle
p of this method is to
introduce a stable substituent (an ethery g
group)
p) onto each free
linked methyl
hydroxyl group of the native glycan.
• The glycosidic linkages, which are much
more labile than the ether-linked methyl
groups, are then cleaved with free
hydroxyl groups at the positions that
were previously involved in a linkage.
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51
Differentiation of Glycan Isomers Using
Tandem MS Analysis of Permethylated Glycans
November 28, 2012
52
Glycosidases Used for Structural
Analysis
Essentials of Glycobiology
Second Edition
Chapter 47, Figure 2
Quantitative Glycomics
• Label-free: Permethylation and MS
• Stable isotope labels for glycomics: based on
diff
differential
ti l stable
t bl isotope
i t
labeling
l b li (CH3I/CD3I) and
d
permethylation
• Reductive Amination Labeling: d0/d4 pyridyl amine
(PA)
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54
Determination of Anomericity
y
• Sequential
q
exoglycosidase
gy
digestions:
g
Cleavage by α- or β-exoglycosidases
y of specific
p
indicates the anomericity
terminal sugar residues.
• Many glycosidases are specific for both
monosaccharide residue and linkage
type, allowing detailed structural
conclusions, although the number of
such enzymes available is limited.
November 28, 2012
55
Glycoproteomics:
y p
Analysis
y
of
Glycopeptides with Glycan Attached
• Mass spectrometric analysis of glycopeptides is
made challenging by the differing chemical properties
of glycans and peptides
peptides.
• The ultimate goal of glycoproteomics is to quantify
p
y of g
glycosylation
y
y
in the p
proteome
the site occupancy
and the structures of glycoforms at each site.
56
Enrichment of Glycopeptides
y p p
and
MS Analysis
• Lectins for affinity capture
• Graphitized carbon solid phase
extraction
• Abundant peptide backbone
dissociation is observed for
glycopeptides using ETD.
ETD CAD results
in preferential fragmentation of the
glycan moiety of glycopeptides
glycopeptides.
November 28, 2012
57
Jonas Nilsson et al. Enrichment of glycopeptides for glycan structure and attachment site identification.
Nature Methods 6, 809 - 811 (2009)
The Informatics Challenges of
Diverse Glycomic Data
• Efforts to correlate large data sets obtained from
glycomic,
l
i transcriptomic,
t
i t i genomic,
i and
d proteomic
t
i studies
t di
have met with several challenges.
• Representation of glycan chemical structures is difficult
because of their complexity and branching patterns. The
use of single alphabet codes, as employed to describe
nucleic acid and amino acid sequences
sequences, is not applicable
to glycans.
p
bioinformatics
• The field is in need of a comprehensive
platform that stores, integrates, and processes data from
glycomic and other “omic” studies and disseminates them
in a meaningful fashion via the Internet to the scientific
community.
November 28, 2012
59
Databases and Bioinformatics
Platforms
•
•
•
•
•
•
•
GlycoSuiteDB,
y
, Sweet,, KEGG GLYCAN
The Consortium for Functional Glycomics (CFG)
EuroCarbDB
National Center for Glycomics and glycoproteomics
Glycomod: all possible compositions of a glycan structure
G
GlycoPep
DB: N-glycopeptide compositional assignment
Cartoonist: automated annotation of N-glycan MALDI TOF
mass spectra with cartoons representing the most
plausible glycan assemblies synthesized by mammals
using 300 manually determined archetypes.
November 28, 2012
60
Databases and Bioinformatics
Platforms
• Peptoonist:
p
automated identification of Nglycopeptides using a combination of MS and MS/MS
data
• Glyco-Peakfinder:
Gl
P kfi d rapid
id assignment
i
t off glycan
l
compositions, is intended to be entirely a de novo
platform for compositional analysis
• SysBioWare: carbohydrate assignment
• NCRR GlycomicsPortal
• SimGlycan
• Accurate Glycan Analyzer
• GlycoWorkbench
Gl
W kb
h
November 28, 2012
61
Summary
• Glycoprotein classes
• Glycoprotein enrichments
• Release of peptides and
glycosite analysis by MS
• Glycomics
y
analysis
y