Glycoproteins Release and Analyze
Transcript of Glycoproteins Release and Analyze
2/22/2013
1
GlycoproteinsRelease and Analyze
Ron Orlando
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Glycoconjugates• Glycolipids• GPI anchors• Proteoglycans
Glycobiology
g y• Protein N‐ and O‐linked glycosylation
o O‐linked GlcNAc• Nucleus, Cytoplasm• Associated with Phosphorylation
o Asn‐linked• Asn‐Xxx‐Ser/Thr Consensus Sequence• Complexity Increases with that of cell
o Ser/Thr‐Linked• Consensus? (proline, other indicators, not reliable)• Hard to release, no good endoglycosidases
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
2
Glycans Modify Proteins in Many Ways
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Importance of the Carbohydrate Chains attached to Glycoproteins
• 50‐90% of proteins are glycosylated
• Often required for biological activity
• Required for proper protein folding
• Protect against proteolysis and thermal denaturationdenaturation
• Participate in the immune response
• Change with condition of the cell/tissue
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
3
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Asn N-Acetylglucosamine
N-and O-linked Carbohydrate Chains of Glycoproteins
X
Ser/ThrN-type carbohydrate-peptide linkage
Ser/Thr
glucosamine
Mannose
Galactose
N-Acetylgalacosamine
Core Oligosaccharides
Se /
X
X
galacosamine
NeuAc
O-type carbohydrate-peptide linkage
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
4
Typical Structures of N‐linked Carbohydrate Chains
N Acet lAsn
High mannose type
Complex type
Asn-
N-Acetylglucosamine
Mannose
Galactose
N-AcetylN Acetylgalacosamine
NeuAcAsn
Hybrid type
Glycoprotein Synthesis, Folding, Modification and Transport
Protein glycosylation begins in the ER and continues in the Golgi. The type and specific glycan structure attached to proteins is highly dependent on cellular architecture enzymecellular architecture, enzyme expression, and chaperone proteins.
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
5
Synthesis and maturation of Asn‐linked oligosaccharides
•All eukaryotes have covalently bound carbohydrates attached through an amide linkage to Asn residues
•Many aspects of the pathway are conserved from yeast to plants and animals
•Common features:» Similar synthesis of the lipid‐linked precursor of the protein‐bound
oligosaccharide» Similar transfer to protein acceptor sequence motif: Asn X Ser/Thr» Similar transfer to protein acceptor sequence motif: Asn‐X‐Ser/Thr» Trimming of all of the glucose residues» Trimming of some of the mannose residues» Extension of the trimmed oligosaccharide by sugar addition in the Golgi
complex» Similar mechanism for acquiring nucleotide sugars into the Golgi complex
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Synthesis of lipid‐linked precursorN‐linked Oligosaccharide precursor synthesized on a lipid substrate
Transferred to protein as it emerges on the lumenal side of the ER membrane
Sugar donors: UDP‐GlcNAc, GDP‐Man, UDP‐GlcSugar donors: UDP GlcNAc, GDP Man, UDP Glc
Acceptor: Dolichol‐phosphate (α‐unsaturated polyisoprenoid))
2/22/2013
6
Synthesis of the lipid-linked precursor for Asn-linked glycosylation
Helenius, A. and Aebi, M. (2004) Ann. Rev Biochem 73, 1019-49
The steps in the synthesis of the lipid-linked precursor and the
Co‐translational glycosylation is the rulefrom yeast to plants and animals.
p pfinal structure are highly conserved.
Even the exceptions are indicative of a highly conserved process:
(From Alberts el al (1994) Molecular Biology of the Cell, 3rd ed.)
2/22/2013
7
Consensus sequence for N‐linked glycosylation
N‐X‐S/T ‐ X cant be P
Recent evidence suggests S/T‐X‐N can also “work”
Protein Folding
Calreticulin-KDEL
Glc II to GolgiComplex
Carbohydrate structures influence glycoprotein folding in the ER
Glc I Glc IIGlc Trans (Parodi enzyme)
(only unfolded glycoproteins)
Glc II
Glc II
ER-associated degradation
(ERAD)9
121110
87 6 5
4
3
21
OST
DolPP Calnexin
Proteasome Degradation+ Amino acids
ER membrane
Cytoplasm
Endoplasmic Reticulum
Timing? Recognition?
2/22/2013
8
Asn-linked Glycoprotein Maturation in the ER and Golgi
Early steps in N‐glycan processing:
• Many of the early steps in the ER are highly conserved in eukaryotes• Play roles in chaperone‐mediated folding, quality control, and disposal of terminally unfolded intermediates
• Critical for maturation of N‐glycans on cell surface and secreted glycoproteins
Endoplasmic Reticulum Golgi Complex
Glc II
G l i GnTI Golgi G TIIB
Legendα1,2-Manα1,3-Manα1,6-Manβ1,4-Manβ1,4-GlcNAcα1,2-Glcα1,3-Glc
ER membrane
OST Glc IGlc II
UGGT
ER Man IGolgi
Man IA/IB/ICGnTI Golgi
Man IIGnTIIB
Dol
P
P
N‐Glycan Branching Reactions
http://www.ccrc.uga.edu/~moremen/glycomics/
2/22/2013
9
Glycan Capping Reactions
http://www.ccrc.uga.edu/~moremen/glycomics/
N li k d l O li k d lN‐linked glycans O‐linked glycans
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
10
O‐Linked Protein Glycosylation
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Glycoprotein Microheterogeneity
• Glycosylation is a co‐/post‐translational modification– Glycans dependent on cellular architecture and individual enzymes
– Cell‐specific– Heterogeneous “glycoforms” at each site of glycosylation
• A glycoprotein purified to “homogeneity” is generally still a distribution of many combinations of glycoformsat each site
• “Microheterogeneity” of glycosylation a mechanism for modulation of protein activity, circulatory half‐life, etc.
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
11
LC‐MS Data for EPG I
Total ion chromatogramnd
ance
(arb
itrar
y un
its)
m/z 204 (HexNAc+) Why is the glycopeptide peak so
Abu
n
4000300020001000Time (sec)
glycopeptide peak so small and broad?
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
s)
Deconvoluted Spectrum of “Pure” Glycopeptide From EPG I: Carbohydrate Heterogeneity
danc
e (a
rbitr
ary
units
162 Da(hexose)
2850 3050 3250 3450 3650 3850 4050 4250molecular weight
Abun
d
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
12
Analysis of Ribonuclease B by ESI-MS
nits
)bu
ndan
ce (a
rbitr
ary
un
Mass to Charge ratio (m/z)
Ab
650 1050 1450 1850
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Analysis of a more heterogeneous glycoprotein by ESI‐MS
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
13
Analysis of Ribonuclease B by MALDI-MSits
)GlcNAc2Man5
Glycoforms ofRibonuclease B
bund
ance
(arb
itrar
y un
i
Non-glycosylatedRibonuclease B
GlcNAc2Man6
GlcNAc2Man7
GlcNAc2Man8
2 4 0
1 3 0 0 0 1 3 5 0 0 1 4 0 0 0 1 4 5 0 0 1 5 0 0 0 1 5 5 0 0 1 6 0 0 0 1 6 5 0 0 1 7 0 0 0
Mass to Charge ratio (m/z)
Ab
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
ΔM 510
More Glycan Heterogeneity Problems
Avidin
ndan
ce (a
rbitr
ary
units
) ΔM = 510M/ΔM = 30
ΔM = 70
Myglobin
Mass to Charge ratio (m/z)
Abu
n ΔM 70M/ΔM = 250
-2 7 0 0
14000 15000 16000 17000 18000 19000
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
14
uni
ts)
MALDI-TOF Data for EPG I
Media Apredictedmass
dan
ce (a
rbitr
ary
Media B
1500 Da
mass
30000 33000 36000 39000 42000
m/z
Ab
und
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Glycoprotein Microheterogeneity
Electrospray mass spectrometry of an intactglycoprotein generally yields a spectrum too complexf l d lfor conventional deconvolution or interpretation
MALDI‐TOF/MS generally too low resolution toreveal any details of glycoprotein structure, butaverage masses of some value
Generally unusual to say anything useful about proteinglycosylation from MS of intact material
• Exceptions: single sites of glycosylation, very low heterogeneity
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
15
Analytical Strategy for Glycoprotein CharacterizationTwo questions are central to glycoprotein analysis:
1. Where on the protein are glycan chains attached?
2 What are the structures of the glycan chains?2. What are the structures of the glycan chains?
Release and Analyze
MS
MS
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Glycosylation Site Identification Release Glycans and Tag Sites
For this type of site mapping one wants to release glycans but “tag” the sites of glycosylation, in this manner making ll l k h• Release glycans
• MS or MSn
• Data Base Search
all sites look the same
MS
MS
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
16
Endoglycosidases can be used to identify location of a glycoprotein in a cell or release glycans for
structural analysis
PNGAse F most common, but it will not cleave glycans with Fuc linked to the 3 position of the core
GlcNAc (present in plants)
EndoH sensitive EndoH resistant
PNGase A will but its not commercially available
Endoglycosidase work much better after proteolytic digestion
ER membrane
Endoplasmic Reticulum Golgi Complex
OST Glc IGlc II
Glc II
UGGT
ER Man IGolgi
Man IA/IB/ICGnTI Golgi
Man IIGnTIIB
Dol
P
P
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
17
100
80
706050
40
90
Rel
ativ
e In
tens
ity
1741.8, zero 18O Calculated Mass from AAsequence
Products of PNGase F digestion
Deglycosylation causes deamidationof glycosylated N producing D, which results in either a 1 or a 3 Da shift if the
H216O
30
20
100
% R
(m/z)
1754.01751.21748.41745.61742.81740.0
70
80
90
100 174
100
80
70
90
ensi
ty
Peg1743.7
one 18O
reaction is performed in H2
16O or H218O
H218O
The + 3 Da shift is easier to identify and eliminates confusion with naturally deamidated N residues
1740.0 1742.4 1744.8 1747.2 1749.6 1752.0Mass (m/z)
0
10
20
30
40
50
60
% In
tens
ity
1741
.72
6050
4030
20
100
% R
elat
ive
Inte
(m/z)
1754.01751.21748.41745.61742.81740.0
Peg
1741.7
zero 18O
This can also be performed with PNGase A
Fragment Ions Observed upon MS/MS Analysis of Tryptic Peptides
y y y y
1 -- 2 -- 3 -- 4 – 5 -- 6 -- 7 -- 8 -- 9 -- (K/R)H+
y9
y8y7
y6 y4y3
y2y5
b6
b5
b4
b3
b2 Modified amino acid
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
18
Incorrect Assignment of Glycosylation Sites
• Naturally occurring sites of deamidation
• Poor mass accuracy
• Data base search routines
• Trypsin
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
N‐linked glycosylation sites identified in a proteomic experiment on E. coli, which does not glycosylate
• Lower m/z accuracy of LTQ leads to more false positives
• Glycosylation sites identified by LTQ‐FT in 16O were either sites of deamidation or selection of the 113C peak as the precursor
• 18O labeling reduces incorrect assignments by reducing mass accuracy demands and eliminating natural deamidation being assigned as a glycosylation site
2/22/2013
19
Example of natural deamidation being identified as a site of N‐glycosylation
Example of mass error causing the incorrect assignment of an N‐linked glycosylation site
2/22/2013
20
Trypsin can also lead to the addition of 18O during PNGase F digestion
Peptides identified as containing N‐linked glycosylation sites Care was not taken to prevent tryptic activity during PNGase F deglycosylation in 18O enriched water
Sites “identified” as being glycosylated are denoted with as N*, and the consensus sequences for N‐linked glycosylation (N‐X‐S/T) are underlined.
Denoted N-linked glycosylation sites Charge XCorr ΔCn
IYGSIPVEFTQLN*FQFLN*VSYN*R(L) 3 5.46 0.71
IYGSIPVEFTQLNFQFLN*VSYN*R(L) 2 4.58 0.10
LQSFDEYSYFHN*R(C) 2 3 48 0 18LQSFDEYSYFHN*R(C) 2 3.48 0.18
NKLEGDASVIFGLN*K(T) 3 4.22 0.12
5 of the 7 sites identified must be incorrect
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
21
30
40
50
60
70
80
90
100
% In
tens
ity
174
100
80
706050
4030
90
% R
elat
ive
Inte
nsity
Peg
1743.7
one 18O
Trypsin can also lead to the addition of 18O digestion
PNGase F digestion in H218O after
removal of trypsin
PNGase F digestion in H218O with trypsin
f b l f
1740.0 1742.4 1744.8 1747.2 1749.6 1752.0Mass (m/z)
0
10
20
30
1741
.72
30
20
100
%
(m/z)
1754.01751.21748.41745.61742.81740.0
Peg
1741.7
zero 18Oafter boiling for 10 min
PNGase F digestion in H218O with trypsin
after resuspending in H216O overnight
70
80
90
100 174
100
80
70
90
tens
ity
1743.7
one 18O
1740.0 1742.4 1744.8 1747.2 1749.6 1752.0Mass (m/z)
0
10
20
30
40
50
60
% In
tens
ity
1741
.69
6050
4030
20
100
% R
elat
ive
Int
(m/z)
1754.01751.21748.41745.61742.81740.0
Peg
1741.7
zero 18O
What we do
• Perform deglycosylation site (N‐linked) experiments with H2
18O.2
• Re‐suspend in H216O for 24 hours after deglycosyalation
• Use a high resolution/accurate mass instruments (Q‐ToF, Orbitrap, FTMS)
• Accept only automated assignments with a false discover rate of 1% or better if visual inspection reveals at least 1 fragment ion containing the modified residue.
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
22
Release of O‐linked glycans: Enzymatic Cleavage
O‐glycanase (endo‐a‐N‐acetyl‐D‐galactosaminidase) from Diplococcuspneumoniae – hydrolyzes O‐glycosidic linkage between GlcNAc and Ser or Throf the disaccharide Galα1‐3GalNAc.
Appears to require unsubstituted residues; so must desialylate first by mild acid hydrolysis or neuraminidase.
Endo‐a‐N‐Acetylgalactosaminidase (Diplococcus pneumoniae)
(O‐GlycanaseTM)
Galß1‐3GalNAcα1‐Ser/Thr‐(Peptide)
Galß1‐3GalNAc + Ser/Thr‐(Peptide)
Limitation: Substitution with sialic acid or other saccharides blocks activity
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Release of O‐linked glycans: β‐Elimination
The conventional alkaline ß‐elimnation has been used in occasion where there are mg quantity of material present. Using this method, the base labile O‐glycosidic linkages b h Gl NA d h S /Th id f h ibetween the GlaNAc and the Ser/Thr residues of the protein are cleaved under mild alkaline conditions. Involves treatment of glycopeptides or glycoproteins with mild alkaline borohydride
Complete release of O‐linked oligosaccharides via β‐eliminationelimination.
Yields stable sugar alditols with destruction of the peptide.
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
23
Alkali-labile O-linked oligosaccharides
NaBH4 in NaOH,CH2OH
NHAc
RO O CH2
NH 2
C OC HNHC O
C H 2OH
NHAc
R OOH
NH 2
C O
NHC O
CH 2+•
C H 2OHOH
C H 2OH
NHAc
R O
NaBH4
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Release of O‐linked glycans: Ammonia‐based ß‐elimination
• Alternative ß‐elimination using ammonia‐based medium has recently been proposed (Yunping Huang , Rapid Commun. Mass Spectrom, 2002, 16, 1199‐1204).)
• Aqueous ammonia has been substituted instead of the NaOH used in the conventional method.
• The ammonia keeps the pH at around 11 which is suitable for ß‐elimnation of O‐glycans. It is also easily removed by evaporation.
• Borane‐ammonia complex (BH3 NH3) has replaced the NaBH4 reducing agent which was the cause of much of the salt.
• The elimination of the NaBH4 allowed the use of only small amount (40ul) of cation exchange resin for removal of the borane.ammonia complex.
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
24
Labeling sites of O‐glycosylation
The draw back for both the ß‐elimination and the hydrazinolysis method has been that the integrity of the protein component is not retained and no information can be obtained on the actual sites of O‐glycosylation.
A recent based catalyzed ß‐elimination method uses the addition of ammonia to the unsaturated amino acid, i.e. OH group is replaced by a NH2 group. The problem is that the difference of 1 mass unit is often difficult to observe.
A base catalyzed ß‐elimination is attractive with being able to label the original Ser/Thr residue so that the site of O‐glycosylation can be determined.
This can be followed by alkylamine labeling by the addition of methylamine or ethylamine
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Differential isotopic tagging of both cysteine and post‐translationally modified ser/thr through β‐elimination/Michael
addition with light (d0) and heavy (d6) DTT.
SICH2 C
O
NH2
β−Elimination
CNH
C
O
CH2
Alkylated Cysteine
Dehydroalanine(or
CNH
C
O
CH2H
CNH
C
O
CH2H
DTT (d0 or d6)
HSCH2CHCHCH2SH
Michael Addition
OH
OH
HSCd2CdCdCd2SH
OH
OH
Light DTT (d0) or Heavy DTT (d6)
O
(GlcNAc or phosphate)
OH OH
O-GlcNAc or O-phosphateModified Serine (or threonine)
CNH
C
O
CH2H β−Elimination
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
25
Analytical Strategy for Glycoprotein CharacterizationTwo questions are central to glycoprotein analysis:
1. Where on the protein are glycan chains attached?
2 What are the structures of the glycan chains?2. What are the structures of the glycan chains?
Release and Analyze
MS
MS
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Analysis of Released Glycans
• Common derivatizations/sample prep.
• MS
• MS/MS and MSn
• Exoglycosidase digestions
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
26
Why Permethylate the Oligosaccharide?
It increase the sensitivity of oligosaccharides for subsequent MS analysis. “Equalizes” the MS response for different glycans
The mass increase is not too much to shift the mass to higher massThe mass increase is not too much to shift the mass to higher mass range and decrease sensitivity.
It allows for diagnostic molecular ions which are easier to interpret than the native oligosaccharides.
Stabilizes negatively charged sugars (sialic acids for example)
M k t d t i t t blMakes tandem mass spectra more interpretable
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
These derivatives give diagnostic MS/MS or CID spectra which are important in obtaining sequence
MS/MS Analysis of Glycans
spectra, which are important in obtaining sequence information on the oligosaccharide.
The cross‐ring cleavages are more prominent in the spectra of Li+, Na+ or K+ adducts rather protonatedmolecular ions.
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
27
CID Fragment ions for N‐glycan Linked Glycans
OOHCH2OH
OH OO
CH2OH
OHO
CH2OH
OH OHO
Y3βY4βY5β
O
OHOH O
OCH2OH
OH
NHAc
OCH2OH
OH
NHAc
O
OOHCH2OH
OH
OH
OO
CH2OH
OH
NHAc
OCH2OH
OH OHOO
CH2
NH COCH2CH
OOH
O OH
NHAc
OHO
NH2
COOH
B2βB1β B3β
B1α B2α B3α
Y4αY5α Y3α
Y2 Y1 Y0
B4 B6C5
p [ ]
100 1580.09
12C
ESI‐MS analysis of released N‐linked glycans from T. cruzi
Show MS/MS capabilities of low abundance ions – glycans fragment very well
40
50
60
70
80
90
Rel
ativ
e A
bund
ance
1566.09
1784.27
1552.091603 18
13C13C
12C
12C12C
12C
* *
1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950m/z
10
20
301603.18
1770.27 1989.451810.36
1521.09 1975.361740.181947.09
1451.00 1931.911712.181498.001417.00 1655.09 1825.27 1902.45
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
28
ESI‐MS analysis of released N‐linked glycans from T. cruzi
Show MS/MS capabilities of low abundance ions – glycans fragment very well
100 1402.87
40
50
60
70
80
90
Rel
ativ
e A
bund
ance
1663.35
900 1000 1100 1200 1300 1400 1500 1600m/z
0
10
20
30 1384.86
1143.71 1508.611473.60 1589.361198.86866.53 939.73 1251.731061.66 1353.591017.63
1125.74
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
MS/MS of Glycans
• Provides non stereochemical sequence and branch points
• Can at times provide linkage information based on the appearance of fragments arising from cross‐ring cleavage
• Information of stereochemistry and anomericity are inferred from database – biosynthetic pathway (may not always be correct)
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
29
Glycosidase Digestions
Endoglycosidases Exoglycosidases
N N
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
On‐target Glycosidase Digestion Procedure
Matrix
MALDI‐MS
MatrixEnzymeSample
MALDIsample region
10 min@ room T
analysisdry
J. Colangelo, R. Orlando, Anal. Chem., 1999, 71, 1479‐1482.J. Colangelo, R. Orlando, Rapid. Commun. Mass Spectrom, 2001, 15, 2284‐2289.
2/22/2013
30
ΔMW = 2 204nits
)
Endoglycosidase/MALDI‐MS analysis of GP‐II (CST)
IntactΔMW 2,204
Abu
ndan
ce (a
rbitr
ary
un
After Release of N-linkedCarbohydrate Chains
2 5 5 0 3 05 0 3 5 5 0 4 05 0 4 5 5 0 5 05 0 5 5 5 0
Mass to Charge Ratio (m/z)
A
2550 3550 4550 5550
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Known Glycoprotein Glycans with a MW Equal to that Found on GP‐II (CST)
NANA α 2-3,6 Gal β 1-3,4 GlcNAc β 1- 2 Man α 1-6
Man β 1-4 GlcNAc β 1-4 GlcNAc
NANA α 2-3,6 Gal β 1-3,4 GlcNAc β 1-2 Man α 1-3
Non-variable Variable Core
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
31
Exoglycosidase/MALDI‐MS analysis of GP‐II (CST)
nits)
Intact- 2 NANA
bund
ance (arbitrary un
After Release α 2-6 NANA
Af R l β 1 4 G l
- 2 Gal
Mass to Charge Ratio (m/z)
A After Release β 1-4 Gal
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Deduced Structure of the Carbohydrate Chain Attached to GP‐II
of Canine Serum Transferrin
NANA α 2-6 Gal β 1-4 GlcNAc β 1-2 Man α 1-6
Man β 1-4 GlcNAc β 1-4 GlcNAc
NANA α 2-6 Gal β 1-4 GlcNAc β 1-2 Man α 1-3
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
32
Advantages of Exoglycosidase
• Can provide linkage, stereochemical, and anomeric configurationconfiguration
• Small amounts of material – enough for 3‐10 additional MS experiments
• Fast – can completely characterize a glycan in an hour or so
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Disadvantages of Exoglycosidase digestion/MS for Analyzing Glycoprotein Glycans
• Limited availability of exoglycosidases
• Cannot always provide complete glycan structures
D t k ll ith i t• Does not work well with mixtures
• Need prior information (database)
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
33
Problem: Identification of Individual GlycoformsEx. Major glycans attached to bovine fetuin
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Problem: Identification of Individual GlycoformsEx. Fully sialated triantennary glycans from fetuin
α2‐6
3 α2‐6α2‐3
3 α2 6
2 α2‐6/1 α2‐3
1 α2‐6/2 α2‐3 3 α2‐3
8 glycoformsfrom sialic acid
diversity
2/22/2013
34
β1‐48 glycoformsfrom sialic acid
Problem: Identification of Individual GlycoformsEx. Fully sialated triantennary glycans from fetuin
β1‐3
8 l f
diversity
16 glycoforms
8 glycoformsfrom sialic acid
diversity
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Problem: Identification of Individual GlycoformsEx. Major glycans attached to bovine fetuin
# of biosynthetically 4 14 16 48possible glycoforms
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
35
RP‐LC/MS of per‐methylated glycans released from fetuinGlycoform separation
XIC of +Q1 MI (4 ions): 1073.9 Da from Sample 1 (F_PMe_N_glycans_SIM_50B_120min) of SJ F Nglycans SIM Nexera 061411.wiff (Turbo Sp...No peak detection if number of points exeeds 1139
Max. 3.0e5 cps.
4.4e64.6e6 3
CGE‐LIF – APTS labeled fetuin glycans
2.0e6
2.2e6
2.4e6
2.6e6
2.8e6
3.0e6
3.2e6
3.4e6
3.6e6
3.8e6
4.0e6
4.2e6
Inte
nsity
, cps
16
5
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115Time, min
0.0
2.0e5
4.0e5
6.0e5
8.0e5
1.0e6
1.2e6
1.4e6
1.6e6
1.8e6
2 49
87
1312
10
1415
11from - Paula Jane Domann, Ana Carmen Pardos-Pardos, Daryl LudgerFernandes, Daniel Ian Richard Spencer, Catherine Mavis Radcliffe, Louise Royle, Raymond Allen Dwek and Pauline Mary Rudd, Separation-based Glycoprofiling Approaches using Fluorescent Labels, Proteomics DOI 10.1002/pmic.20070064
RP‐LC/MS of per‐methylated glycans released from fetuinRemoval of sialic acids decreases chromatographic complexity
XIC of +Q1 MI (4 ions): 1073.9 Da from Sample 1 (F_PMe_N_glycans) of F_PMe_SIM Nexera062111.wiff (Turbo Spray) Max. 1.4e5 cps.
2.2e6
2.4e6
2.6e6
2.8e6
3.0e6
3.2e6
3.4e6 NP LC – fluorescence , 2‐AB labeled fetuin glycans
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115Time, min
0.0
2.0e5
4.0e5
6.0e5
8.0e5
1.0e6
1.2e6
1.4e6
1.6e6
1.8e6
2.0e6
Intensity, cps
21.92 26.4728.144.58 43.9340.1720.27
XIC of +Q1 MI (2 ions): 1033.3 Da from Sample 1 (AF_Re_PMe_062111) of AF_Re_PMe_SIM Nexera062111.wiff (Turbo Spray) Max. 3.0e6 cps.
4.6e6
4.8e6
Chemical release of sialic acids
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40Time, min
0.0
2.0e5
4.0e5
6.0e5
8.0e5
1.0e6
1.2e6
1.4e6
1.6e6
1.8e6
2.0e6
2.2e6
2.4e6
2.6e6
2.8e6
3.0e6
3.2e6
3.4e6
3.6e6
3.8e6
4.0e6
4.2e6
4.4e6
Inte
nsity
, cps
10.74
1.43 8.34 13.9813.619.20
from - Paula Jane Domann, Ana Carmen Pardos-Pardos, Daryl LudgerFernandes, Daniel Ian Richard Spencer, Catherine Mavis Radcliffe, Louise Royle, Raymond Allen Dwek and Pauline Mary Rudd, Separation-based Glycoprofiling Approaches using Fluorescent Labels, Proteomics DOI 10.1002/pmic.20070064
400
2/22/2013
36
XIC of +Q1 MI (7 ions): 829.3 Da from Sample 2 (AF_Nglycan_Re_Beta1-3_control_PMe_48h_SIM) of AF_Exoglycosidase_111411.wiff (Turb... Max. 2412.5 cps.
2200
2400
2600
2800
3000
3200
3400
3530
Exoglycosidase Digestion of Asialofetuin Glycanswith β1‐4 Galactosidease
5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0Time, min
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
Inte
nsity
, cps
6.385.62 11.10 14.006.66 14.887.38 12.488.54 13.12 18.428.19 9.26 17.91 19.9810.46 17.7711.9710.15 18.5716.1415.92 17.145.33 5.76
XIC of +Q1 MI (7 ions): 829.3 Da from Sample 6 (AF_Nglycan_Re_Beta1-4l_PMe_48h_SIM) of AF_Exoglycosidase_111411.wiff (Turbo Spray... Max. 1070.0 cps.
6000
6500
7000
7273
Before
6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
Inte
nsity
, cps
6.30
11.16 19.618.84 11.915.07 9.97 13.3310.825.42 19.236.94 7.74 15.55 18.059.228.05 15.127.08 12.85 15.7713.75 16.30 17.77
After
Exoglycosidase Digestionof Asialofetuin Glycans
with β1‐3 Galactosidease
XIC of +Q1 MI (7 ions): 829.3 Da from Sample 2 (AF_Nglycan_Re_Beta1-3_control_PMe_48h_SIM) of AF_Exoglycosidase_111411.wiff (Turb... Max. 2412.5 cps.
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3530
nten
sity
, cps
Before
5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0Time, min
0
200
400
600
800
1000
1200
1400
1600In
6.385.62 11.10 14.006.66 14.887.38 12.488.54 13.12 18.428.19 9.26 17.91 19.9810.46 17.7711.9710.15 18.5716.1415.92 17.145.33 5.76
1.0e4
1.1e4
1.2e4
1.3e4
1.4e41.4e4
After
5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0Intensity, cps
15.227.668.335.02 11.69 15.495.676.33 12.016.96 13.5713.33 16.1710.848.939.6310.53 14.36
2/22/2013
37
Enzymatic deglycosylation of proteins (or peptides, etc.) using PNGase F releases glycans to yield a free reducing terminus (alditol) that is readily labeled by amines via the formation and reduction of a Schiff’s base
HILIC Analysis of Released and Labeled Glycans
Many amines have been applied to labeling glycans, in the current work Procainamide is favored.
O
Standard Analysis Conditions2.1 x 150 Penta‐HILIC, 80%B to 55%B in 25 minB:100%AcNA: 50 mM Ammonium Formate pH 4.45 (FA titration)0.6 mL/min, 60C400‐2000 @0.33s/0.1s each, +4.0 kV/12.5L/min, 250C DL
Mass: glycan + (235.325 – “O”)= Glycan + 219.32
H2N
NH
N
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
HILIC‐LC/MS of tagged glycans released from FetuinDatafile Name:[email protected] Name:D1000Sample ID:1L-4
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5mVDetector A 300nm
42.9
68
45.1
41
591
48.6
50
55.0
97
56.9
71
62.0
81
63.8
42
64 peaks total, 19 not assigned masses93.7% of UV signal assigned
60000
65000
70000
2:1114.10(+)(20.00)2:931.40(+)(20.00)2:1077.00(+)(20.00)2:1842.00(+)(20.00)2:1259.70(+)(20.00)2:1696.50(+)(20.00)2:1551.00(+)(20.00)2:1405.30(+)(20.00)2:1222.70(+)(20.00)2:BPC(+)(10.00)
1/10
77
55.1
25/1
551
57.0
04/1
551
598/
1405
48.6
58/1
405
7/14
05
42.9
79/1
223
45.1
56/1
223
25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 60.0 62.5 65.0 67.5 70.0 72.5 min-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
25.6
84
26.6
43 27.0
8527
.621
28.0
68
31.5
5131
.991
32.3
90 32.9
22
34.3
9134
.960 35
.284
35.6
08
36.6
0237
.000
39.2
4939
.680
39.9
6540
.640 41
.020
41.3
79 41.8
82 42.1
8742
.523
44.5
73
45.6
19 46.2
9546
.547
.258
48.2
20
49.2
60
50.1
0650
.541
52.0
08
53.2
2453
.946
54.2
60
55.7
7356
.503
58.3
4459
.078
59.6
9660
.185
61.5
66
63.3
79
65.1
0665
.439
68.3
04
25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 60.0 62.5 65.0 67.5 70.0 72.5 min
20000
25000
30000
35000
40000
45000
50000
55000
25.6
80/9
31
32.3
63/1
077
32.9
35/1
077
34.9
80/1
077
35.2
80/1
077
35.6
11
39.6
75/1
260
39.9
08/1
260
41.4
30/1
260
41.8
94/1
260
62.1
02/1
697
63.8
68/1
697
53.2
33/1
551
53.9
70/1
551
55.7
78/1
551
59.0
93/1
551
46.5
48.0
45/1
405
50.1
68/1
405
50.5
6 7
55.1
33/1
551
57.0
08/1
551
40.0
28/1
223
40.6
73/1
223
42.2
18/1
223
25.6
80/9
31
32.3
63/1
077
32.9
30/1
077
33.6
45/1
842
34.6
95/1
077
34.9
95/1
077
35.2
76/1
077
35.6
09/1
077
39.6
75/1
260 39
.908
/126
0
40.6
73/1
223
41.4
23/1
26041
.889
/126
042
.218
/122
3
42.9
78/1
223
45.1
55/1
223
46.5
97/1
405
47.2
50/1
405
48.0
48/1
405
48.6
62/1
405
49.5
60/1
405
50.1
63/1
405
50.5
66/1
405
53.2
33/1
551
53.9
70/1
551
55.1
19/1
551
55.7
78/1
551
57.0
01/1
551
59.0
93/1
551
61.5
83/1
697
62.1
02/1
697
63.8
70/1
697
2/22/2013
38
XIC of +Q1 MI (5 ions): 1076.5 Da from Sample 14 (SIM_Alpha1_Bovine_g1) of 031912.wiff (Turbo Spray)No peak detection if number of points exeeds 1096
Max. 1.6e5 cps.
NeuAc vs NeuGCHILIC‐LC/MS of tagged glycans released from α1 acid glycoprotein (bovine)
No peak detection if number of points exeeds 1096
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
7.5e5
8.0e5
8.5e5
9.0e5en
sity
, cps
38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5Int
Conclusions – LC‐MS
HILIC of glycans tagged at the reducing termini and RP of per‐methylated glycans both appear to be
bl f l i f i i l fcapable of resolving some of isomeric glycoforms
Additional work is needed to identify the components that are being separated.
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
39
Glycomics
• Identification from all glycans released from ll ti i tcells, tissue, organism, etc.
• Everything we just talked about for analyzing glycoproteins can be used to analyze mixtures of glycoproteinsof glycoproteins
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Glycomics necessitates development of an entire analyticaland informatics scheme parallel to that of proteomics:
Proteomics methods are still largely inadequate but we have high‐throughput analytical methods and informatics tools.
For glycomics, we still lack:1. Adequate methods for specific detection of glycoproteins or
glycopeptides in complex mixtures2. High‐throughput methods for sequence analysis of glycans3. Adequate glycan or glycan‐binding‐protein microarraysq g y g y g p y4. Automation of glycan or glycopeptide MS spectra5. An adequate carbohydrate structure database or ontology6. Data warehousing and curation tools for CH2O‐related data7. Database search capability for MS identification of glycans8. Informatics tools for linkage of CH2O data with proteomics
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
2/22/2013
40
Example of a typical workflow for the analysis of
glycoprotein glycans
Permethylated Glycans from wildtype Drosophila embryos
K. Aoki, M. Perlman, J. Lim, R. Cantu, L. Wells, M. Tiemeyer, Journal of Biological Chemistry, 2007, 282, 9127‐9142.
2/22/2013
41
LTQ‐FT MS/MS spectrum of N‐glycans cleaved with PNGase F from rhOVGP1
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013
Questions ?
Mass Spectrometry of Glycans and Glycoproteins ABRF Workshop 2013