glycoproteins release and analyze

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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


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Glycans Modify Proteins in Many Ways


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


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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


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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.


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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


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))

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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.)

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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?

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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/

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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


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O‐Linked Protein Glycosylation


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.


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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?


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


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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


Analysis of a more heterogeneous glycoprotein by ESI‐MS


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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


Ab


ΔM 510

More Glycan Heterogeneity Problems

Avidin

ndan

ce (a

rbitr

ary

units

) ΔM = 510M/ΔM = 30

ΔM = 70

Myglobin


Abu

n ΔM 70M/ΔM = 250

-2 7 0 0

14000 15000 16000 17000 18000 19000


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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


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


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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


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


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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


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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


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Incorrect Assignment of Glycosylation Sites

• Naturally occurring sites of deamidation

• Poor mass accuracy

• Data base search routines

• Trypsin


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

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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

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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


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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.


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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


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.


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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


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.


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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


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


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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


Analysis of Released Glycans

• Common derivatizations/sample prep.

• MS

• MS/MS and MSn

• Exoglycosidase digestions


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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


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.


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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


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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


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)


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Glycosidase Digestions

Endoglycosidases Exoglycosidases

N N


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.

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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


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


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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


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


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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


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)


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Problem: Identification of Individual GlycoformsEx. Major glycans attached to bovine fetuin


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

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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


Problem: Identification of Individual GlycoformsEx. Major glycans attached to bovine fetuin

# of biosynthetically 4 14 16 48possible glycoforms


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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


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.


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


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


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


Questions ?


glycoproteins release and analyze

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