M. Osman SheikhSimone Kurz

Glycoanalytics:

release, reducing end tagging, LC/GC-analysis, linkage analysis

BCMB8130, GlycobiologyFeb 2, 2017, CCRC

• Glycosylation is a non-template driven process

• Many building blocks are isomers/epimers

• Not one single linkage of building blocks to one another

• Majority of glycans are non-linear (branched)

• Free glycans are extremely hydrophilic

• Microheterogeneity is the norm on glycoproteins

• Glycosidic linkage to amino acid is labile

Biological/Technical Challenges

O

O

O

Requirements for StructuralDetermination of a Carbohydrate

-Identification of sugars.-Stereochemistry of each sugar.-Types of linkages.-Types of ring structures.-Anomeric configuration of each sugar.-Sequence of the different sugar residues.

AB

C

? How/Where !?

Levels of characterization of a (glyco)protein

Is it even glycosylated?

Carbohydrate Profiling (Monosaccharide analysis, Glycomics)Characterizes carbohydrates, but not locations or site

specific heterogeneity.

Glycosylation Site Mapping (Glycoproteomics)Identifies location(s) and site-specific heterogeneity

but may not give complete structures

Complete Characterization

Difficulty LevelLow

High

Glycoanalytics

Old approach – periodic acid/Schiff (PAS) staining(see, e.g., Zacharius et al. (1969) Anal Biochem 30, 148)

• Immerse gel with 12.5% trichloroacetic acid, Rinse,

• Immerse in 1% periodic acid (oxidising agent), Rinse and then immerse in fuchsin-sulphite in dark

• Wash with metabisulphite• Pink bands are suggestive of

glycosylation

The periodic acid/Schiff reagentstains vicinal diol groups foundmainly on peripheral sugars andsialic acids

Is my protein glycosylated?

https://www.thermofisher.com/order/catalog/product/24562

+ HIO4 =

1-2-glycol Aldehyde

+ HIO4 =

1-amino-2-hydroxy Aldehyde

A number of different lectins are used in glycobiology. These are mainly ofplant origin and are often used in biotinylated forms (detection with streptavidinor anti-biotin) or, for purification, attached to agarose beads

There are also anti-carbohydrate antibodies:anti-Lewis, anti-Tn, anti-α-Gal, anti-core α1,3-Fuc ...

Lectin specificity – despite much work, one of the big unknowns and oftenpoorly described!

Detection by glycan binding proteins (e.g. Lectins)

Lectin Binding preferenceWheat germ agglutinin GlcNAc/GalNAc/...Elderberry bark (Sambucus nigra) α2,6-sialic acid

Maackia lectin α2,3-sialic acid

Aleuria aurantia lectin fucose

Ricinus and Erythrina lectins β-galactoseConcanavalin A mannose/biantennary

Is my protein glycosylated?

Is my protein glycosylated?Glycan epitope screening using anti-carbohydrate antibodies

FIGURE 45.5Examples of different glycan antigens recognized by specific monoclonal antibodies. The antigens have the structures shown within the boxed area and are named as indicated. Usually, the antigen shown in the box can be linked to almost any glycan and antibodies will still recognize the antigen. (Essentials of Glycobiology 2nd Ed. Chapter 45)

P… PlasmaH… HaemocytesP+… αHexNAc‘ase treated P A… blood group A salivaB… blood group B saliva

Kurz S. et. al J Biol Chem. (2013) Aug 23;288(34):24410-28

Eastern Oyster Crassostrea virginica:

Is my protein glycosylated?Glycan epitope screening using anti-carbohydrate antibodies

Anti-O-Linked N-Acetylglucosamine antibody [RL2] (Abcam)

Geisler C, Jarvis DL. J Biol Chem. 2012 Mar 2;287(10):7084-97.Expression and purification of recombinant Sf-GNT-I and -II. The His6-tagged ectodomains of Sf-GNT-I and -II were expressed in recombinant baculovirus-infected Sf cells and purified from the extracellular fraction by nickel affinity chromatography, as described under “Experimental Procedures.” Samples of the purified proteins were treated with buffer alone (−) or PNGase-F (+), and then equal amounts of the untreated or treated proteins were analyzed by SDS-PAGE with Coomassie Brilliant Blue staining (CBB, left panel) or immunoblotting with an antiserum specific for His6 (α-6xHis, right panel). The calculated molecular masses of the recombinant His6-tagged Sf-GNT-I and -II ectodomains minus the signal peptides are 49.4 and 57.1 kDa, respectively.

• TFMS (trifluoromethanesulphonic acid) this destroys O-glycosidic bonds

• PNGase F (N-glycosidase) removes N-glycans whole (as long as they lack core a1,3-fucose)...Can also use PNGAse A

• Endoglycosidases (Endo F1, Endo H, specific for different types of N-glycan, one core GlcNAc retained on peptide)

Protein deglycosylation (change in SDS-PAGE mobility)Is my protein glycosylated?

Carbohydrate Profiling (Monosaccharide analysis, Glycomics)

Characterizes carbohydrates, but not locations or site specific heterogeneity.

To analyse N- or O-glycans, can examine masses of glycopeptides(peptide mapping by MALDI-TOF MS, Top-down sequencing)

OR

Cleave/Release the glycans from the polypeptide- remove by hydrazine (N2H4)- remove enzymatically (peptide:N-glycosidases)- remove by β-elimination (esp. O-glycans)- Hydrogen Fluoride [Hydrofluoric Acid, HF(aq)] to cleave phosphodiester-linked sugars

Step 1: Release glycans

Step 2: The choice is then to analyse with a label (fluorescent or radioactive) or without a label

Typical labelling methods often involve reducing the reducing-terminal sugar (i.e., the ring of the sugar unit that was directly attached to the protein is opened)

E.g., reduction with 3H-labelled borohydride (radiolabelling) or subsequent reaction with pyridylamine, aminobenzaminidine acid or another fluorescent group

Release of N- and O-linked glycans

Peptide-N-Glycosidase F, also known as PNGaseF, is a glycosidase that cleaves between the innermost GlcNAc and Asn residues of high mannose, hybrid, and complex

oligosaccharides from N-linked glycoproteins

Step 1: Release glycans, N-glycans

Trypsinize glycoproteins prior to enzymatic release of N-glycans…

EndoF1 Cleaves between two GlcNAc residues; Leaves one GlcNAc attached to Asn.Cleaves high-mannose and hybrid N-glycans. Does not cleave complex N-glycans.Can cleave in native or non-denaturing conditions.

EndoF1 will cleave sulfated high mannose oligosaccharides whereas Endoglycosidase H will not.

EndoH Also cleaves between two GlcNAc residues; Leaves one GlcNAc attached to Asn.Cleaves high-mannose and hybrid N-glycans, with and without core fucosylation. Does notcleave complex N-glycans. Also cleaves dolichol-linked structures.

PNGase A hydrolyzes N-glycan chains from glycoproteins/peptides regardless of the presence of xylose or fucose. [x = H or Man or

GlcNAc].

PNGase A differs from PNGase F in

that it cleaves N-linked glycans with or without α(1,3)-linked

core fucose residues.

https://www.neb.com/products/p0707-pngase-a

Step 1: Release glycans, O-glycans

Adapted from Esko, J

O-glycosidic linkage is sensitive to alkali (regardless of stereochemistry)

-elimination

GalNAc

Ser

Step 1: Release glycans, O-glycans

1. Reductive amination

2. Permethylation

Step 2: Derivatization of released glycans

Technique Fluorescent label

HPAEC2-Aminobenzoic acid (2-AA)

2-Aminobenzamide (2-AB)

Capillary Electrophoresis (CE)

9-Aminopyrene-1,4,6-trisulfonic acid (APTS)

2-Aminobenzoic acid (2-AA)

Gel Electrophoresis(FACES)

2-Aminoacridone (AMAC)

2-Aminobenzoic acid (2-AA)

7-Amino-1,3-naphthalenedisulfonic acid (ANDS)

8-Aminonaphthalene-1,3,6-trisulfonic acid (ANTS)

HPLC

2-Aminobenzoic acid (2-AA)

2-Aminobenzamide (2-AB)

2-Aminopyridine (AP; PA)

Mass spectrometry (MS)

2-Aminobenzamide (2-AB)

2-Aminobenzoic acid (2-AA)

2-Aminopyridine (AP; PA)

Common fluorophores used in glycan labeling

modified from: http://www.sigmaaldrich.com/technical-documents/articles/biology/glycobiology/glycan-labeling.html

1. HPLC methods such as Dionex HPAEC-PAD (in high pH, hydroxyls are ionised, and so anion-exchange can be used)

2. GLC/GC-MS (gas chromatography/mass spectrometry)

3. Electrophoresis method (FACE – fluorescence-assisted carbohydrate electrophoresis)

4. Capillary Electrophoresis (CE)

5. HPLC methods in which the sugars are derivatised

Carbohydrate Profiling (Monosaccharide analysis, Glycomics)

Monosaccharide analysisHPAED-PAD

• Carbohydrates – nonchromophoric. Max wavelength for pyranoses, such as glucose, is 189nm, thus poorly detected by UV absorption.

• Can be detected by refractive index detectors, however not very sensitive and stable…also incompatible with gradient elution.

• Mid 1980s – Dennis Johnson at Iowa State University developed an oxidative electrochemical detector for monitoring carbohydrates by LC…Commercialized by Dionex and others.

• HPAEC-PAD: High performance anion-exchange chromatography with pulsed amperometric detection

Monosaccharideanalysis example

HPAEC-PADHigh performanceanion-exchangechromatography withpulsed amperometricdetection

Works since hydroxylgroups of sugarsionize in high pH(pKa in range of 12-14)

Strong acid hydrolysis

Mild acid hydrolysis

HPAE-PAD Detection of Monosaccharides

HPAE-PAD Detection of Monosaccharides

Stalnaker SH, et al. J Biol Chem. 2011 Jun 17;286(24):21180-90.

O

OH

O

OH

H+

HO

C OHO

O

OH

HO

OH

HO

C OCH3

O

CH3OHOCH3

O

OH

HO

OH

HO

C OCH3

O

+(CH3)3SiX

OCH3

O

OTMS

TMSO

OTMS

TMSO

C OCH3

O

OCH3

O

OTMS

TMSO

OTMS

TMSO

C OCH3

O

OCH3

+ furanose form

Compositional Analysis Using Derivatized Glycansoften done by gas-liquid chromatography (GLC) Separation- Parastoo Azadi’s Summer Course

O

OH

O

OH

H+ O

OH

HO

OHOH

HO HONaBD4

CHOH

OH

OH

HO

OH

HO

D

Ac2O/Pyr.

CHDOAc

COAc

AcOCH

COAc

CHOAc

CH2OAc

Preparation of Alditol Acetates:

Preparation of Trimethylsilyl (TMS) Methyl Glycosides

Acid hydrolysis, Reduction, Peracetylation

Methanolytic cleavage, TMS reagentRuiz-Matute et al. Journal of Chromatography B (2011) Derivatization of carbohydrates for GC and GC–MS analyses

TMS=

Determination of the StereochemicalConfiguration of Glycosyl Residues

O

OT MS

OT MS

OT MS

CH2OT MS

OMe

2,3,4,6-tetra- O -trimethylsilyl-methyl -D -glucoside

Enantiomers: Identical physical properties, unable to separate.

O

OT MS

OT MS

OT MS

CH2OT MS

O

OT MS

TMSO

OT MS

CH2OT MS

CH3

CH2

CH3

O

O

OT MS

TMSO

OT MS

CH2OT MS

MeO

CH3

CH2

CH3

O

2,3,4,6-tetra- O -trimethylsilyl-methyl -L -glucoside

2,3,4,6-tetra- O -trimethylsilyl-2-(-)-butyl -D -glucoside

2,3,4,6-tetra- O -trimethylsilyl-2-(-)-butyl -L -glucoside

Diastereomers: Different physical properties, able to separate.

Adapted from Parastoo Azadi

Incorporation of an optically pure chiral aglycone (e.g., a [–]-2-butyl group), in combination with trimethylsilylation, allows the GLC separation of the D and L pair of isomers and thus determination of the absolute configuration of each

monosaccharide.

0

1000000

2000000

3000000

4000000

5000000

6000000

Ara

Ara Gal

Gal

Gal

Gal

Inositol

Man Glc

Time (minutes)10 302015 25

Glycosyl Composition Analysis using TMS

Xyl Glc

Parastoo Azadi

4 Gal peaks alpha, beta, D and L

Reference D.C.Dejongh, T. Radford, J.D.Hribar, S. Hanessian, M. Bieber, G. Dawson, and C.C. Sweeley, J. Am. Chem. Soc. 91, 1728-1740

Glycosyl Composition Analysis using AA or TMS…a comparison

Pros Cons

Ruiz-Matute et al. Journal of Chromatography B (2011) Derivatization of carbohydrates for GC and GC–MS analyses

TMS

A

A

FACE – fluorescence-assisted carbohydrate electrophoresis

1. Label glycans with negatively charged fluorophore

2. Resolve glycans using PAGE3. Detect and quantify using fluorescence

imagers/CCD cameras.

Fig. 1. ANTS- and ANDS-labeled glucose oligosaccharide standards. The same amounts of glucose oligomers were labeled with ANDS (net charge, −2) or ANTS (net charge, −3). The intensity of ANDS signal is about five times more than that obtained with ANTS, but ANTS-labeled oligosaccharides migrate faster. In this figure, the G6 oligomer is 20 pmol. Gao N. Methods. 2005 Apr;35(4):323-7.

Capillary Electrophoresis

• Separates ions based on their electrophoretic mobility with the use of an applied voltage.

• Neutral species…not affected, only ions move with the electric field.

• If two ions are the same size, the one with greater charge will move the fastest.

• For ions of the same charge, the smaller particle has less friction and overall faster migration rate.

• High separation efficiency• Small sample volume (1-10µL)• Fast separation (min - sec)• Predictable selectivity• Full automation• Quantification/good dynamic range• Reproducibility/robustness• Multi-capillary option (CAE, “Capillary Array

Electrophoresis”)• Multi-spectral imaging• Can couple to mass spectrometer • Requires labeling (reductive amination) and standards

Capillary Electrophoresis

Capillary Electrophoresis

Each sugar yields the same detector response, such that their relative quantities can be directly compared.

CE separation of (A) the mild acid hydrolysis product of fetuin after aldolase treatment. (B) The strong acid hydrolysis product after reacetylation and (C) monosaccharide standards. Capillary: 27 cm x 25 um bare fused silica; Buffer: 240 mM borate (pH 9.0); Field Strength: 740 Volts/cm; Injection: 0.5 psi for 5; Detection: Laser-induced fluorescence (excitation at 488 nm, emission at 520 nm).

https://sciex.com/therapeutic-protein-characterization/carbohydrate-labeling-and-analysis

Questions so far??

Technique Fluorescent label

HPAEC2-Aminobenzoic acid (2-AA)

2-Aminobenzamide (2-AB)

Capillary Electrophoresis (CE)

9-Aminopyrene-1,4,6-trisulfonic acid (APTS)

2-Aminobenzoic acid (2-AA)

Gel Electrophoresis(FACES)

2-Aminoacridone (AMAC)

2-Aminobenzoic acid (2-AA)

7-Amino-1,3-naphthalenedisulfonic acid (ANDS)

8-Aminonaphthalene-1,3,6-trisulfonic acid (ANTS)

HPLC

2-Aminobenzoic acid (2-AA)

2-Aminobenzamide (2-AB)

2-Aminopyridine (AP; PA)

Mass spectrometry (MS)

2-Aminobenzamide (2-AB)

2-Aminobenzoic acid (2-AA)

2-Aminopyridine (AP; PA)

Common fluorophores used in glycan labeling

modified from: http://www.sigmaaldrich.com/technical-documents/articles/biology/glycobiology/glycan-labeling.html

HPLC-separation of fluorescently-tagged glycans

• Separation is not always necessary, but offers advantage to reduce sample complexity, including elucidation of isomeric structures!

• Offline (HPLC fractionation first, then MS analysis) vs. Online (HPLC coupled to MS) approaches

Native glycan PA-labeled glycan

Stationary phases: polar having either plain silica or organic compounds such as amino, cyano, etc., groups bound to silica based supports. Mobile phases: non-polar such as hexane, heptane, etc. with small quantity of polar modifiers such as methanol, ethanol, isopropanol, etc.

Stationary phases: non polar alkyl hydrocarbons such as C-8 or C-18 chains bound to Silica or other inert supports.Mobile phase: polar

HPLC-separation of released glycans

1. Normal phase or Hydrophilic interaction chromatography (HILIC) separation by size and polarity

Zauner G et. al Electrophoresis. (2011) Dec;32(24):3456-66

2. Reverse phase separation by hydrophobicity (good for isomer separation)

Vreeker GC and Wuhrer M. Anal Bioanal Chem. (2017) Jan;409(2):359-378

3. Anion-exchange chromatography anionic structures (e.g. sialic acid or sulfate), weak (WAX), strong (SAX) and HIAX

Domann P et. al Proteomics. (2007) Sep;7 Suppl 1:70-6Neville DC et. al J Proteome Res. (2009) Feb;8(2):681-7

4. Porous graphitized carbon (PGC) isomers and anionic glycans due to hydrophobic interactions and electronic forcesPGC is powerful for native and reduced glycans(Note: PGC also used for desalting or enrichment of anionic glycans prior to MS)

Kolarich D et. al Methods Mol Biol. (2015) 1321:427-35Stavenhagen K et. al Chromatographia. (2015) 78(5-6):307-320

• Day-to-day variation and choice of stationary phase influences retention times on LC-separation need of standards

• To fully elucidate glycan structures, multidimensional approaches are required (HPLC combined with MS and MSn, NMR, exoglycosidase treatments, etc.)

Assignment of glycan structures based on their retention time and mass:

mass + retention time = structure

Assignment of glycan structures in terms of glucose units

RP-HPLC

NP-HPLC

• Mix of PA-labeled glucose oligomers prepared from dextran hydrolysate

• Differences in separation using Reverse phase (RP) and Normal phase (NP)-HPLC

• Run oligomer-mix as standard prior to analysis of glycan samples

Tomiya N. et. al Anal Biochem. (1988) May 15;171(1):73-90.

Elution of high-mannose glycans in NP- and RP-HPLC

RP-HPLC

NP-HPLC

• 1D: Fractionation by NP-HPLC

• 2D: Fractionation by RP-HPLC

• 3D: NMR of fractions

Elution positions based on glucose units are different for 2-AB, 2-AA and PA-

labeled glycans!!

Tomiya N. et. al Anal Biochem. (1988) May 15;171(1):73-90.Tomiya N. et. al Anal Biochem. (1991) Feb 15;193(1):90-100.

Examples of isomeric glycans

• Isomer = identical atomic composition arranged in a different structure

• Isobar = different atomic composition occurring at nominal (unit) mass resolution usually not a significant problem in structural characterization at MS profile stage. However, isobaric fragments may be formed during fragmentation, but identification requires higher mass resolving power and mass accuracy

RP-separation of N-glycans (PNGase F released)

Elution patterns of PA-labelled mosquito N-glycans by RP-HPLC

Distinct elution patterns of fucosylated PA-labelled mosquito N-glycans using a RP-Amide column (e.g. core α1,3 before core α1,6)

RP-separation of N-glycans (PNGase A released)

• Every collected fraction dried, resuspended in water and subjected to MALDI-TOF MS/MS labor-intensive!!!

• In MALDI-TOF only singly charged ions

• Interesting masses are manually subjected to MS/MS

Matrix-assisted laser-desorption/ionisation time-of-flight (MALDI-TOF)

???

MS/MS fragmentation

• In MALDI-TOF MS/MS predominantly B and Y ions

• In ESI-MS B/Y, C/Z and Cross-ring cleavages (A, X ions) of permethylated glycans possible

Domon and Costello Biochemistry. (1988) Mar 8;27(5):1534-43

Reducing end

Non-reducing end

GlycoWorkbench – a helpful tool in glycomics

Ceroni A et. al J Proteome Res. (2008) Apr;7(4):1650-9.

Freely available for multiple operation systems (Windows, MacOS, and Linux) and can be downloaded here: https://code.google.com/archive/p/glycoworkbench/downloads#makechanges

Analysis of Hex3 HexNAc3 Fuc1 (H3N3F1) isomers

β1,2 FDL

α1,2/3 ManLoss of HexNAc: ∆m/z=203

Loss of Hex: ∆m/z=162

Kurz, S. et. al J Proteomics. (2015) Aug 3;126:172-88

MS analysis of isomeric glycan structures

β1,2 FDL

α1,2/3 Man

β1,2 FDL

α1,2/3 Man

Exoglycosidase sequencingExamples of exoglycosidases removing terminal sugars

Fucosidase (α-1-3,4) for Lewis type from Xanthomonas sp

Fucosidase (α-1-3,4) from almond meal

Fucosidase (α-1-6) from bovine kidney

Galactosidase (ß-1-3(4)) from bovine testes

Galactosidase (ß-1-4) from Bacteroides fragilis

Galactosidase (ß-1-4,6) from jack bean

Galactosidase (α-1-3) from green coffee bean

Galactosidase (α-1-3,4,6) from green coffee bean

Mannosidase (all α-) jack bean

Mannosidase (α-1-2) from Aspergillus saitoi

Mannosidase (α-1-2,3) from Xanthomonas manihotis

Mannosidase (α-1-6) recomb.

N-acetylgalactosaminidase (α-1-4(3)) from chicken liver

N-acetylglucosaminidase (all ß HexNAcs) from jack bean

N-acetylglucosaminidase (ß1-2,3,4,6) from Xanthomonas manihotis

Neuraminidase (a-2-3, or-6, >>2-8) from Clostridium perfringens

• Does not work on permethylated glycans!!!

• Not all exoglycosidases are specific and/or available

• Steric hindrance can influence the access of the glycosidase to the sugar (e.g. α1,6 mannosidase can’t remove α1,6-linked mannose, if α1,3-linked is still present)

• Many glycosidases do not accept additional modifications on the sugars (e.g. methyls, sulfates, phosphates) removal of these modifications (e.g. methanolic HCl, hydrofluoric acid treatments) prior to exoglycosidase treatments

Restrictions in exoglycosidase sequencing

α1,6-man resistant

Permethylation - another common way for glycan and linkage analysis

Increases volatility of the glycans and improves sensitivity in MS

1. Glycosyl Linkage analysis of partially methylated alditol acetates by GC-MS analysis

2. Permethylation of released N- and O-glycan pools analyzed by ESI-MS/MSn looking for cross-ring cleavages

CH3I

O

OH

OH

O

O

OHHO

HOOH

OO

HO

HO

OH

OR

OOH

HOHO

OH

O O

OCH3

OCH3

O

O

OCH3

H3COH3CO

OCH3

OO

H3CO

H3CO

OCH3

OR

OOCH3

H3COH3CO

O

H3CO

R1OH B-R1O- R2I

R1OR2

2 M TFA

NaBD4

Ac2O/Pyr

CHDOAc

OAc

MeO

OMe

OAc

CH2OMe

CHDOAc

OMe

AcO

AcO

OAc

CH2OMe

CHDOAc

MeO

OMe

OAc

CH2OMe

OMe

+ +

Ether synthesis

CH3SCH3

O

O-CH2SCH3K+

KH

CHDOAc

MeO

OMe

OAc

CH2OMe

MeO

+

Parastoo Azadi

1. Glycosyl Linkages by Preparing Partially Methylated Alditol Acetates (PMAAs)

#1

#2

#3

#4

#1 #3 #4#2

R

OMeH

OMeH

R'

R

C O+ MeH

.+ +

R

C OMeH .

=

R'

CH OMe

R'

CH O+ Me

. +

=.

R

C OAcH

+

R'

CH O+ Ac

+

.

R'

CH OAc

R

C O+ AcHR

OAcH

OAcH

R'

. +

. +

>>.

R

C OMeH

+

R'

CH O+Ac

+.

R'

CH OAc

R

C O+ MeHR

OMeH

OAcH

R'

Parastoo Azadi

Primary fragments for PMAAs

Primary Fragments

CHDOAc

OMe

MeO

OMe

OAc

CH2OMe

118205

162161

45

Secondary FragmentsCH

C OMeH

C OAcH

CH2 OMe

O+ Me

- HOAc

CH

C OMeH

CH

CH2OMe

O+Me

m/z205 m/z145

Parastoo Azadi

Primary and Secondary Fragments for PMAAs of terminal Glucose

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

10 30

Time (minutes)

2015 25

Inositol

3,6-Gal

6-Gal

4-Glc

3-Gal

t-Gal

t-Ara(f)

t-Ara(p)t-Glc 5-Ara(f)

4-Xyl(p)

4-Gal

6-Glc

Intensity

t-Man

Parastoo Azadi

Glycosyl Linkage Analysis by GC-MS

2. Glycomics of permethylated glycans

Adapted from Morelle W and Michalski JC. Nat Protoc. (2007) 2(7):1585-602.

Enzymatically released glycans(e.g. N-glycans)

Chemically released and reduced glycans(e.g. O-glycans)

Non-reduced permethylated glycan

Reduced permethylated glycan

+16

Advantages of Permethylation in MS

• Can tell reducing end from internal or branch glycan• Leaves a “scar” (OH instead of OCH3) where linkage was• Permethylation and cations facilitate cross ring fragments:

• Li and heavy metals > Na+ > H+

219 204 236

Reducing end

Non-reducing end

10% sample in1 mM NaOH in 50% MeOH10 pmol external standard (malto-oligosaccharide)

Mehta N. et. al J Proteome Res. (2016) Sep 2;15(9):2969-80

Glycan analysis by Direct Infusion-NSI-MS

Automated MS/MS analysis Total Ion Mapping vs. Top10 approaches

Glycan analysis by Direct Infusion-NSI-MS cont’d

Full MS profile (MS1 profiling):− Detect molecular ions in m/z 500-2000− Positive ion mode [M+Na]+, [M+2Na]2+, [M+3Na]3+, [M+4Na]4+

− Acquisition in profile mode to determine the charge state− Only composition information e.g. NeuAc1Hex5HexNAc4Fuc1

Automated MSn analysis (Top10 Peak Picker, Total Ion Mapping)− Oligosaccharide sequence− Specific epitopes− Positive ion mode− Acquisition in centroid mode

Top10 Peak Peaker: data-dependent MS2

Total Ion Mapping (TIM): data-independent MS2

MS profiling / MS1

Fragment MS / MS2

[M+2Na]2+

Cross-ring fragmentation

Anthony, RM. et. al Science. (2008) Apr 18;320(5874):373-6.

α2,3

α2,6

Cross-ring fragmentation facilitated using for example Lithium containing infusion buffer for MS analysis

Monoisotopic (all-C12) sodiated mass=2792.3833Double charge C12 sodiated mass ((2792.3833-23)/2)+23=1407.6916Triple charge C12 sodiated mass ((2792.3833-23)/3)+23=946.1278Quad charge C12 sodiated mass ((2792.3833-23)/4)+23=715.3458

But since the highest peak for this structure will be the 1 C13 peak of 2793.3833The highest DC =1407.69+0.50=1408.19TC =946.13+0.33=946.46QC=715.35+0.25=715.6

As the structures get bigger the largest peak shifts to C13

Multiple charge states in NSI-MS

Singly charged

Doubly charged

Triplycharged

Quadcharged

C12 2792.383 1407.692 946.1278 715.3458

C13 2793.383 1408.192 946.4611 715.5958

2C13 2794.383 1408.692 946.7944 715.8458

The most intense peak here is the C13 peak

C13 peak 1408.19 z=2 mwt = ((1408.19 -23)*2)+23=2793.38

C12 1408.19 z=2 mwt=2792.36

Diff of 1.02 D

2C13 1408.19 z=2 mwt=2794.37

A diff of 2 D from monoisotopic mwt

Multiple charge states + isotope

sc dc tc qc

C12 2792.383 1407.692 946.1278 715.3458

C13 2793.383 1408.192 946.4611 715.5958

2C13 2794.383 1408.692 946.7944 715.8458

2C13C13C12

Multiple charge states + isotope

The most intense peak here is the C13 peak

C13 peak 946.46 z=3 mwt = ((946.46-23)*3)+23=2793.38

C12 946.12 z=3 mwt=2792.36

Diff of 1.02 D

2C13 946.79 z=3 mwt=2794.37

A diff of 2 D from monoisotopic mwt

sc dc tc qc

C12 2792.383 1407.692 946.1278 715.3458

C13 2793.383 1408.192 946.4611 715.5958

2C13 2794.383 1408.692 946.7944 715.8458

2C13C13C12

946.46 z=31408.20 z=2No 715.6

Multiple peaks same structure

sc dc tc qc

C12 2792.383 1407.692 946.1278 715.3458

C13 2793.383 1408.192 946.4611 715.5958

2C13 2794.383 1408.692 946.7944 715.8458

Quantitative strategies (absolute vs. relative)

Absolute quantitation• UV/Fluorescence (HPLC, CE) calibration curves needed to

correlate intensity with absolute quantity for 1:1 (label/glycan)

• MS use of internal standards in pmol

Relative quantitation • Normalized to most intense peak height/area

• Stable isotopic labeling

• in vitro: 12C- and 13C-Iodomethane labeling• in vivo: IDAWG

• Tandem mass tag approaches• Hydrazide tag (INSIGHT) • Amine tag (QUANTITY)

Moh ES et. al Proteomics Clin Appl. (2015) Apr;9(3-4):368-82; Yang S et. al Sci Rep. 2015 Nov 30;5:17585Walker SH et. al J Am Soc Mass Spectrom. 2013 Sep;24(9):1376-84

Quantification of glycans

1. Manually average intensities/charge

2. Use programs (e.g. Xtract) to deconvolute the spectra

Average intensity=(sc/1)+(dc/2)+(tc/3)+(qc/4)

Aoki K. et. al J Biol Chem. 2007 Mar 23;282(12):9127-42

Isotope labeling with light/heavy iodomethane

J. Proteome Res., 2009, 8 (8), pp 3816–3823

Quantitative strategies

Quantitative strategies: IDAWG

J. Proteome Res., 2009, 8 (8), pp 3816–3823

Quantitative strategies: IDAWG

Quantitative strategies

Labeling strategy

Glycomics

Label-free TIM/Prevalence

In-vitro 13C-CH3I or DC3I PermethylationUV/Fluorescence (peak area vs. standards)

In-vivo IDAWG

MS2 ??? TMT-approaches

• There are a multiple ways of analyzing glycoconjugates

• Combination of methods probably best approach

• Methods of choice are based on laboratory-specific preferences and the research questions being targeted

Take Home Points