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M. Osman SheikhSimone Kurz
Glycoanalytics:
release, reducing end tagging, LC/GC-analysis, linkage analysis
BCMB8130, GlycobiologyFeb 2, 2017, CCRC
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• 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
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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 !?
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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
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Glycoanalytics
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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
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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?
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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)
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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)
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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?
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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
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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
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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…
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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.
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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
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Step 1: Release glycans, O-glycans
Adapted from Esko, J
O-glycosidic linkage is sensitive to alkali (regardless of stereochemistry)
-elimination
GalNAc
Ser
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Step 1: Release glycans, O-glycans
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1. Reductive amination
2. Permethylation
Step 2: Derivatization of released glycans
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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
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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)
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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
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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
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HPAE-PAD Detection of Monosaccharides
Stalnaker SH, et al. J Biol Chem. 2011 Jun 17;286(24):21180-90.
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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=
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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.
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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
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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
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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.
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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.
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• 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
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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
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Questions so far??
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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
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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
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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
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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
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• 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
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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.
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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.
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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
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RP-separation of N-glycans (PNGase F released)
Elution patterns of PA-labelled mosquito N-glycans by RP-HPLC
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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)
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• 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)
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???
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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
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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
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Analysis of Hex3 HexNAc3 Fuc1 (H3N3F1) isomers
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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
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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
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• 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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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MS profiling / MS1
Fragment MS / MS2
[M+2Na]2+
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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
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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
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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
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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
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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
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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
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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)
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Aoki K. et. al J Biol Chem. 2007 Mar 23;282(12):9127-42
Isotope labeling with light/heavy iodomethane
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J. Proteome Res., 2009, 8 (8), pp 3816–3823
Quantitative strategies
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Quantitative strategies: IDAWG
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J. Proteome Res., 2009, 8 (8), pp 3816–3823
Quantitative strategies: IDAWG
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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
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• 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