-Techniques in Glycobiology-

-Techniques in Glycobiology-
Introduction to Glycan Analysis -Techniques in Glycobiology- NHLBI CardioPEG – Gerald W.Hart, August 19, 2013 Funded by NHLBI P01HL107153

Techniques in Glycobiology

Topics Overview of Glycoproteomic and Glycomic Approaches
Composition Analysis of Glycans. – Labs 8/21 & 9/4 Radiochemical and Bioorthogonal Labelling of Glycans – Lab 8/30 Detection of Glycoproteins in Polyacrylamide Gels Lectin Arrays to Analyze Glycoproteins – Lab. 10/11 Antibody Detection of Specific Glycans in Gels Detection of Specific Glyosphingolipids on TLC.- Lab. 10/7 &10/9 Methods for Isolation of Glycoproteins. Enzymatic and Chemical Release of Glycans from Glycoproteins. – Lab 8/23 Labeling Released Glycans – Lab 8/30 Methods to Separate Released Glycans –Lab 9/11 Glycosidases & Structural Analyses – Lab 9/18 Methylation Analysis for Linkage Determination – Lab. 8/26 MALDI-TOF Profiling of Glycans – Lab 10/14 Nuclear Magnetic Resonance Spectroscopy – Labs 10/16 & 10/18 GPI-Anchors & Glycosaminoglycans – Lab 9/25 Glycosyltransferases As Probes- Lab 9/27

Types of Glycoproteomics – Require Different Methods:
Figure 1. Glycoproteomics covers three major aspects differing in their sample preparation and analysis requirements: (1) In the protein-focused approach the glycan moiety is used to enrich glycopeptides from complex mixtures before enzymatically cleaving off the glycans and analysing the deglycosylated peptides. These analyses provide valuable insight into which peptides and thus which proteins were initially glycosylated. However, glycan-specific information is lost in this approach. (2) Glycan-focused glycoproteomics (or glycomics) characterises the glycan moieties after their removal from the peptide. Glycomics provides deeper insights into the particular structural features of the glycans, but this information can rarely be directly linked to individual proteins/peptides. (3) Glycoprotein-focused glycoproteomics is joining these sets of information, making use of the information obtained from the protein-focused and glycan-focused approaches. This allows us to link glycan and protein information, providing an additional level of information in studying the biological functions of glycoproteins. J. Mass Spectrom. 2013, 48, 627–639

Many Options for Glycoprotein Analyses:
NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 |

Level of Analysis & Approach is Determined by Biological Question:

Rough Estimates of Glycans by Colorimetric Assays (Lab. #1) Wed
Rough Estimates of Glycans by Colorimetric Assays (Lab. #1) Wed. August 21, 2013 Useful as a starting place to estimate the quantity of glycans in a tissue or cell. Phenol-Sulfuric Acid Assay for hexoses & pentoses. MBTH Assay for Hexosamines & Acetylhexosamines. Carbazole Assay for Uronic Acids BCA Assay for Reducing Sugars Analysis of Free Sialic Acids From Glycoconjugates by the Thiobarbituric acid assay.

Compositional Analysis by HPAEC-PAD
HPAEC-PAD=High Performance Anion Exchange Chromatography with Pulsed-Amperiometric Detection

Compositional Analysis by GC-MS of Alditol Acetates
Monosaccharides Reduce Peracetylate (acetic anhydride in pyridine)  GC-MS Alditols  1 peak per monosaccharide

Radiolabeling strategies for the detection of glycans
Chapter 47, Figure 1 Radiolabeling strategies for the detection of glycans Essentials of Glycobiology Second Edition FIGURE Radiolabeling strategies for the detection of glycans. Metabolic labeling with [3H]- or [14C]-monosaccharides allows detection of any glycoconjugate into which the monosaccharide is incorporated. 35SO4 can be used to trace glycosaminoglycans (GAGs), and labeled myo-inositol or ethanolamine can be used to follow GPI anchors. An example of the chemical introduction of a label using sodium [3H]-borohydride is also shown, and this technique can be applied to cell-surface glycoconjugates or to partially purified mixtures. Radiolabeled glycoconjugates can then be separated by gel electrophoresis (for glycoproteins or proteoglycans) or TLC (for glycolipids), the labeled compounds being detected by autoradiography. Radiolabeling is an efficient and sensitive method, but it is decreasing in use due to safety considerations.

Metabolic Labeling of Glycoproteins with Radioactive Sugars
Martin D. Snider Current Protocols in Cell Biology (2002) Nice discussion of issues: Competition with glucose – need to compromise. it is advisable to use these Precursors (open squares) in medium with reduced Glc (0.1 mg/ml). Lowering the Glc concentration further is not advisable because Glc starvation can affect glycan synthesis. cells must not exhaust the supply of Glc or other nutrients in the medium. “*Catch 22” – need to lower glucose, but you don’t want to alter glycosylation! Figure Pathways of sugar metabolism in mammalian cells. Compounds in boxes are precursors that can be taken up and incorporated into glycoconjugates. Pathways for conversion into the nucleotide sugar donors for biosynthesis are shown. Sugars in open boxes compete with Glc for uptake and incorporation. These are incorporated efficiently and can be used in the labeling procedures (see Basic Protocol and Alternate Protocol). Sugars in shaded boxes do not compete with Glc. They are incorporated less efficiently and should be used only in long-term labeling experiments (see Alternate Protocol). Abbreviations used: Fru, fructose; Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcA, glucuronic acid; GlcNAc, N-acetylglucosamine; GlcNH2, glucosamine; Man, mannose; ManNAc, N-acetylmannosamine; NeuAc, N-acetylneuraminic acid (a sialic acid); P, phosphate; UDP, uracil diphosphate; Xyl, xylose.

Analysis of glycans using the bioorthogonal chemical reporter strategy
Figure 1 | Analysis of glycans using the bioorthogonal chemical reporter strategy. (a) The per-O-acetylated azido sugars, represented here in generic form, passively diffuse across the cell membrane. In the cytosol, the acetyl groups are removed by promiscuous intracellular esterases, the azido sugar is activated to a nucleotide sugar donor (X . uridine diphosphate for GalNAz and GlcNAz, cytidine monophosphate for SiaNAz or guanosine diphosphate for 6AzFuc) and appended onto glycans in the secretory pathway (GalNAz, SiaNAz, 6AzFuc) or in the cytosol (GlcNAz). (b) The chemical structures of azido sugars discussed in this protocol and the glycan subtypes labeled: per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz), per-O-acetylated N-azidoacetylgalactosamine (Ac4GalNAz), per-O-acetylated N-azidoacetylglucosamine (Ac4GlcNAz) and per-O-acetylated 6-azidofucose (Ac46AzFuc). (c) The Staudinger ligation: triaryl phosphines react selectively with alkyl azides to form an amide bond to the alkyl substituent, such as an azido sugar. Scott T Laughlin & Carolyn R Bertozzi VOL.2 NO.11 | 2007 | NATURE PROTOCOLS

Essentials of Glycobiology
Chapter 48, Figure 3 Metabolic and covalent labeling of glycans for in vivo imaging of the glycome Essentials of Glycobiology Second Edition Drawback is low efficiency of incorporation

The bioorthogonal chemical reporter strategy for imaging glycans.
Fig. 2. The bioorthogonal chemical reporter strategy for imaging glycans. (A) Glycans can be metabolically labeled with unnatural sugars bearing a chemical reporter group. The chemical reporter group, typically an azide or terminal alkyne, can be detected in a second step by covalent reaction with a probe. (B) Bioorthogonal reactions used to visualize chemical reporters appended to unnatural sugars (R). Azides can be detected by Staudinger ligation with triaryl phosphines, resulting in the formation of an amide linkage between the reporter and the probe. Azides and terminal alkynes can be detected by reaction with each other via CuAAC, forming in a triazole linkage between the reporter and probe. The azide can also be detected by Cu-free click chemistry with strained cyclooctynes. This latter reaction avoids the use of a cytotoxic metal catalyst. Fig. 3. Azide- and alkyne-bearing monosaccharides used for metabolic labeling of glycans. Sialic acids may be labeled with N-azidoacetylneuraminic acid (SiaNAz), ManNAz, 9-azido N-acetylneuraminic acid, and alkynyl Man- NAc. GalNAc-containing glycans and O-GlcNAc-labeled glycans may be labeled with azides by using GalNAz. O-GlcNAc-labeled glycans may also be labeled with GlcNAz. Fucose-containing glycans may be labeled with 6AzFuc or alkynyl fucose. Structures are shown in peracetylated form, which are typically used for metabolic labeling experiments. After cell entry by passive diffusion, the acetyl groups are cleaved by cytosolic esterases. Laughlin & Bertozzi 12–17 ! PNAS ! January 6, 2009 ! vol. 106 ! no. 1

Imaging of Glycans in Living Zebrafish!
Zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at63 hpf to detect newly synthesized glycans – Temporal resolution during development. Fig. 6. Application of the bioorthogonal chemical reporter strategy for in vivo imaging of glycans. (A) Zebrafish embryos were treated with azidosugars during their development, resulting in metabolic labeling of glycans with azides. The azides were visualized by reaction with fluorescent DIFO reagents. (B) An example of a zebrafish embryo metabolically labeled with Ac4GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at 63 hpf to detect newly synthesized glycans. (Left) Single z-plane brightfield image. (Left Center) z-projection of DIFO-647 fluorescence. (Right Center) z-projection of DIFO-488 fluorescence. (Right) z-projection of DIFO-647 and DIFO-488 fluorescence merge. (Scale bar, 100 !m.) Laughlin & Bertozzi 12–17 ! PNAS ! January 6, 2009 ! vol. 106 ! no. 1

Detection of Glycoproteins in SDS-PAGE
Figure 1. Overview on protein detection procedures in a proteomic experiment based on 2-DE. The relationships between qualitative and quantitative assessment of a sample’s components are emphasized. Examples are given for the main alternative procedures. aa, amino acid; AB, amido black; PR, Ponceau Red, RuBP, ruthenium II tris (bathophenanthroline disulfonate) or other reversible stain. Proteomics 2006, 6, 5385–5408

Figure 4. Outline of the procedures for glycoprotein investigation in a proteomic experiment based on 2-DE. Under ‘detection’, italics for reagents to be used on gels, underlined for reagents to be used on blots, italics underlined for reagents to be used both on gels and on blots. Proteomics 2006, 6, 5385–5408

FIG. 1. A comparison of staining patterns of human erythrocyte membranes separated bySDS PAGE in gels with different degrees of cross-linking. Coomassie blue (CB), “Stainsall” (SA), and periodic acid-Schiff (PAS) staining of erythrocyte membranes are compared in a 5, 7.5 or 10% Tris-acetate buffered (TA, pH 7.4) polyacrylamide gel electrophoresis (PAGE) system containing 0.1% sodium dodecyl sulfate (SDS). Arabic and Roman numerals indicate the proteins and glycoproteins of human erythrocyte membrane numbered according to Fairbanks et al. (9). The colors seen in the SA stained gels are indicated alongside the 10% SA gel: red(R); purple (P); blue (B). The lipid containing dye front is shown by (*). * Early work on RBC Surface Glycoproteins * periodic acid-Schiff (PAS) staining ANALYTICAL BIOCHEMISTRY 71, (1976)

Selective Staining of Glycoproteins on 2D-Gels:
SYPRO Ruby total protein stain Fig. 1 Total protein and glycoprotein- stained 2-D gels. Whole saliva was subjected to isoelectric focusing on a pH 3–10 nonlinear IPG strip, which was then resolved in the second dimension on a 12.5% SDSPAGE gel. The resulting gels were either stained with SYPRO Ruby total protein stain (a) or Pro-Q Emerald 488 glycoprotein stain (b). Labeled are the protein spots that were identified from the Sypro Ruby-stained gel either by MALDI-TOF MS or LC-MS/MS in our previous work [8] or by LC-MS/MS in this work Pro-Q Emerald 488 glycoprotein stain – Proprietary dye reacts with periodate reacted glycans. Clin Proteom (2009) 5:52–68

Lectin Blotting is a Powerful Tool to Detect Glycoproteins:
Clin Proteom (2009) 5:52–68

DSL Lectin UEA I Lectin AAL Lectin LEL Lectin
Fig. 2 2-D lectin blots. Twodimensional gels containing whole saliva were transferred and probed with either lectin DSL (a), LEL (b), UEA I (c), AAL (d), or WGA (e). The spots were matched to the Sypro Ruby-stained gel shown in Fig. 1a and scored for having high, medium, low, or no reactivity (Supplemental Table) Clin Proteom (2009) 5:52–68

OMICS A Journal of Integrative Biology Volume 14, Number 4, 2010
FIG. 2. Summary of lectin microarray technologies. Lectin microarrays have been fabricated using various immobilization technologies (1) using lectins derived from multiple sources (2). The binding of glycoconjugates from a diverse set of analytes of biological samples (3) have been detected using multiple detection approaches (4). Lectin microarrays provide a rapid, sensitive, and high-throughput screening tools for glyco-profiling resulting in important biomedical applications (5). Simultaneous application of lectin microarrays with complementary glycomics technologies holds potential for decoding the ‘‘glycocode’’ and development of new and effective diagnostics and therapeutics. SAM, self-assembled monolayers; SELDI, surface enhanced laser desorption ionization; NHS, N-hydroxysuccinimide esters; Au, gold-coated surfaces.

Detection of Glycoproteins by Glycan-Specific Antibodies:
Eg. Pan-Specific antibody to O-GlcNAc O-GlcNAc is Abundant on Nuclear & Cytosolic Proteins

Detection of Glycosphingolipids by Antibodies After TLC:
Figure 1. Work flow diagram of the multiple TLC immunostaining procedure combined with multicoloring and matched with IR-MALDIo- TOF mass spectrometry. (I) Separation of a GSL mixture on a silica gel precoated TLC plate. (II) Visualization of GSLs by multiple immunostaining combined with multicoloring. Three different GSL structures are localized sequentially in three immunostaining cycles. In each of the single immunostaining rounds, the TLC plate is overlaid with a primary antibody with defined GSL specificity, followed by incubation with an enzyme-conjugated secondary antibody. Visualization of the desired GSL is generated upon incubation with a chromogenic substrate. Alkaline phosphatase-labeled secondary antibodies in conjunction with Fast Red (1) and BCIP (2) revealed red- and blue-colored GSL bands, respectively; horseradish peroxidase in combination with DAB (3) gave black labeled GSL bands. Bound antibodies were denatured and the conjugated enzymes were inactivated by heat treatment after each immunostaining cycle (described in more detail in the Experimental Section). (III) Structural characterization of GSLs in Fast Red-, BCIP-, and DAB-stained bands after multiple immunostaining by IR-MALDI-o-TOF-MS directly on the TLC plate. Anal. Chem. 2009, 81, 9481–9492

Multiple immunostaining combined with multicoloring
of TLC-separated neutral GSLs from human erythrocytes Figure 3. Multiple immunostaining combined with multicoloring of TLC-separated neutral GSLs from human erythrocytes. (A) Total GSL amounts of 8 μg were applied for orcinol staining (orc) and 4 μg for cumulative immunostain. Multiple TLC immunostaining was performed by three sequential immunostaining rounds on the same chromatogram (see Figure 1) with anti-Gb3Cer (1), anti-LacCer (2), and anti-Gb4Cer antibodies (3). Fast Red-AP, BCIP-AP, and DAB-HRP stains resulted in red-, blue-, and black-colored bands of Gb3Cer, LacCer, and Gb4Cer, respectively. (B) Red-colored Gb3Cer bands were scanned at 520 nm, blue-stained LacCer at 650 nm, and black-labeled Gb4Cer at 750 nm. The predominant fatty acid chain length of the GSLs reflected by the densitometric signals are indicated. Anal. Chem. 2009, 81, 9481–9492

Typical Protocol Used to Analyze Glycoproteins:
H. Geyer, R. Geyer / Biochimica et Biophysica Acta 1764 (2006) 1853–1869

Affinity methods for isolation of glycoproteins.
Methods for isolation of glycoproteins. (a) Antibody and lectin capture reagents have been used to isolate glycoproteins [6,31]. (b) An enzyme can be used to install a ketosugar that can be selectively modified with an aminooxy reagent [13!!]. Azidosugars can be incorporated by metabolic labeling and detected using either (c) the Staudinger ligation [23!!,26] or (d) a [3+2] Huisgen cycloaddition [24,25]. Glycoproteins can be isolated on the basis of their chemical reactivity: (e) geminal diols react selectively with boronic diesters displayed on magnetic beads [11,12!], or (f) can be converted to aldehydes that react with immobilized hydrazide [8!,29,32]. Current Opinion in Chemical Biology 2007, 11:52–58

Unnatural sugars used in the isolation of glycoproteins.
Unnatural sugars used in the isolation of glycoproteins. In metabolic labeling strategies, naturally occurring sugars (shown on the left) can be replaced by chemically tagged analogs (shown on the right): (a) N-azidoacetylglucosamine (GlcNAz) [18]; (b) N-azidoacetylgalactosamine (GalNAz) [19]; (c) 2-ketogalactose (KetoGal) [39]; (d) N-azidoacetylmannosamine (ManNAz) [17]; (e) N-levulinoylmannosamine (ManLev) [16]; (f) N-thioglycolylmannosamine (ManNTGc) [22]; (g) azidofucose [20!,21!]; (h) N-azidoacetyl sialic acid (SiaNAz) [40]; (i) N-levulinoyl sialic acid (SiaLev) [41]; (j) N-thioacetyl-neuraminic acid (Neu5TGc) [22]; (k) 9-aryl-azide sialic acid (9-AAz-NeuAc) [33!!]; (l) 5-aryl-azide sialic acid (5-Aaz-NeuAc) [41]. The chemical handles enable glycoprotein isolation. Current Opinion in Chemical Biology 2007, 11:52–58

Options for Releasing Glycans From Proteins:
L. R. Ruhaak & G. Zauner & C. Huhn & C. Bruggink & A. M. Deelder & M. Wuhrer Anal Bioanal Chem (2010) 397:3457–3481

Endoglycosidases for N-Glycans:

PNGase F works best on Denatured Proteins or Glycopeptides,
But Does Not Release All N-Glycans Equally. (PNGase A from Almonds cleaves all N-Glycans as long as peptide has COOH and amino terminus)

Several Endo-Glycosidases For Releasing N-Glycans
Are Available Commerically PNGase F useful in Site Mapping by MS Sigma-Aldrich Chemical Deglycosylation Strategies

Unlike PNGase F, O-Glycosidase is Highly Specfic:
Sigma-Aldrich Glycoprotein Handbook

Enzymatic De-Glycosylation of O-Glycopeptides
Requires Multiple Enzymes. Sigma-Aldrich Chemical Deglycosylation Strategies

Alkaline ß-Elimination releases Ser/Thr-linked Sugars:
[3H]Gal [3H]Gal(1-4)GlcNAcitol GlcNAc -Elimination O | NaOH/NaBH4 Often done with borohydride to prevent ‘peeling’.

Differential glycan analysis using isotope labeling and mass spectrometry.
Figure 6. Differential glycan analysis using isotope labeling and mass spectrometry. a) Schematic of a typical isotope labeling experiment for quantitative mass spectrometric analysis of glycans. b) Examples of isotopic labeling reactions. The reactive open-chain aldehyde form of the glycan is shown. ACS CHEMICAL BIOLOGY VOL.4 NO.9 • 715–732 • 2009•

BEMAD Site Mapping Replacement of O-GlcNAc with DTT
100 1314.3 PSVPVSerGSAPGR O-GlcNAc DTT BAP 1247.4 1421.7 m/z 1100 1650 MALDI-TOF Replacement of O-GlcNAc with DTT Using b-elimination/michael addition Alkaline induced b-Elimination C N H O CH2 (Serine-O-GlcNAc) (dehydroalanine) GlcNAc Strategy for O-GlcNAc/O-Phosphate site mapping Relative intensity HSCH2CHOHCHOHCH2SH (DTT) Michael Addition Relative intensity 1. Provides tag for Affinity enrichment 2. Tag is stable in mass spectrometer C N H O CH2 DTT (or BAP) Relative intensity BEMAD Site Mapping Wells et al. Molecular & Cellular Proteomics (2002)

Performic acid treatment
Control Cells Experimental Cells Performic acid treatment <--Trypsin--> O-GlcNAc-peptides O-phosphate-peptides Add Controls: Alkaline Phosphatase Alkaline Phosphatase O-GlcNAcase O-GlcNAcase BEMAD Heavy DTT BEMAD Light DTT Mix Heavy and Light DTT labeled O-GlcNAc-peptides Heavy and Light DTT labeled O-phosphate-peptides Thiol Enrichment Thiol Enrichment Johns Hopkins LC-MS/MS LC-MS/MS

Combination of Enzymatic Tagging with ‘Click-It’
Chemistry & Solid-Phase BEMAD to Map Sites: J. Am. Chem. Soc., 125 (52), , 2003. BEMAD replaces the labile modification with a stable nucleoplilic group

Hydrazine hydrolysis has been found to be effective in
the complete release of unreduced O- and N-linked oligosaccharides. –Destroys the Peptide! Hydrazinolysis Hydrazine hydrolysis has been found to be effective in the complete release of unreduced O- and N-linked oligosaccharides. Selective and sequential release of oligosaccharides can be accomplished by initial mild hydrazinolysis of the O-linked oligosaccharides at 60 °C followed by N-linked oligosaccharides at 95 °C. Hydrazine hydrolysis leaves the glycan intact but results in destruction of the protein component. Glycan release is accomplished by the addition of fresh anhydrous hydrazine to a salt-free, freshly lyophilized glycoprotein sample. The volume of hydrazine should produce a protein concentration of 5 to 25 mg/ml. The reaction mixture should be capped immediately. For the release of both N- and O-linked glycans incubation should be for 4 hours at 95 °C. For the selective release of O-linked glycans, incubation at 60 °C for 5 hours is suitable. Sigma-Aldrich Chemical Deglycosylation Strategies Anhydrous Hydrazine = Rocket Fuel!!

Strategies for the analysis of released glycoprotein-glycans.
H. Geyer, R. Geyer / Biochimica et Biophysica Acta 1764 (2006) 1853–1869

Periodate Oxidation to Label Glycans:
Bayer, E.A., et al., Meth. Enzymol., 184, 415 (1990).

Labeling of glycans. A 2-Aminobenzoic acid (2-AA) labeling via reductive amination, B 1-phenyl-3-methyl-5-pyrazolone labeling via a Michael-type addition, C labeling with phenylhydrazide, and D glycan permethylation L. R. Ruhaak & G. Zauner & C. Huhn & C. Bruggink & A. M. Deelder & M. Wuhrer Anal Bioanal Chem (2010) 397:3457–3481

L. R. Ruhaak & G. Zauner & C. Huhn & C. Bruggink & A. M. Deelder & M
L. R. Ruhaak & G. Zauner & C. Huhn & C. Bruggink & A. M. Deelder & M. Wuhrer Anal Bioanal Chem (2010) 397:3457–3481

H. Geyer, R. Geyer / Biochimica et Biophysica Acta 1764 (2006) 1853–1869

Methods to Separate Released Glycans:

Tools of Glycome Profiling

HPLC of Glycans Normal-Phase Chromatography of Underivatized Glycans:
A Type of HILIC HPLC of Glycans Normal Phase Chromatography Carbohydrate Research, 94 (1981) C7-CV

Analysis of N-linked oligosaccharides from human "1-acid
Glycoprotein - 2-AB-labeled oligosaccharides HPAEC Fig. 6 Analysis of N-linked oligosaccharides from human "1-acid glycoprotein. 2-AB-labeled oligosaccharides obtained were analyzed by high-pH anion-exchange chromatography with fluorescence detection (a) or high-performance LC (HPLC) with an amide-80 column and fluorescence detection (b). (Reproduced from [168] with permission) RP-HPLC L. R. Ruhaak & G. Zauner & C. Huhn & C. Bruggink & A. M. Deelder & M. Wuhrer Anal Bioanal Chem (2010) 397:3457–3481

Normal Phase Chromatography of 2-AB labeled Glycans:
Glycoprotein Analysis Manual Sigma-Aldrich

ammonia-based B-elimination at both 40C and 60C.
Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia-based B-elimination at both 40C and 60C. Figure 1. Comparison of fetuin digested with PNGaseF and fetuin that has undergone ammonia-based B-elimination at both 40C and 60C. The pattern of the charged N-linked oligosaccharides is similar, and at 60C ammonia-based elimination appears to fully remove the N-linked oligosaccharides. In the neutral region, O-linked oligosaccharides are visible in the ammonia-based β elimination chromatograms. 2-AA Labeling: Samples were labeled with 2-AA using 2-AA Labeling Kits (QA-Bio), following the directions provided. After labeling, 450 μl DI water was added to the samples, and the samples were dialyzed against ≥400 mL DI water overnight using 500 Da MWCO cellulose acetate membranes (The Nest Group). After dialysis, samples were dried under vacuum and resuspended in 200 μl DI water. Chromatography: Buffer A was 2% glacial acetic acid and 1% diethylamine (Sigma) in acetonitrile. Buffer B was 5% glacial acetic acid and 4% diethylamine in HPLC grade water, adopted from Anumula and Dhume (Glycobiology, 1998). A multistep gradient was used: from 0 to 2 minutes, Buffer A was held at 70%, from 2 to 80 minutes, Buffer A decreased lin- early to 5%, from 80 to 95 minutes, Buffer A was held at 5%. The flow rate was held constant at 0.2 ml/min. Fluorescent data was acquired over the entire period, and chromatograms were created using Chromeleon software. Oligosaccharide Profiling of O-‐linked Oligosaccharides Labeled with 2 Aminobenzoic Acid (2-‐AA) Elisabeth A. Kast and Elizabeth A. Higgins -‐ GlycoSolu􀆟ons Corpora􀆟on, Worcester, MA

Reverse-phase HPLC with fluorescence detection of 2-AB-labeled glycans
RNAase B Ovalbumin Fig. 7 Reverse-phase HPLC with fluorescence detection of 2-ABlabeled glycans released from a 10 μg RNase B, b 30 μg ovalbumin, and c 30 μg fetuin. The structures of the labeled oligosaccharide peaks are shown in d. Species that were coeluted in the same fluorescence peak are indicated by letters a and b. The peaks corresponding to the species that were eluted early (less than 20 min) containing two or more sialic acids are not shown. Circles mannose, squares GlcNAc, diamonds galactose, triangles fucose, stars sialic acid, dashed lines " linkage, solid lines ! linkage, vertical lines 1–2 linkage, horizontal lines 1–4 linkage, forward slashes 1–3 linkage, backslashes 1–6 linkage; wavy lines unknown linkage. (Reproduced from [180] with permission) Fetuin L. R. Ruhaak & G. Zauner & C. Huhn & C. Bruggink & A. M. Deelder & M. Wuhrer Anal Bioanal Chem (2010) 397:3457–3481

Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorption/ionization (MALDI)–Fourier transform ion cyclotron resonance (FTICR)–MS Fig. 8 Analysis of 2-AA-labeled total plasma N-glycans by matrix assisted laser desorption/ionization (MALDI)–Fourier transform ion cyclotron resonance (FTICR)–MS. Samples were prepared as described previously [34], desalted by porous graphitized carbon solid-phase extraction, and analyzed by MALDI-FTICR-MS using a 2,5-dihydroxybenzoic acid matrix. The low-mass range (top) and the high-mass range (bottom) were measured using different ion-transfer times. The inset shows the isotope pattern of registered glycan species. Yellow circles galactose, green circles mannose, blue squares N-acetylglucosamine, purple diamonds sialic acid, red triangles fucose L. R. Ruhaak & G. Zauner & C. Huhn & C. Bruggink & A. M. Deelder & M. Wuhrer Anal Bioanal Chem (2010) 397:3457–3481

A natural glycan microarray approach with reductively aminated glycans
Fig. 9 A natural glycan microarray approach with reductively aminated glycans. a Glycans are derivatized, b fractionated by HPLC, c analyzed by MALDI time of flight MS(/MS), d immobilized on microarray epoxide slides, e assayed for protein interaction, and f the data obtained are interpreted. (Reproduced from [202] with permission) L. R. Ruhaak & G. Zauner & C. Huhn & C. Bruggink & A. M. Deelder & M. Wuhrer Anal Bioanal Chem (2010) 397:3457–3481

Ion-Exhange Chrom. – Separate Glycans by Charge
Ion-Exhange Chrom. – Separate Glycans by Charge. DEAE-Sephadex Separation of Glycans with 1, 2 or 3 Sialic acids Size After Desialylation FIG. 6 (left). Ion exchange chromatography on DEAE-Sephadex of Bio-Gel P-&fractionated oligosaccharides derived from site 2. A, a mixture of oligosaccharide standards either untreated (-) or neuraminidase- treated (- - -) was fractionated by DEAE-Sephadex A-25. The eluting gradient of NaCl was begun at fraction 21. The numbers aboue the peaks represent the number of sialic acid residues on the oligosaccharides contained in the designated peaks. Peak 1 is composed of a fucosylated, biantennary oligosaccharide derived from IgM; peak 2 is a mixture of an oligosaccharide with the identical backbone and source as in peak I and a nonfucosylated, tetraantennary oligosaccharide derived from al-acid glycoprotein; and peak 3 is composed of an nonfucosylated, triantennary oligosaccharide derived from fetuin. B, site 2, peak III, oligosaccharides derived from 5 h-labeled H-2Kk analyzed as in A. C, site 2, peak IV, oligosaccharides derived from 5 h-labeled H-2Kk analyzed as in A. D, site 2, peak V, oligosaccharides, untreated. The peaks of radioactivity for the breakthroughs of treated oligosaccharides for A, B, and C were 1040, 633, and 687 counts/min, respectively. FIG. 7 (center). Ion exchange chromatography on DEAE-Sephadex of Bio-Gel P-6-fractionated oligosaccharides derived from site 1. A, site I, peak I, oligosaccharides derived from 5 h-labeled H-2Kk either untreated (-) or neuraminidase-treated (- - -); B, site I , peak II, oligosaccharides analyzed as in A; C, site 1, peak Ill, oligosaccharides analyzed as in A; D, site I , peak IV, oligosaccharides analyzed as in A. The peaks of radioactivity for the breakthroughs of treated oligosaccharides for A, B, C, and D were 1109, 886, 848, and 832 counts/min, respectively. FJG. 8 (right). Gel filtration chromatography of asialo-oligosaccharides on Bio-Gel P-6. Mixtures (1:l) of two neuraminidase-treated samples derived from 5 h-labeled H-2Kk devoid of sialic acid (Figs. 6 and 7) were cochromatographed on Bio-Gel P-6. A, site 2, peak III, and site 2, peak IV, oligosaccharides; B, site 1, peak III, and site 1, peak IV, oligosaccharides; C, site 1, peak I, and site I , peak II, oligosaccharides; D, oligosaccharides from site 2, peak V, treated with a-mannosidase and re-chromatographed.

Gel Filtration Chromatography – Separate Oligosaccharides by Size.
Beta -Galase Beta-Hexosaminidase Alpha-Mannosidase Beta-Mannosidase Beta-Hexosaminidase Alpha-Fucosidase

Dionex with Pulsed-Amperometeric Detection – A Breakthrough in the mid-80s
At pH 13 Carbohydrates ionize and Become Charged molecules. J. Chromatogr. A 720 (1996)

Derivatization of Glycans for Electrophoresis

Capillary Electrophoresis of Glycans
as published in BTi October 2004

Fluorophore-Assisted Carbohydrate Electrophoresis:
From Glyko

Fluorophore-Assisted Carbohydrate Electrophoresis (FACE)

Hydrophilic Interaction Liquid Chromatography (HILIC)
(Formally called “Normal Phase” Chromatography) Figure 1. Glycan separation comparison of 3.0 and 1.7 mm sorbent HPLC columns. Ten picomoles of fetuin 2-AB-labeled glycans was separated using 3.0 mm TSK gel Amide-80 column in Alliance 2695 (top) and 1.7 mm BEH Glycan column in UPLC system (bottom). Sialylated biantennary and triantennary glycans including positional isomers were baseline resolved using 1.7 mm column in UPLC system in 45-min gradient time. The UPLC separation was done in gradient 65–55% B in 45 min at 0.5 mL/min using 2.1mm150 mm, and the HPLC separation was done in 65–55% B in 50min at 0.45 mL/min using 2.0mm150 mm. The column temperature was at 401C on both runs. Peak assignment was performed on the basis of HILIC-ESI-MS experiments. Notably, to our knowledge the Man5 glycan reported here has hitherto not been reported for fetuin and might represent a contamination. The peaks labeled with same numbers indicate the presence of isomers. Taken from [19] with permission.

HPLC-HILIC profile of N-glycans released from heavy chain
human serum IgG Figure 3 | HPLC-HILIC profile of N-glycans released from heavy chain human serum IgG. This profile was obtained using a methodology adapted from ref. 48. The scale of glucose units (GU) is based on the elution of the 2-AB–labeled glucose ladder. Only the major species have been shown in this particular figure, but the whole nested family of 36 different structures, including arm-specific isomers, can be identified using this technique. NATURE CHEMICAL BIOLOGY | VOL 6 | OCTOBER 2010 |

Hydrophilic Interaction Liquid Chromatography (HILIC) Many Stationary Phases

Glycosidases used for structural analysis
Chapter 47, Figure 2 Glycosidases used for structural analysis Essentials of Glycobiology Second Edition FIGURE Glycosidases used for structural analysis. (Left) A biantennary N-glycan is shown with exoglycosidases that can be used to remove each monosaccharide sequentially. Exoglycosidases act only on terminal sugars. Also shown are endoglycosidases that remove the intact N-glycan. N-Glycanase cleaves the GlcNAc-Asn bond, releasing the N-glycan and converting asparagine into aspartate. Endoglycosidase F cleaves between the core N-acetylglucosamine residues and therefore leaves an N-acetylglucosamine attached to the protein. (Right) Endoglycosidase H (Endo H) cleaves between the core N-acetylglucosamine residues of oligomannose or hybrid N-glycans that have at least four mannose residues as shown. Endo H does not act on complex N-glycans.

Exoglycosidase Sequencing using HILIC of 2AB-Labeled Glycans

Chapter 47, Figure 3 Essentials of Glycobiology Second Edition A simple example of methylation analysis, showing a structural motif that may be found in the polysaccharide glycogen Methyl Ether is Acid Stable 100% RXN Reduction Eliminates Anomeric Mixtures Methylation Analysis is Used to Determine Linkages. Enzymes or NMR are used to Determine Anomericity. FIGURE A simple example of methylation analysis, showing a structural motif that may be found in the polysaccharide glycogen. An α1-4- linked glucose chain has an α1-6-linked glucose branch. Successive steps of methylation, hydrolysis, reduction, and acetylation result in a set of compounds in which the linkage positions can be identified by their acetylation. For the sequence illustrated, the terminal α1-6 glucose gives rise to glucitol acetylated at C-1 and C-5; the two α1-4 glucose units on either side of the branch point are acetylated at C-1, C-5, and C-4, and the branch-point α-glucose is acetylated at C-5, C-1, C-4, and C-6. Methylation analysis cannot give details of sequence or anomericity, but when combined with NMR spectroscopy, it has been used to elucidate the structures of many complex and unusual glycans.

Data from a glycomics study of N-glycans from mouse kidney
Chapter 47, Figure 6 Data from a glycomics study of N-glycans from mouse kidney Essentials of Glycobiology Second Edition Assumptions Based Upon Known Pathways Typically Confirmed by MS/MS and enzymes. FIGURE Data from a glycomics study of N-glycans from mouse kidney (courtesy of Anne Dell, Imperial College London). (Top) MALDI-TOF profile of neutral N-glycans released from kidney extracts by peptide N-glycosidase F and permethylated. The glycan structures indicate the most probable sequences attributable to each major molecular ion. (Bottom) Part of the evidence for these assignments is provided by fragment ion data derived from ESI-MS/MS experiments. In this experiment, the doubly charged molecular ion at m/z [M + 2Na]2+, which corresponds to the singly charged MALDI-TOF signal at m/z 2837 [M + Na]2+, was selected for collision-induced decomposition. Sequence-informative fragment ions arising from the antennae and core are shown in the cartoon. Glycosidic bonds that have been cleaved during the MS/MS process are “tagged” by the presence of a hydroxyl group (HO–). All other hydroxyl groups on the glycan carry methyl groups because the sample was permethylated prior to analysis.

Example of MALDI-TOF Profiling of Glycans
MALDI-TOF mass spectra of (a) Glycodelin-A (GdA), m/z 1500–4500; (b) Glycodelin-A, m/z 3650–3800; (c) bovine pregnancy-associated glycoproteins (PAG), m/z 2900–5900. Panels a and c cover equivalent mass spans (3000 Da) and for clarity only some of the major signals are annotated. Note that the PAG N-glycan profile is dominated by a single component at m/z [29]. In contrast, the GdA spectrum [28] showed a high level of heterogeneity and more than 130 components are observed over this m/z 3000 range. This is exemplified by the expanded data in Panel b. The peaks which are labelled with ‘x’ are either derived from known contaminants (b) or are derivatisation artefacts (c). Current Opinion in Structural Biology 2009, 19:498–506

1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans
Chapter 47, Figure 4 1H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans Essentials of Glycobiology Second Edition NMR is the most Powerful Method For Glycan Structure Determination. Provided 2 things are True: Enough Material Oligosaccharide Is pure! FIGURE H-NMR spectrum of a mixture of two trisialyl triantennary N-glycans obtained as alditols from bovine fetuin by hydrazinolysis, followed by purification on HPAEC (Table 47.1) and subsequent reduction with sodium borohydride. The spectrum was recorded at 500 MHz, using a solution of 100 μg of the glycan mixture in 0.7 ml of D2O in a 5-mm NMR tube at pH 6.5 and 23°C.

Chapter 47, Figure 5 NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F Essentials of Glycobiology Second Edition FIGURE NMR and MS data used in the determination of the structure of the complex pneumococcal capsular polysaccharide 17F. Mild base treatment of the polysaccharide specifically breaks the phosphodiester linkage between rhamnose (Rha) and arabinitol and removes O-acetylation to give an oligosaccharide with the structure shown in the box. p denotes a pyranose ring. (A) 1H-NMR spectrum of the intact polysaccharide (at 70°C). Prominent, well-resolved signals come from the anomeric protons (H-1), the H-6 of rhamnose, and the methyl protons of O-acetyl groups. The signal from rhamnose D H-2 is shifted downfield (to the left) because of an O-acetyl group at D C-2. Residue G is an alditol with no anomeric center; all of its signals are in the crowded central region and are hard to assign. (B) 1H-NMR spectrum of the oligosaccharide (at 30°C). The signals from the oligosaccharide are sharper: The O-acetyl methyl signal has disappeared and the D H-2 signal is no longer shifted. Residue A is usually phosphorylated at C-3; the good resolution of the oligosaccharide spectrum allows identification of a small signal (A′) from H-1 of the small proportion of A phosphorylated at C-2. (C) Part of the HSQC spectrum used to assign 13C signals of the glycan (rhamnose H-6/C-6 signals omitted). This two-dimensional spectrum has 1H and 13C chemical shifts as X and Y axes. The guidelines (for the anomeric signals) show how 13C shifts may be determined from this spectrum once the 1H spectrum is assigned. These and other NMR spectra, taken together, can provide information on the number and type of residues in the repeating unit and their anomeric configuration, ring size (pyranose [p] or furanose [f]), linkage positions, substitution positions, and sequence. (D) The MALDI spectrum showing a molecular ion at m/z 1473 [M + Na]+ corresponding to a permethylated glycan of the composition PdeoxyHex3Hex3pent-ol. (E) The ESI-MS/MS spectrum of m/z 748 [M + 2Na]2+ showing fragment ions that define the sequence shown in the cartoon in panel F.

Complete Structural Analyses of Glycans Requires Several
Approaches: MS, NMR, Enzymes, Fractionation. Ion Trap MS A Powerful tool For Structural Determination of Glycans. Figure 6 MS5 of the monosialo fragment Tri(OH)3Neu1, m/z 962, CE = 31, 28, 26, 26. MS conditions were as described for Figure 1. The oligosaccharide structure and fragmentation pathway are shown at the top of the figure. Published in: Douglas M. Sheeley; Vernon N. Reinhold; Anal. Chem. 1998, 70, DOI: /ac Copyright © 1998 American Chemical Society

GPI-Anchored Proteins Are Extracted into the Detergent Phase
Using Two-Phase Separation with TritonX-114:

Basic Structure of GPI-Anchors:

Strategy for Analysis of GPI-Anchors:

Chemical and enzymatic reactions of GPI anchors
FIGURE Chemical and enzymatic reactions of GPI anchors. Nitrous acid deamination of the GlcN residue generates the sugar 2,5-anhydromannose (AHM) that can be radiolabeled by sodium borotritide reduction or fluorescently labeled by reductive amination with 2-aminobenzamide (2-AB). Following dephosphorylation with cold aqueous hydrogen fluoride (HF), the labeled glycans can be conveniently sequenced with exoglycosidases. (CRD) Cross-reacting determinant. Essentials of Glycobiology Second Edition Chapter 11, Figure 3

Characterization of Glycosaminoglycans:
Bacterial Eliminases Are Powerful Tools: Sequential Degradation Followed by Gel Filtration. 2. Other Separation Methods.

FACE Analysis of GAG-Derived Disaccharides:

HPLC separation of CS-derived saturated and unsaturated
disaccharides labeled with 2AB FIG. 2. HPLC separation of CS-derived saturated and unsaturated disaccharides labeled with 2AB. CS-derived saturated tetrasaccharides Tetra-C1–C4 were individually digested with chondroitinase AC-II and derivatized with 2AB. The four digests and 2ABderivatized unsaturated CS-disaccharides were mixed and analyzed by HPLC as described in the legend to Fig. 1. The HPLC conditions were the same as those for the experiments shown in Fig. 1.

Glycosyltransferases as Probes:

Galactosyltransferase Labeling of Living Cells  O-GlcNAc

Conclusions: There are now a multitude of powerful methods to analyze glycoconjugates. However, most of the existing technology requires a fairly high level of skill and knowledge in analytical chemistry. Existing Technology is still unable to completely define the molecular species of a glycoprotein with more than one glycosylation site. Major advances in Separation Technologies and in Mass Spectrometry have had a huge impact upon the field.

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