1. Glyco-challenged Glyco-literate
GlycoTest
Range:
Avg: 75 ; Mean:81
Glyco-challenged Glyco-literate

2. GlycoAnalytics Complex Carbohydrate Research Center,
Department of Biochemistry & Molecular Biology
University of Georgia

5. Compositional Analysis Using Derivitized Glycans
often done by GLC Separation
—Azadi Summer Course

6. HPAE-PAD Detection of Monosaccharides

7. GLC/GC Analysis
--Also taught in Azadi’s Summer Courses

8. Tandem Mass Spectrometry-Based Approaches
For the Characterization of Glycopeptides
NCRR Biomedical
Glycomics

9. Biological/Technical Challenges
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

10. Glycan Capping Reactions

11. All MS do one thing: Measure m/z

12. Site Mapping and Characterization -Glycans, N-linked Sites, O-linked SitesQ N G S W E K A F S Y D P R S T P G L C I T I H C A N G R E S S M K Q N V S L

13. Glycan Release/Permethylation of Glycans
Trypsin digestion of protein
Enzymatic release of N-glycans
b-elimination for O-linked- additon of NaOH
resulting in release of glycan structure from hydroxyl of Ser or Thr
Reduction with NaBH4 prevents re-attaching of glycan
Permethylation of glycan- OH→OMe
Addition of MeI
Analyzed permethylated glycans by applying MSn fragmentation as needed to completely determine the structure
J. Am. Chem. Soc. (2003) 125(52):

14. Glycan Analysis

15. LC-nSI/MS/MS or Direct Infusion
Buffer A
Buffer B
HPLC
Capillary Column
Peptide
mixtures
LTQ ion trap
Mass spectrometer
Protein
Peptides sequenced,
Proteins Identified
1. Enolase
EEALDLIVDAIK
2. P
yruvate kinase
NPTVEVELTTEK
3. Hexokinase
4. Hypusine
5. BMH1
IEDDPFVFLEDTDDIFQK
APEGELGDSLQTAFDEGK
QAFDDAIAELDTLSEESYK
Database
Nucleotide
ESTs
Predicted MS/MS
TurboSEQUEST
Cross-Correlation
Comparison
Automated MS/MS MS 2 5 M a r 3 1 # 8 R T : . 9 4 A V N L 6 E 7 + c S I F u l m s [ - ]
542.5
1082.7
729.5
1486.1
CID
MS/MS

16. Permethylated Glycans from wildtype Drosophila embryos

18. POMGnT1 appears to be essential for O-Man Extension

20. GRITs..CCRC way

23. Relative quantification between 2 samples
of released and permethylated N-glycans
via isotope labeling with light/heavy iodomethane

25. +1 Dalton
J. Proteome Res., 2009, 8 (8), pp 3816–3823

26. O-linked Glycan Mixture N-linked Glycan Mixture
Amide-15N L-glutamine
“GLN-15”
Amide-14N L-glutamine
“Gln-14”
Light Medium
Heavy Medium
Harvest & Combine
Mixed Protein Powder
Homogenization and delipidation
Peptide Mixture
O-linked Glycan Mixture
N-linked Glycan Mixture
Tryptic digestion
C18 reverse phase
PNGase F digestion
-elimination
Permethylated Glycans
Permethylation
Deionization
Characterization by MS/MS
Quantification by Full MS
Mass Spectrometry
Heavy
GLN-15
Light
Gln-14

31. : Figure 5 A. [M+Na]+: 1705.862 and 1706.854 m/z (mono) * hDE = 1.74
1707
1708
1709
1710
1711
1712
1713
1714 10 20 30 40 50 60 70 80 90
100
m/z
Relative Abundance : *
hES
hDE
= 1.74
----- Meeting Notes (11/23/11 15:08) -----
add transition slide before

32. : Figure 5 B. [M+Na]+: 1432.741 and 1435.735 m/z (mono) hDE = 0.23 hES
1431
1432
1433
1434
1435
1436
1437
1438
1439
1440 10 20 30 40 50 60 70 80 90
100
m/z
Relative Abundance
hES
hDE
= 0.23 : *

33. (Ratio=hDE/hESCs) (Prevalence > 1%)
Table 3: Relative quantification of O-glycan expression levels in hESCs and hDE using IDAWG
(Ratio=hDE/hESCs) (Prevalence > 1%)
Structure
m/z
Prevalence
Ratio CV
983.52
21.78%
1.11
20.20%
20.22%
1.09
14.42%
17.60%
2.64
30.21%
895.46
8.20%
0.87
13.56%
and
534.29
6.76%
1.04
17.20%
5.81%
0.64
61.95%
4.93%
2.54
29.48%
4.70%
0.41
37.70%
2.12%
1.75
49.39%
1.99%
0.99
29.78%
779.42
1.03%
0.79
26.42%
----- Meeting Notes (11/23/11 15:08) -----
cv?

34. Pulse-Chase experiment designed for dynamic IDAWG
Heavy Media
Light
Media
X 4
X 3
X 2
0 hr
12 hr
24 hr
36 hr
X 1
Harvest
Release N- and O-linked glycans
Permethylation
Light
Media
Light
Media
Complete Labeling
X 1
X 1

35. 0hr-- >95% Heavy Relative Abundance m/z 1259.622 1260.626 1261.628
1255
1256
1257
1258
1259
1260
1261
1262
1263
1264 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100
m/z
Relative Abundance

36. 6hr Relative Abundance m/z 1259.626 1260.629 1258.631 1257.633
1255
1256
1257
1258
1259
1260
1261
1262
1263
1264 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100
m/z
Relative Abundance

37. 12hr Relative Abundance m/z 1259.622 1257.629 1258.629 1256.630
1255
1256
1257
1258
1259
1260
1261
1262
1263
1264 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100
m/z
Relative Abundance

38. 24hr Relative Abundance m/z 1256.630 1257.631 1258.631 1259.621
1255
1256
1257
1258
1259
1260
1261
1262
1263
1264 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100
m/z
Relative Abundance

39. 36hr Relative Abundance m/z 1256.629 1257.630 1258.631 1259.622
1255
1256
1257
1258
1259
1260
1261
1262
1263
1264 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100
m/z
Relative Abundance

40. Proportion of remodeling at 50% degradation time (%)
50% degradation time and proportion of remodeling at 50% degradation time for 9 major O-glycans
Structure
50% degradation time
（hours）
Proportion of remodeling at 50% degradation time (%)
Rep1
Rep2
18.6
19.4
1.6
1.1
20.7
22.6
0.0
7.7
8.4
16.2
15.6
9.0
9.7
14.9
14.1
26.8
25.4
20.4
18.1
16.9
21.2
18.4
19.8
9.3
21.3
25.3
21.5
22.1
27.9
27.0
----- Meeting Notes (11/23/11 15:28) -----
o-man short half life
no difference in terms of half life but remodeling for non and sialyted

41. Site Mapping and Characterization -Glycans, N-linked Sites, O-linked SitesQ N G S W E K A F S Y D P R S T P G L C N I T I H C A G R E S S M K Q N V S L
Focus on Site-Mapping

42. PNGase F Treatment and N-linked Glycosylation Site-mapping
N-X-S/T
Secreted Proteins from Adipocytes (insulin responsive vs insulin resistant)
Sulfated glycoprotein 1 precursor (SGP-1) gi|
(K)TVVTEAGNLLKDN#ATQEEILHYLEK (K)FSELIVNN#ATEELLVK
(K)LVLYLEHNLEKN#STKEEILAALEK
lipoprotein lipase gi|
(R)TPEDTAEDTCHLIPGLADSVSNCHFN#HSSK
vimentin gi|
(R)QVQSLTCEVDALKGTN#ESLER
Follistatin-related protein 1 precursor gi|
(K)GSN#YSEILDK
Haptoglobin gi|
(K)VVLHPN#HSVVDIGLIK
(K)NLFLN#HSETASAK
(K)CVVHYEN#STVPEKK
Adipsin gi|673431
(K)LSQN#ASLGPHVRPLPLQYEDK
Decorin gi|
(R)ISDTN#ITAIPQGLPTSLTEVHLDGNK
Hemopexin gi|
(R)SWSTVGN#CTAALR
56 proteins with 83 N-linked sites

43. N-linked Site Mapping from ConA-enriched glycopeptides
from Drosophila heads--272 sites mapped from 197 Proteins
Pileup of the 272 N-linked Sites to Determine Consensus beyond N-X-S/T
Concerns: PNGaseF/A, Deamidation, C-terminal O-18

44. PGIP N-linked Site Mapping with PNGase F/A
PNGase F (red = coverage) 3 sites identified
DLCNPDDKKV LLQIKKAFGD PYVLASWKSD TDCCDWYCVT CDSTTNRINS LTIFAGQVSG QIPALVGDLP YLETLEFHKQ PNLTGPIQPA IAKLKGLKSL RLSWTNLSGS VPDFLSQLKN LTFLDLSFNN LTGAIPSSLS ELPNLGALRL DRNKLTGHIP ISFGQFIGNV PDLYLSHNQL SGNIPTSFAQ MDFTSIDLSR NKLEGDASVI FGLNKTTQIV DLSRNLLEFN LSKVEFPTSL TSLDINHNKI YGSIPVEFTQ LNFQFLNVSY NRLCGQIPVG GKLQSFDEYS YFHNRCLCGA PLPSCK
PNGase A (red=coverage) 7 sites identified
DLCNPDDKKV LLQIKKAFGD PYVLASWKSD TDCCDWYCVT CDSTTNRINS LTIFAGQVSG QIPALVGDLP YLETLEFHK Q PNLTGPIQPA IAKLKGLKSL RLSWTNLSGS VPDFLSQLKN LTFLDLSFNN LTGAIPSSLS ELPNLGALRL DRNKLTGHIP ISFGQFIGNV PDLYLSHNQL SGNIPTSFAQ MDFTSIDLSR NKLEGDASVI FGLNKTTQIV DLSRNLLEFN LSKVEFPTSL TSLDINHNKI YGSIPVEFTQ LNFQFLNVSY NRLCGQIPVG GKLQSFDEYS YFHNRCLCGA PLPSCK
High Stringency Filter

45. Cell May 28;141(5):
Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints.
Zielinska DF, Gnad F, Wiśniewski JR, Mann M.
>6,000 Sites Mapped from Mouse Organs
>99% of Sites Match N-X-S/T(C), X is not Pro,
~1% of sites used Cys in place of S/T
Small percentage of sites (<1%) were N-X-V or NG
(Real of False-Positives???)

46. O-Glycopeptides GalNAc Man Fuc Glc Gal Xyl GlcNAc Ser/Thr Ser/Thrb
Ser/Thr
Ser/Thr
Ser/Thr
Ser/Thr
Hyl
Ser
Hyp
Ser/Thr
No PNGaseF equivalent for O-Glycans
O-Glycanse has very restricted specificity

49. --Can enrich glycopeptides before --Can map via the dehydro-amino acid
Glycan
DTT
CH2 O
Michael Addition H
CH2 H
b-Elimination
CH2 C C C N H C N H C
DTT N H C
OH- O O O
--Can enrich glycopeptides before
--Can map via the dehydro-amino acid
--Can enrich the peptides after

50. Concerns: Specificity, efficiency, recovery
Differential isotopic tagging of both cysteine and post-translationally modified
ser/thr through b-elimination/Michael addition with light (d0) and heavy (d6) DTT. O
ICH2 C
NH2 S H
CH2
b-Elimination C N H C C N H O
CH2
DTT (d0 or d6)
CH2
Michael Addition O C
Alkylated Cysteine N H C
Light DTT (d0) or Heavy DTT (d6) O OH OH
(GlcNAc or phosphate)
Dehydroalanine
(or
HSCH2CHCHCH2SH
HSCd2CdCdCd2SH O OH OH
b-Elimination H
CH2 C N H C O
O-GlcNAc or O-phosphate
Modified Serine (or threonine)
Concerns: Specificity, efficiency, recovery

51. Quantifying Peptides with D0- and D6-DTT
Theoretical: 1 to 1 ; Experimental 1 to 0.98, 1 fmol scale

53. Site Mapping and Characterization -Where we want to be!Q N G S W E K A F S Y D P R S T P G L C N I T I H C A G R E S S M K Q N V S L
(75%) (25%)

54. Alpha-Dystroglycan (O-Man/O-GalNAc-initiated glycans)

55. N- and O-linked glycan structures determined by
“Total Ion Mapping” with manual interpretation
Full MS and TIM spectra were obtained for mouse ES, EB, and ExE
Spectra analyzed, glycans identified, and quantitated
Directed MSn analysis performed as necessary to clarify structures
Expressed as “% of total” glycan profile
Method described in: Aoki, K., Perlman, M., Lim, J. M., Cantu, R., Wells, L., and Tiemeyer, M. (2007) J Biol Chem 282,

56. Figure 2. Released O-Man and O-GalNAc linked glycans

57. Identification of Glycans using TIM-MS

58. Pseudo Neutral Loss Activated Data Dependant MS3 for Glycopeptide Mapping
MS Survey Scan
MS survey scan
MS/MS scan
Neutral loss? No
Yes
Top N peaks?
MS/MS/MS scan
SEQUEST ID
If we have purified protein and glycomics data

59. Figure 3. Fragmentation of O-GalNAc a-DG glycopeptide

63. Assignment of Glycan Structure by Residue
JBC 2010, 285:24882

64. Assignment of Modified Glycopeptide
Assignment of the phosphorylated trisaccharide structure to a
specific glycopeptide of alpha-dystroglycan using MSn approaches. Displayed
is a MS3 spectra demonstrating the presence of the phoshotrisaccharide .
Science :88

65. Another O-Glycan site mapping solution
High Collision Dissociation (HCD) Fragmentation Generating oxonium ions
Electron Transfer Dissociation (ETD) Fragmentation Targeting Modified peptides
Peptide Sequencing and Modification Site Mapping
JPR :4088

66. HCD spectra PSVPV S GSAPGR GlcNAc HexNAc-66+1, 138.05 b7, 827.43
y9-HHexNAc,
b3-H2O,
PSVPV S GSAPGR y6 y7
GlcNAc
MH-HexNAc+2,
HexNAc+1,
y9-HexNAc+2,
MH-HexNAc,
HexNAc-H2O+1,
b2-H2O,
y6,
y7,
S+HexNAc
290.12m/z
HexNAc-78+1,
b3,
y7-HexNAc,
y10-HexNAc+2,
HexNAc-60+1,
y9,
y10-HexNAc,
y8-HexNAc,
y11-HexNAc,
y3,
MH+2,
y5,
y10,
y11,
MH,

67. D HCD (zoomed in) b2, 154.548 201.858 PGGSTPVSSANMM 138.054 [C7H8O2N]+
HexNAc+1
[C8H14O5N]+
[C8H12O4N]+
[C8H10O3N]+
[C6H10O3N]+
[C7H8O2N]+
[C6H8O2N]+
HCD (zoomed in) D
PGGSTPVSSANMM
GlcNAc
b2,
110
130
150
170
190
210
230
250
m/z 10 20 30 40 50 60 70 80 90
100
Relative Abundance

68. ETD spectra PSVPV S GSAPGR GlcNAc MH+2, 657.51 MH, 1314.58z7 z6
MH-H2O,
z6,
z7,
S+HexNAc
290.01m/z
z5,
c11,
z10,
z10-H2O,
z4,
c11-HexNAc,
z7-HexNAc,
z11,
z8,
y11,
y9,
y8+3,
y2,
b6,

69. Simultaneous O-Man and O-GalNAc Analysis by ETD MS/MSZ3 Z4
Z5^
Z7*
Z6^
Z8* Z9
Z10
(Z10) C8
+3, m/z
+1, m/z
Ac-IRT*T*T^S^GVPR-NH2
* Man ^ GalNAc

70. Take Home Points Quantitative Glycomic Platforms Exist
Automation of Analysis for HTG well under development
Glycopeptide analysis can use glycomics and proteomics to confine the search space and for structural determination
Pseudo-NL MSn approaches work for direct glycopeptide analysis
ExD (ETD, ECD, etc.) are very promising for glycopeptides
HCD-triggered ETD approaches for mining deep and can aid in confident assignment