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

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

5. HPAE-PAD Detection of Monosaccharides

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

7. HPAEC GLC
Derivatives No Yes
Sensitivity >10pmol >lpmol
Reproducibility Fair Good
Interface to MS Not easily Yes
Column life 2 – 3 years 2 – 3 years
Column cost $800 $350
Machine cost $20-35,000 $7-10,000
Maintenance easy easy
Miscellaneous eluants need He

8. Tandem Mass Spectrometry-Based Approaches
For the Characterization of Glycopeptides
Lance Wells, Complex Carbohydrate Research Center,
Department of Biochemistry & Molecular Biology, and Chemistry
University of Georgia
NCRR Biomedical
Glycomics

9. Current Efforts: Glycopeptides
Site Mapping and Characterization -Glycans, N-linked Sites, O-linked Sites M P 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
Current Efforts: Glycopeptides

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

11. LC-nSI/MS/MS---Protein Identification
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

12. The Glycan Problem??: Neutral Loss
Fragmentation of GlcNAc-CTD by CID-MS/MS
Parent ion 535 ([M+2H+] + GlcNAc) y8
NL 7.62e6
b ions
866.3
100
Y--S--P--T--S--P--S--K 95 90
y ions
Poor Fragmentation
No Site Information 85
O-GlcNAc (204) 80 75
Relative Abundance 70 65 60 55 50 45 40 35
GlcNAc 30 25
203.8
410.3 y6
819.2 20 b2
616.2 15
433.8 b8
250.9 10
476.8
848.4 5
200
300
400
500
600
700
800
900
1000
1100
1200
m/z

13. Fragmentation of GlcNAc-CTD by CID-MS/MS/MS
Parent ions 535/866
NL 2.88e6
b ions y6 b8
616.2
848.2
Y--S--P--T--S--P--S--K 50
y ions
Ion-Trap MS Allows
MSn 45
Once sugar is off,
fragmentation is good.
MS/MS/MS works well for
Sequencing, not site mapping 40 35 30
Relative Abundance 25 y3
331.0 20
830.2 15
598.2 y5 10 y4 b5 b7
518.0
804.3
418.1
536.1 y7 b6
720.1
738.1
499.9 y8 5
313.0
488.0
580.2
703.1
400.0
633.1
684.4
866.4
250
300
350
400
450
500
550
600
650
700
750
800
850
900
m/z

14. Site Mapping and Characterization -How Unsatisfying for Complex Glycans!Q 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
Lance Wells
CCRC, UGA

15. 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):

16. 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

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

18. Characterizing Plant Protein PGIP
N-linked glycoprotein
7 Putative Sites of Glycosylation
Collaboration with Carl Bergmann’s and Mike Tiemeyer’s group at the CCRC

19. LC-MS/MS Analysis of O-18 labeled PGIP

20. N-linked Site-Mapping on PGIP with PNGaseF
Xcorr= gi|169684  @ = +3

21. 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

22. PNGF
PNGA
LTQ
Permethylated
1505.0
1331.7
MALDI

23. PNGaseF

24. 1331
PNGaseF
M3N2X
GlcNAc-OMe
-Xyl, -H2O
GlcNAc-OMe
-GlcNAc-
- MeOH

25. 1505
PNGaseA

26. PNGaseA 1054.55 M3N2XF GlcNAc-OMe - MeOH -Fuc or -Xyl-H2O

27. 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

28. Quantifying Peptides with D0- and D6-DTT

29. Other Promising Approaches: ECD/ETD

30. N-Glycans in Drosophila Embryos
Collaboration with Mike Tiemeyer, CCRC
Evidence for Complex, Hybrid Structures
Changes During Embryogenesis

31. Glycan Analysis

32. Permethylated Glycans from wildtype Drosophila embryos

35. Hypoglycosylation and Congenital Muscular Dystrophy
Current Opin. Strct. Bio. (2003) 13:621-30
Glycoconjugate J. (2004) 21:3-7

36. Alpha-Dystroglycan/Dystophin Complex

37. Comparison of relative abundance

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

39. 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%)

40. Alpha-Dystroglycan

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

42. Identification of Glycans using TIM-MS

43. 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

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

48. Assignment of Glycan Structure by Residue

49. 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

50. Where we are going next…..
NL triggered MSn
“non-NL” triggered MSn
Further exploring ECD/ETD capabilities
Enrichment Chromatography and HILIC
Quantification strategies

51. Glycan Analysis

53. N- and O-glycan structures determined by “Total-ion monitoring”
Full MS and TIM spectra obtained
Spectra analyzed, glycans identified, and quantitated
Identification based on accurate mass and fragmentation
Expressed as “% of total” or “Fold change from standard sample”
Method described in: J Biol Chem (2007) 282,

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

55. +1 Da *

56. 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

57. A. [M+Na]+: 1099.562 m/z (mono) Relative Abundance m/z 724.37 398.18
847.97
Dalton-MES-O #
401
RT:
9.68
AV: 1
NL:
5.62E3 T:
ITMS + c NSI Full ms2 [ ]
350
400
450
500
550
600
650
700
750
800
850
900
950 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100
724.5
847.4
472.4
398.4

58. B. [M+Na]+: 1101.556 m/z (mono) Relative Abundance m/z 725.37 399.18
849.97 *
Dalton-MES-HND3-O #
402
RT:
9.74
AV: 1
NL:
5.34E3 T:
ITMS + c NSI Full ms2 [ ]
350
400
450
500
550
600
650
700
750
800
850
900
950 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100
725.6
473.3
849.5

59. G. [M+Na]+: 1256.636 + 1259.627 m/z (mono) Amide-15N-GLN = 0.83
1255
1256
1257
1258
1259
1260
1261
1262
1263
1264
m/z 10 20 30 40 50 60 70 80 90
100
Relative Abundance
Amide-14N-Gln
Amide-15N-GLN
= 0.83
94% incorporation

60. Glycoanalytics -Glycan Characterization and Site Mapping
--robust methods
-- sensitivity & quantification in progress
-Intact glycopeptide analysis
--very difficult, only pure samples, in infancy