1. Tandem Mass Spectrometry-Based Approaches
For the Characterization of Glycans and Glycopeptides
Lance Wells, Jae-Min Lim, Dan Sherling, Bryan Woosley,
Dawei Lin, Ron Orlando, Enas El-Karim, Chin-Fin Teo,
Olga Stuchlik, Michael Tiemeyer& Carl Bergman
Complex Carbohydrate Research Center,
Department of Biochemistry & Molecular Biology
University of Georgia

2. 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

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

4. 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

5. 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

6. 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

7. What We Really Need? A method that allows for mapping of sites
A method that allows for enrichment of modified peptides
A method that can be used to do quantitative mass spectrometry (isotope labeling of PTM)

8. BEMAD Methodology for Sites of O-Glycosylation

9. b-elimination/Michael addition with light (d0) and heavy (d6) DTT.
Differential isotopic tagging of post-translationally modified ser/thr through
b-elimination/Michael addition with light (d0) and heavy (d6) DTT. O
CH2 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)

10. Quantifying Peptides with D0- and D6-DTT

11. LTQ: BEMAD (Light/Heavy) Quantified by LC-MS (200 amol) Theoretical: 1:1, Experimental: 1:0.88

12. 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
CH2 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)

13. Comparison of Quantitative ICAT vs BEMAD* * **
*= site of phosphorylation, ** = site of O-GlcNAc Modification
Quantification for both ICAT and BEMAD differed by <25%
Collaboration with Keith Vosseller in Alma Burlingame’s Laboratory

14. BEMAD and Neutral Loss Approaches for Other O-Glycans
O-Man (not extended) on fungal proteins
Complex O-Glycosylation

15. Neutral Loss and BEMAD Mapping of O-Man Sites on PGC

16. BEMAD for Mapping Complex O-Glycosylation

17. ECD Fragmentation Site-Mapping on an LTQ-FT

19. N-linked glycosylation on Secreted Adipocyte Proteome Site mapping using PNGaseF + 18O Water
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
Cyclophilin C-associated protein MAMA/CyCAP gi|
(K)GLN#LTEDTYKPR
(R)ALGYEN#ATQALGR
RIKEN cDNA D05 gene gi|
(K)MELKN#QSRLQEPAAR
Cathepsin L gi|929719
(R)AEFAVAN#DTGFVDIPQQEK
PPBG/ Cathepsin A gi|
(R)LDPPCTN#TTAPSNYLNNPYVR
Relative Quantification Possible by 16O and 18O Water

20. 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

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

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

23. 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

24. Glycan Release/Permethylation of Glycans
Trypsin digestion of a-DG
b-elimination- 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):

25. PNGF
PNGA
LTQ
Permethylated
1505.0
1331.7
MALDI

26. PNGaseF

27. 1331
PNGaseF
M3N2X
GlcNAc-OMe
-Xyl, -H2O
GlcNAc-OMe
-GlcNAc-
- MeOH

28. 1505
PNGaseA

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

31. Permethylated Glycans from wildtype Drosophila embryos

34. Site Mapping and Characterization -How unsatisfying for complex glycoproteins!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

35. Alpha-Dystroglycan a test case for mapping sites to structures

36. Fragmentation of O-glycans
-SA
-Hex
-Hex
-Hex-HexNAc + Na

37. Glycan Structures Observed

38. MS3 – Pseudo Neutral Loss
Overall survey scan
8 most intense peaks were selected
Fragmentation was induced upon these peaks
If result from fragmentation produced neutral loss, additional fragmentation was induced when the loss of a neutral from table was observed
Fragmentation was repeated resulting in MS3
Taking a different approach, our laboratory recently described a new strategy for ion dissociation of phosphopeptides in an ion
trap mass spectrometer.25 The method, termed Pseudo MSn, induces constitutive collisional-activation of common neutral loss
fragments following activation of the precursor ion, converting the dominant neutral loss product ions into a variety of structurally
informative fragments. The Pseudo MSn approach is distinguished from the MS3 method, since product ions produced during the
initial activation of isolated precursor ion and all subsequent neutral loss activations are simultaneously stored. Thus, the
resulting Pseudo MSn mass spectrum comprises fragments derived from multiple precursor activations (a “composite” spectrum).
In this report, we further characterize the method and its utility as applied to phosphopeptide ion dissociation.

39. Pseudo Neutral Loss Activated Data Dependant MS3
MS Survey Scan
MS survey scan
MS/MS scan
Neutral loss? No
Yes
Top N peaks?
MS/MS/MS scan
SEQUEST ID

40. Use of Pseudo Neutral Loss Method
Full Scan at 5.81 minutes

41. MS/MS of
Neutral loss of SA

42. Identification of O-GalNAc Glycosylated Peptides
- SA
- Hex
- HexNAc
(R)TPRPVPR(V) SA
Hex
HexNAC
[M+2H]2+
[M+H]+

43. Assignment of Isobaric Glycans
- SA
- Gal
- GalNAc
(R)TPRPVPR(V) SA
Gal
GalNAC
[M+2H]2+
[M+H]+

44. Full Scan at 52.38
Full Scan at minutes

45. MS/MS of 895.6
-SA

46. Identification of O-Man Glycosylated Peptides
-Hex
-HexNAc
[M+2H]2+
[M+H]+
(R)LETASPPTR(I) SA
Hex
HexNAc

47. Assignment of O-Man Isobaric Glycans
-Gal
-Man
-GalNAc
[M+2H]2+
[M+H]+
(R)LETASPPTR(I) SA
Gal
GalNAc
Man

48. How can we isolate the exact site of glycosylation?
For peptide with only one Ser or Thr residue within sequence site of glycosylation is obvious
For peptides containing more that one Ser or Thr residue assignment is difficult…
Identify glycosylated peptide based upon addition of parent mass and combined neutral losses
Observe glycosylation of b & y ions
BEMAD-b-elimination and Michael addition of DTT

49. Identifying the Site O-GalNAc Glycosylation by b & y ions
b2+HexNAc
b3+HexNAc
[M+H]+
[M+2H]2+ SA
Hex
HexNAC b5 b6 b2 b3 b4
(R)T P R P V P R (V) y4 y6 y5 y3 y2 y1

50. Identifying the Site O-Man Glycosylation by b & y ionsSA
Hex
HexNAc
-Hex
(R)LETASPPTR(I)
[M+H]+
b5+Hex
[M+2H]2+
b6+Hex
-HexNAc
-Hex

51. Assignment of Sites of Glycosylation
a-DG contains 114 Ser/Thr residues which are potential sites of O-linked glycosylation
Observed peptides that contained 72 possible acceptor sites of glycosylation
Confidently assigned glycan structures to 22 of the observed glycan receptor sites
Out of the sites identified as being glycosylated
10 O-Man & 15 O-GalNAc sites