1. A Fully Automated Workflow for Glycopeptide Analysis
Julian Saba1, Rosa Viner1, Paul Shan2, Lei Xin2, Sergei Snovida3, Edward Bodnar4, Kay-Hooi Khoo3 and Hélène Perreault4
1Thermo Fisher Scientific, San Jose, CA, USA; 2Bioinformatics Solutions Inc., Waterloo, ON, Canada; 3Institute of Biological Chemistry, Academia Sinica; Taiwan; 4University of Manitoba, Winnipeg, MB, Canada

2. Introduction: Why Are We Interested?
50% of all mammalian proteins are glycosylated
Glycopeptides play important roles in cell development, differentiation and tumorigenesis
Despite its ubiquity and biomedical importance, only a small fraction of O- and N-linked glycosylation sites have been mapped and characterized to date

3. Why Are Glycopeptides So Difficult To Analyze By Mass Spectrometry?
Oligosaccharides can be much larger than the peptide backbone
Glycosylation influences chromatograpihc elution properties compared to nonglycosylated peptides
Using standard HPLC solid phases may not result in chromatographic resolution (i.e. co-eluters)
Conventional CID preferentially cleaves glycosidic bonds or peptide-glycan linkages
CAD

4. Why Are Glycopeptides So Difficult To Analyze By Mass Spectrometry?
Modifications are often highly heterogeneous
May dilute the peptide signal compared to nonglycosylated peptides
There are no clearly defined mass shifts as those for acetylation, oxidation, and phosphorylation
Hard to search for peptides in protein databases using MS/MS spectra

5. Glycopeptide Identification Requires CAD and ETD
Serotransferrin T
CAD: glycan composition identification
ETD: peptide/glycosylation site identification

6. Localization of the Glycosylation Site by ETD
Challenges:
ETD performance decreases for ions with m/z >1000
N-linked glycopeptides are often beyond the range unless
the peptide backbone is short
asialylated glycan
ETD fragmentation efficiency is low
It’s sequence and charge dependent

7. Our Strategy: How to Improve ETD Performance for Gycopeptides
Dynamic range
Sample enrichment
Multiple strategies
Optimize HPLC conditions
Sample specific LC column
Increase precursor charge state- Introduce additional charge groups
TMT labeling
Metal adducts 7

8. Glycoproteomic Strategies
Taking from what is already out there and combining
Pan, S. et al., MCP

9. Our Glycoproteomics Workflow

10. Glycopeptide Enrichment Strategies

11. Glycopeptide Enrichment Strategies
Graphite
SAX
TiO2
ZIC-HILIC
Cellulose
Amide 80
Poly-
Hydroxyethyl A
TiO2-Graphite

12. Comparison of ZIC-HILIC vs TiO2 for Enrichment of Human Serum Sialylated Glycopeptides
*PNGaseF/H2O18 Treated

13. Enrichment Efficiency of TiO2 vs ZIC-HILIC for Sialylated N-Linked Glycopeptides T from Histidine-Rich Glycoprotein in Human Serum
NL: 2.53E4
1. Hex5HexNAc4Neu5Ac2
2. dHexHex5HexNAc4Neu5Ac2
3. dHex2Hex6HexNAc5Neu5Ac2
4. dHexHex6HexNAc5Neu5Ac3
5. Hex5HexNAc4Neu5Ac1
6. dHex2Hex5HexNAc4Neu5Ac1
7. dHex3Hex5HexNAc4Neu5Ac1
100 90
R=43614
z=4 80
R=35905
B=47.95
z=2 70 7
B=107.24 60
R=34109 50
z=?
R=36682 40
B=86.22
z=2
B=97.07 30 1 2
R=38281
R=42792
z=1 6 20
z=?
B=76.67
R=31405
R=30808 10
B=58.13
z=?
z=?
Relative Abundance
B=118.64
B=130.74 1
R=41325
NL: 3.60E4
100
z=4
B=17.93 90 80 4 70 60 3 7 2 50
R=38541 40
z=3
B=20.50
R=38520
R=35548
R=34985 30
R=40047
R=33713
z=4 5
z=3
z=3 20
z=4
B=20.08 6
z=3
B=16.48
B=10.47
B=18.59
B=6.43
R=32244 10
z=2
B=0.89
900
1000
1100
1200
1300
1400
1500
1600
1700
m/z

14. Identification of TiO2 Enriched N-Linked Glycopeptides T from Histidine-Rich Glycoprotein in Human Serum
HCD
ETD

15. T274 is a novel glycosylation site
Identification of TiO2 Enriched O-Linked Glycopeptides T from Histidine-Rich Glycoprotein in Human Serum
ETD
ETD
T274 is a novel glycosylation site

16. Enrichment of N- and O-Linked Sialylated Glycopeptides from Histidine-Rich Glycoprotein (P04196) in Human Serum
Peptide
ZIC-HILIC
TiO2
SAX
HSHNNNSDLHPHK 4 10
SSTTKPPFKPHGSR 3
VENTTVYYLVLDVQESDCSVLSRK 2
VIDFNCTTSSVSSALANTK
# Glycopeptides

17. Summary of All Enrichment Strategies
Sixteen different enrichment strategies on protein/peptide level were evaluated:
Lectins: WGA, ConA ( at the protein level)
HILIC: ZIC, Cellulose, Amide80, PolyHydroxyethyl A
Miscellaneous: TiO2, Graphite, TiO2-Graphite, SAX
Total number of glycoproteins identified 101
Total number of glycopeptides identified 706
Total number of sites 307
All strategies provide some complementarities, most orthogonal are SAX and TiO2

18. Improving ETD Fragmentation with Isobaric Labeling Approach via Tandem Mass Tags (TMT)

19. Tandem Mass Tags - TMT
A family of amine reactive isobaric MS/MS tags based on identical chemical structure *
126 Da *
127 Da`
The addition of the basic TMT groups increases the average charge state of the
precursor ion
improves ionization
chromatographic separation
improves ETD fragmentation of acidic glycopeptides.
type of isobaric mass tag.
Several different forms of a molecule have the same mass and can be independently attached to a peptide or protein via a reactive group.
Mass Reporter (Tag) and the Mass Normaliser are connected via a Cleavable Linker, which has the tendency to break preferentially during MS/MS fragmentation conditions.
By selective placement of isotopes such as 13C, 15N or 18O at certain positions of the Tag and the Mass Normaliser, the mass of the Tag can be varied to produce series of Mass Reporters, typically in 1 Dalton increments, while keeping the mass of the entire label constant.
Mass Reporters: m/z 126, 127, 128, 129, 130, 131

20. Increases the Average Charge State of the Precursor Ions+5 +4
Fetuin T54-85
-TMT +6 +7
+TMT

21. - TMT( m/z , 4+)
Serotransferrin T
CID
ETD

22. + TMT( m/z 1036.41, 4+)- Improves ETD Fragmentation Even for the Same Charge State Precursor
Serotransferrin T
CID
ETD
Same peptide but with TMT label, cysteine was alkylated with iodoacetic acid.

23. Identified TiO2 Enriched Human Serum Glycosites With and Without TMT-Labeling
151 71

24. Mass Spectrometry Acquisition Strategy
Thermo Scientific LTQ Orbitrap Velos with ETD

25. Countless Possibilities for PTM Analysis +HCD/ETD, NL(product) ETD
‘Pick and Mix’ method setup
Instrument
Fragmentation
Method
Acquisition Method
LTQ Family
CID/PQD MSn
NL MS3, MSA
LTQ ETD Family
+ ETD
+NL ETD, CID/ETD, DDDT
Orbitrap ETD Family
+ HCD
+HCD/ETD, NL(product) ETD

26. Discovery of Glycopeptides
Traditional approaches:
In source decay- Pseudo-MS3 of Oxonium ions
Peterman, S. et al., JASMS (2006) 17,
Low mass oxonium ions from HCD(PQD) MS/MS Spectra
Snovida, S. et al., Carbohydr Res. (2010) 345,
Kuster, B. et al., JASMS (2011) 22,
Glycopeptide’s precursors identified in a post-acquisition fashion !

27. The Utility of Oxonium Ions – Importance of Mass Accuracy
Huddleston, M.J. et al., Analytical Chem. (1993) 65,
Jebanathirajah, J. et al., JASMS (2003) 14, 777–784

28. Our Approach - On the Fly Identification of Glycopeptides
HCD Accurate Mass Product Dependent ETD (HCD-PD-ETD)

29. Our Approach- On the Fly Identification of Glycopeptides
Streamlines data analysis
Improves dynamic range and duty cycle

30. Hex4HexNAc4Neu5AcNeu5Gc dHexHex4HexNAc4Neu5Gc2
Looking for Needle in a Haystack – Targeting Low-Abundance Glycopeptides
36.71
Unenriched 12 protein mixture digest
100
Base Peak Chromatogram
47.22 90 80
55.74
34.05 70
47.85 60
32.03
57.23
75.13
Relative Abundance 50
62.83
26.75 40
78.58
85.47
105.65 30 20
1.94
25.27
88.68
111.47 10
19.96
105.06
124.15
Hex4HexNAc4Neu5AcNeu5Gc dHexHex4HexNAc4Neu5Gc2
bAGP
QNGTLSK
40.11
XIC for m/z
from HCD spectra
100
Ovomucoid 90 80
dHex2Hex7HexNAc6Neu5Ac2 70 60
CNFCNAVVESNGTLTLSHFGK
34.69 50 40 30
40.67 20
48.51 10
89.36 10 20 30 40 50 60 70 80 90
100
110
120
Time (min)

31. Looking for Needle in a Haystack – Targeting Low-Abundance Glycopeptides
Number of glycopeptides identified
Total number of ETD spectra acquired
Percentage of ETD spectra identified
224
160
Higher-Energy Collisional Dissociation Accurate Mass Product Dependent Electron Transfer Dissociation – HCD-PD-ETD

32. Data analysis

33. Current Solution

34. Proteome Discoverer Software + GlycoMaster = Solution for Glycopeptide Identification

35. =V*VLHPN(2204.57)YSQVDIGLIK*(P00738:HPT_HUMAN)
Sequence tag: SQVDIGLIK
Charge 4 precursor

36. Software Evaluation: Training Set Results
Sample
10 standard protein digest mixture containing following glycoproteins human serotransferrin, chicken ovalbumin; and chicken ovomucoid as contaminant protein 25 6

37. Summary
A complementary of ZIC-HILIC and TiO2enrichment strategies for glycopeptide analysis was demonstrated
Introduction of a novel instrumental control within the LTQ Velos Orbitrap software called HCD-PD-ETD analysis. This approach increases the overall productivity for MS analysis of glycopeptides
Streamlines data analysis
Improves dynamic range and duty cycle
GlycoMaster automates the analysis of the data acquired from the combined fragmentation techniques
A fully automated workflow has been presented that minimizes some of the pain points associated with enrichment, MS acquisition and data interpretation

38. Acknowledgments Thermo Fisher Scientific, San Jose, CA Terry Zhang
Scott Peterman
Eric Hemenway
David Horn
Bernard Delange
John Syka
Jae Schwartz
Vlad Zabrouskov
Amy Zumwalt
Sucharita Dutta
Thermo Fisher Scientific, Rockford, IL
John Rogers