1. Carbonyl-Reactive Tandem Mass Tags for MS-Based Quantitative Glycomics
Sergei I. Snovida1; Julian Saba2; Rosa Viner2; John C. Rogers1
1Thermo Fisher Scientific, Rockford, IL;
2Thermo Fisher Scientific, San Jose, CA

2. Derivatization and Labeling of Glycans for MS
Mass spectrometry
Essential for glycan analysis (detection, composition and structure, quantitation)
Native glycans don’t ionize very well
Need to derivatize/label to improve sensitivity
Chemistry:
Permethylation
structural (linkage) analysis
Reducing end labeling
UV-fluorescence detection; improves LC/CE separation
DMSO
NaOH (s), MeI
NH2R
reduction

3. Derivatization and Labeling of Glycans for MS
Mass spectrometry
Essential for glycan analysis (detection, composition and structure, quantitation)
Native glycans don’t ionize very well
Need to derivatize/label to improve sensitivity
Chemistry:
Permethylation
structural (linkage) analysis
Reducing end labeling
UV-fluorescence detection; improves LC/CE separation
MS-based quantitation is difficult
no standards for all glycoforms
difficult to normalize data
Heavy isotope labeling methods
a promising approach
DMSO
NaOH (s), MeI
NH2R
reduction

4. Derivatization and Labeling of Glycans for MS
Mass spectrometry
Essential for glycan analysis (detection, composition and structure, quantitation)
Native glycans don’t ionize very well
Need to derivatize/label to improve sensitivity
Chemistry:
Permethylation
structural (linkage) analysis
Reducing end labeling
UV-fluorescence detection; improves LC/CE separation
MS-based quantitation is difficult
no standards for all glycoforms
difficult to normalize data
Heavy isotope labeling methods
a promising approach
Fluorescence-based quantitation
well established
Why use MS-based approach?
DMSO
NaOH (s), MeI
NH2R
reduction

5. Glycan Quantitation by LC-Fluorescence
Reaction:
NaBH3CN +
glycan
2-AB
Schiff base
Reduced amino-glycan

6. Glycan Quantitation by LC-Fluorescence
Reaction:
NaBH3CN +
glycan
2-AB
Schiff base
Reduced amino-glycan
Clean-up
(HILIC)
Clean sample
+ internal standard (IS)

7. Glycan Quantitation by LC-Fluorescence
Reaction:
NaBH3CN +
glycan
2-AB
Schiff base
Reduced amino-glycan
Retention time or glucose units
Fluorescence IS ∫
Clean-up
(HILIC)
Clean sample LC
HILIC/PGC
+ internal standard (IS)
accurate relative quantitation of glycans within the sample
relatively inexpensive
depends entirely on separation
still need MS analysis to verify glycoforms

8. Glycan Quantitation by LC-Fluorescence
Relative quantitation of multiple samples using UV/fluorescence LC
Sample #1
Sample #2
Sample #3
Sample #4
Sample #5
Sample #6
independent experiment is required for each sample
still need MS analysis to verify glycoforms

9. Thermo Scientific aminoxyTMT Reagent Family
Thermo Scientific™ aminoxyTMT™ Reagent Structure
HCD

10. Thermo Scientific aminoxyTMT Reagent Family
Thermo Scientific™ aminoxyTMT™ Reagent Structure
Thermo Scientific™ aminoxyTMTsixplex™
Isobaric Reagents
HCD

11. Thermo Scientific aminoxyTMT Reagent Family
Thermo Scientific™ aminoxyTMT™ Reagent Structure
Thermo Scientific™ aminoxyTMTsixplex™
Isobaric Reagents
HCD
Reaction: +
glycan
aminoxyTMT reagent
Stable oxime product
90% MeOH
0.1% AcOH

12. Thermo Scientific aminoxyTMT Reagent Family
Thermo Scientific™ aminoxyTMT™ Reagent Structure
Thermo Scientific™ aminoxyTMTsixplex™
Isobaric Reagents
HCD
Reaction: +
glycan
aminoxyTMT reagent
Stable oxime product
90% MeOH
0.1% AcOH
1. Acetone quench
Clean sample
2. HILIC clean-up
reaction is very quick
reduction step is not required
product is stable
MS-ready sample in ~90 minutes

13. Relative Quantitation with aminoxyTMT Reagents
unknown samples #1 #2 #3 #4 #5 #6
TMT126
TMT127
TMT128
TMT129
TMT130
TMT131
Acetone quench
HILIC clean-up
MS-ready sixplex sample

14. Relative Quantitation with aminoxyTMT Reagents
unknown samples #1 #2 #3 #4 #5 #6
TMT126
TMT127
TMT128
TMT129
TMT130
TMT131
Acetone quench
HILIC clean-up MS
MS-ready sixplex sample

15. Relative Quantitation with aminoxyTMT Reagents
unknown samples
Reporter ions
(126,127,128,129,130,131 m/z) #1 #2 #3 #4 #5 #6 #1 #2 #3 #4 #5 #6 +
TMT126
TMT127
TMT128
TMT129
TMT130
TMT131
Acetone quench
HILIC clean-up
MS/MS MS
MS-ready sixplex sample
ratios are related to relative amounts
better precision
less individual sample handling
relative quantitation between samples only

16. Combining LC-fluorescence and aminoxyTMT Methods
LC-fluorescence data
unknown samples #2 #1 #3 #4 #5
TMT126
TMT127
TMT128
TMT129
TMT130
TMT131 +
Sixplex sample
Standard reference
sample
Std

17. Combining LC-fluorescence and aminoxyTMT Methods
LC-fluorescence data
unknown samples #2 #1 #3 #4 #5
TMT126
TMT127
TMT128
TMT129
TMT130
TMT131 +
Sixplex sample
Standard reference
sample
Std
Reporter ions
Std #1 #2 #3 #4 #5 + MS
MS/MS
relative quantitation between samples
relative quantitation within each sample
a very powerful technique

18. Labeled Glycan Ionization
Native unlabeled Glc7
100µg/mL
Glc7 (unlabeled)
Glc7 (unlabeled)
[M+2Na]2+
[M+Na]+
ESI solution: 50% ACN, 50 µM NaOH
Thermo Scientific™ Velos™ Pro
Mass Spectrometer
aminoxyTMT Reagent-labeled Glc7 2µg/mL
Glc7 (labeled)
[M+H+Na]2+
20-fold increase in S/N
No residual reagent
Glc7 (labeled)
[M+H]+

19. Labeled Glycan HCD Fragmentation
Trap-HCD m/z [M+H]+
Reporter ion
*Y1
*Y6
*Y5
*Y4
*Y3
*Y2
*Y1 B1 B2 B3 B4 B5 B6
H+ reporter ion
*Y2
Trap-HCD m/z [M+H+Na]2+
Reporter ion
-CO Y6 Y5 Y4 Y3 Y2 Y1 B1 B2 B3 B4 B5 B6
H+ reporter ion Y3 Y2 Y4 B5
[M+ Na-reporter-CO]+
*Y1 Y6 Y1 B3 B4 Y5 B2 B6 B1

20. Unlabeled vs. Labeled Glycan HCD Fragmentation
Reporter ions
Y (Na+)
[M+Na]+
B (Na+)
0,2A
[M+ Na-reporter-CO]+
C (Na+)
unlabeled
aminoxyTMT Reagent-labeled
Trap-HCD m/z
[M+2Na]2+
Trap-HCD m/z
[M+H+Na]2+
Structural information is preserved after labeling

21. Chromatography of Labeled Glycans: HILIC LC-MS
Sample: bovine thyroglobulin N-glycans
Contaminant
peptides
8.0E6
Column: Accucore Amide
(2.6µm, 150Å; 75µm×150mm)
Flow rate: 450 nL/min
Mobile phase A: water 0.1% FA
Mobile Phase B: acetonitrile 0.1% FA
0 min – 95%B
40 min – 60%B
50 min – 60%B
aminoxyTMT-labeled glycans
(from ~0.6 µg protein on column)
6.0E6
Native (unreduced) glycans
(from ~2.5 µg protein on column)
Can achieve good separations using HILIC

22. Quantitative Accuracy and Interferences
Reporter ions
20:16:12:8:4:1
1:4:8:12:16:20
1:2:5:5:2:1
5:2:1:1:2:5
Measured ratios match actual ratios for all glycoforms

23. Quantitative Accuracy and Interferences
Reporter ions x x x x
Reporter
ion
Interference
Res: 15,000
Res: 30,000
Res: 60,000 x x x x vs vs
HR/AM instruments can resolve these interferences

24. Quantitation Using Velos Pro Ion Trap InstrumentMS
[M+3H]3+
[M+2H+Na]3+
Reporter ions
Y1-ion
Trap-HCD MS2

25. Quantitation Using Velos Pro Ion Trap InstrumentMS
[M+3H]3+
[M+2H+Na]3+
Reporter ions
Actual ratios
1:2:5:5:2:1
Y1-ion
Trap-HCD MS2
Low resolution leads to poor quantitative accuracy due to interferences

26. Targeted MS3 on Velos Pro
DDA/targeted
MS2 (HCD or CID)
MS3 (HCD)
[M+3H]3+
Y1-ion
“clean”
reporter
ions

27. Targeted MS3 on Velos Pro
DDA/targeted
MS2 (HCD or CID)
MS3 (HCD)
[M+3H]3+
Y1-ion
1.4E4
“clean”
reporter
ions
Actual ratios
1:2:5:5:2:1
3.9E2
Targeted MS3 improves quantitative accuracy
Drawback – loss in sensitivity (~10-fold)

28. mAbs Analysis Case Study #1
Combined sample mass spectrum
Deglycosylate, label, mix, clean
1/10
A280
Anti-c-Myc
hybridoma
IgGs
G0F
[M+H+Na]2+
G1F
G2F
Reporter ions
HCD
Extracted glycan MS profiles
Reporter ion relative intensities
Culture
Mouse

29. mAbs Analysis Case Study #2
N-glycans
TMT129
TMT126
TMT127
TMT130
TMT128
TMT131
Combined sample

30. mAbs Analysis Case Study #2
G0F
G1F
G2F
HCD
Reporter ions
N-glycans
N-glycans
N-glycans
TMT126
TMT129
TMT127
TMT130
TMT128
TMT131 MS
Combined sample
Can analyze multiple replicates in the same experiment for better statistics

31. Summary
LC-UV/fluorescence and TMT-based quantitation can be complementary
Increase throughput with aminoxyTMT reagents
No significant interferences in the reporter ion region
Overall improvement in MS sensitivity with aminoxyTMT label approach
Structural elements are preserved in labeled glycans
Ideal for analysis of biotherapeutics and biomarker discovery

32. Acknowledgments Thermo Fisher Scientific Ryan Bomgarden Chris Etienne
Kay Opperman
University of Wisconsin (Madison, WI, USA)
Lingjun Li
Xuefei Zhong
Yan Liu
Texas Tech University (Lubbock, TX, USA)
Yehia Mechref
Shiyue Zhou
Yunli Hu
Academia Sinica (Taipei, Taiwan)
Kay-Hooi Khoo
Ming-Yi Ho
Shui-Hua Wang
Chia-Wei Lin
Premier Biosoft
SimGlycan team
Shonali Paul
Sanjib Meitei
Proteome Sciences
Ian Pike
Karsten Kuhn
Technische Universität München (Munich, Germany)
Bernhard Küster
Hannes Hahne
Patrick Neubert