material measurement laboratory mass spectral database of glycans and glycopeptides in therapeutic...
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Material Measurement Laboratory
Mass Spectral Database of Glycans and Glycopeptides in Therapeutic DrugsMaria Lorna A. De Leoz, Xinjian (Eric) Yan, Xiaoyu (Sara) Yang, Lisa Kilpatrick, Yuxue Liang, Michael Tarlov, Stephen Stein
Therapeutic Monoclonal Antibodies are Widely Used in Medicine
• Used as treatment for cancer and several other diseases
• $20B market/year• >30 approved mAbs• 6/10 best-selling drugs, 2012
2mAbs – monoclonal antibodies
Therapeutic Monoclonal Antibodies Are Glycosylated
• Glycans are attached to the protein molecules
• Changes in glycosylation may affect drug safety and efficacy
• Glycosylation highly heterogeneous
• Glycan analysis remains a formidable challenge
Adapted from Oxford Glycobiology Institute
Glycans
Glycans
Fab
Fc
Monoclonal Antibody Therapeutic Protein
3
Protein denaturation
Glycoprotein
N-glycan releaseN-glycan releaseDecantate
Precipitate
Ethanol precipitation
Solid phase extractionLiquid chromatographyMass spectrometry
N-GLYCANS ARE RELEASED FROM PROTEINS
De Leoz, M.L.A.; Young, L.J.T.; An, H.J.; Kronewitter, S.; et al., Mol Cell Proteomics 2011; 10, M110.002717.De Leoz, M.L.A.; Young, L.J.T.; An, H.J.; Kronewitter, S.; et al., Mol Cell Proteomics 2011; 10, M110.002717.
N-GLYCANS ARE ANALYZED BY MASS SPECTROMETRY
• Can analyze using ion trap or orbitrap MS• Sample injected through LC or direct infusion • Several MS/MS activation methods
• Higher-energy C-trap Dissociation (HCD)• Collisionally-Induced Dissociation (CID by ion trap or orbitrap)• Electron Transfer Dissociation (ETD)
• High mass resolution • Max at 125,000 at m/z 400 (i.e. it can distinguish peaks at m/z
400.0000 and 400.0032)• High mass accuracy
• < 5 ppm error routinely
ESI-LTQ Velos/Orbitrap MS
5
*
*
*
* *
* **
10% ACN (+)
20% ACN (+)
40% ACN (-)
Rela
t ive Inte
ns i
t y
Hexose (Hex) N-acetylhexosamine (HexNAc) Fucose (Fuc) Sialic Acid (NeuAc)
De Leoz, M.L.A.; Young, L.J.T.; An, H.J.; Kronewitter, S.; et al., Mol Cell Proteomics 2011; 10, M110.002717.De Leoz, M.L.A.; Young, L.J.T.; An, H.J.; Kronewitter, S.; et al., Mol Cell Proteomics 2011; 10, M110.002717.
N-Glycan Profiling of Serum Glycoproteins
STARTERGLYCAN MS/MSLIBRARY
NGA42
G0NGA2F1
G0F
NGA3B1,2
G0BNA2G1F1
G1F
A1F1
G2FSNA2F1
G2F
A2F1
G2FS2NGA2B2
G0B
NGA21
G0NGA4B2
G0B
1Purchased; 2Kindly provided by the Consortium for Functional Glycomics through Dr. Jim Paulson
Glycan Consensus Spectrum
Y4, Y4
B4
[M+Na]+
Y3, Y3
Y4/Y4B3, B4/Y4, B4/Y4
0,2A5
0,2A40,2A4/Y40,2A4/Y4
B4/Y3
B4/Y3
B3/Y4, B3/Y4
B3/Y3, B3/Y3
-H2O
2,4A5
[M+2Na]2+-(2H2O+CH2O)
B4
Y4 Y4
Y3Y3 B3
Mannose N-acetylglucosamine
[M+2Na]2+
HCD, 55eV
Glycopeptide Consensus Spectrum
b2y2 y4 b5 b6
[M+2H]2+
-(2H2O+CH2O)
(TKPREEQYNSTYR)=p
y1
0,2A1
oxonium
oxonium
oxonium
p-NH3
-H2O
-H2O -H2O -H2O p-NH3
-NH3
2,4A1
m/z 963.417, z=3HCD, 62eVp p p
p p
p
pp
p