N-GLYCAN ANALYSIS: COMBINING THE POWER OF A NOVEL …€¦ · glycan confirmation. UNIFI Platform...
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TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2015 Waters Corporation INTRODUCTION A highly efficient MS tag, RapiFluor-MS TM (RFMS) label was used to label released N-glycans. RFMS glycan label improves MS and MS/MS signals allowing detailed characterization of low abundant glycans released from biotherapeutics. 1 We also present a holistic approach for detailed glycan analysis. Characterization and profiling that combines HILIC-LC/Fluorescence/ QTOF MS for data acquisition and a customized scientific library for glycan confirmation. UNIFI Platform Solution GlycoWorks RapiFluor- MS N-Glycan Kit Analysis. N-GLYCAN ANALYSIS: COMBINING THE POWER OF A NOVEL GLYCAN LABEL AND CUSTOMERIZED SCIENTIFIC LIBRARY FOR CONFIDENT GLYCAN ASSIGNMENT Mark Hilliard 1 , Niaobh McLoughlin, 1 Pauline M Rudd 1 , Matt Lauber 2 and Ying Qing Yu 2 1 NIBRT, Dublin, Ireland, 2 Waters Corporation, Milford, MA EXPERIMENTAL References 1. “Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection”. Anal. Chem., 2015, 8(10), pp 5401–5409 RESULTS N-Glycan characterization and profiling result using NIST mAb standard CONCLUSION Novel RFMS labeling methodology enables: Tagging released glycans in minutes. Enhanced MS and MSMS signals for LC/MS analysis of glycans. Glycan analysis workflows in UNIFI software enables: Routine glycan profiling and characterization by incorporating a curated Glycan library in the analytical workflow. Glycan DDA data from UNIFI can be imported to SimGlycan for further structure characterization. Figure 2. N-Glycan analysis workflows. (A) Glycan identification using scientific library database inside UNIFI software. The assignment is based on the best matched retention times (calibrated using Dextran ladder) and the accurate mass from the QTOF MS. (B) Glycan MSMS in data dependent acquisition mode. The processed spectra lists can be exported into SimGlycan software for identification. Additional confirmation of monosaccharide sequence and linkage can be carried out by enzyme array digestions. N-Glycan sample preparation from Biotherapeutic proteins: Figure 1. NIST mAb standard (15 μg total) was used to generated the RFMS labeled glycans. After labeling the RFMS-Glycans were subjected to UPLC/FLR/ QTOF MS analysis. Sample amount required for the analysis is less than 5 pmol of glycans. Glycan Analysis Workflows in UNIFI Software 1. Thorough glycan structure analysis using Exoglycosidase Array 2. Customized scientific library generated using the structure information and the calculated mass value Figure 3. Unique glycan analysis tools inside UNIFI software. 1-3 show how to curate a glycan scientific library using calibrated retention times in Glucose Unit value and the accurate mass information. The scientific library can be used routinely to confirm glycan assignment as part of the profiling analysis. In addition, the integrated FLR peak areas are used for accurate relative% calculations since the labeling is stoichiometric. Processed DDA (data dependent analysis) processed in UNIFI can be imported to SimGlycan software for further identification. Glycan F(6)A2G2Sg1 is shown here as an example to illustrate that both analyses were consistent. A. Glycan Scientific Library (RT + MS Confirmation) UPLC ACQUITY H-Class bio UPLC ACQUTIY Fluorescent Detector UPLC ACQITY BEH Glycan Column (2.1 x 150 mm) Xevo G2-XS QTof MS UNIFI Processed Glycan DDA Data .mzml and .LCS are compatible with SimGlycan Data Export B. Glycan DDA Dextran ladder FLR Retention time Calibration Curve 5 Analyte FLR XIC Scientific Library Search GU5 GU10 GU15 3. Routine Analysis Using the Curated Library FLR BPI MS XIC (M+2H)2+ (M+3H)3+ MSMS (CID) alpha (1,3)-Gal (Ga1) NeuGc (Sg1) SimGlycan ID Quantitation + ID UNIFI processed MSMS spectrum CFG nomenclature Oxford nomenclature likely assignments FA2G2Ga1Sg1 Tag mass 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 528.186 366.137 204.086 673.226 1733.671 835.276 1368.562 1206.501 882.401 1571.626 1895.747 2567.927 2260.845 2421.880 Tag (M +H) + Tag Tag Tag Tag Tag Tag Tag Tag Fucose GlcNAc Manoses Galactose NeuGc MSMS
Transcript of N-GLYCAN ANALYSIS: COMBINING THE POWER OF A NOVEL …€¦ · glycan confirmation. UNIFI Platform...
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2015 Waters Corporation
INTRODUCTION A highly efficient MS tag, RapiFluor-MSTM (RFMS) label was used to label released N-glycans.
RFMS glycan label improves MS and MS/MS signals allowing detailed characterization of low abundant glycans released from biotherapeutics.1
We also present a holistic approach for detailed glycan analysis. Characterization and profiling that combines HILIC-LC/Fluorescence/QTOF MS for data acquisition and a customized scientific library for glycan confirmation. UNIFI Platform Solution GlycoWorks RapiFluor-MS N-Glycan Kit Analysis.
N-GLYCAN ANALYSIS: COMBINING THE POWER OF A NOVEL GLYCAN LABEL AND CUSTOMERIZED SCIENTIFIC LIBRARY FOR CONFIDENT GLYCAN ASSIGNMENT Mark Hilliard1, Niaobh McLoughlin,1 Pauline M Rudd1, Matt Lauber2 and Ying Qing Yu2 1NIBRT, Dublin, Ireland, 2 Waters Corporation, Milford, MA
EXPERIMENTAL
References
1. “Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection”. Anal. Chem., 2015, 8(10), pp 5401–5409
RESULTS N-Glycan characterization and profiling result using NIST mAb standard
CONCLUSION Novel RFMS labeling methodology enables: Tagging released glycans in minutes. Enhanced MS and MSMS signals for LC/MS
analysis of glycans. Glycan analysis workflows in UNIFI software enables: Routine glycan profiling and characterization
by incorporating a curated Glycan library in the analytical workflow.
Glycan DDA data from UNIFI can be imported to SimGlycan for further structure characterization.
Figure 2. N-Glycan analysis workflows. (A) Glycan identification using scientific library database inside UNIFI software. The assignment is based on the best matched retention times (calibrated using Dextran ladder) and the accurate mass from the QTOF MS. (B) Glycan MSMS in data dependent acquisition mode. The processed spectra lists can be exported into SimGlycan software for identification. Additional confirmation of monosaccharide sequence and linkage can be carried out by enzyme array digestions.
N-Glycan sample preparation from Biotherapeutic proteins:
Figure 1. NIST mAb standard (15 µg total) was used to generated the RFMS labeled glycans. After labeling the RFMS-Glycans were subjected to UPLC/FLR/QTOF MS analysis. Sample amount required for the analysis is less than 5 pmol of glycans.
Glycan Analysis Workflows in UNIFI Software
1. Thorough glycan structure analysis using Exoglycosidase Array
2. Customized scientific library generated using the structure information and the calculated mass value
Figure 3. Unique glycan analysis tools inside UNIFI software. 1-3 show how to curate a glycan scientific library using calibrated retention times in Glucose Unit value and the accurate mass information. The scientific library can be used routinely to confirm glycan assignment as part of the profiling analysis. In addition, the integrated FLR peak areas are used for accurate relative% calculations since the labeling is stoichiometric. Processed DDA (data dependent analysis) processed in UNIFI can be imported to SimGlycan software for further identification. Glycan F(6)A2G2Sg1 is shown here as an example to illustrate that both analyses were consistent.
A. Glycan Scientific Library (RT + MS Confirmation)
UPLC ACQUITY H-Class bio UPLC ACQUTIY Fluorescent Detector UPLC ACQITY BEH Glycan Column (2.1 x 150 mm) Xevo G2-XS QTof MS
UNIFI Processed Glycan DDA Data
.mzml and .LCS are compatible with SimGlycan
Data Export
B. Glycan DDA
Dextran ladder
FLR
Retention time Calibration Curve
GU 5
GU 15
GU 10
Analyte
FLR
XIC
Scientific Library Search
GU
5
GU
10
GU
15
3. Routine Analysis Using the Curated Library
FLR
BPI MS
XIC
(M+2H)2+
(M+3H)3+
MS
MS
(C
ID)
alpha (1,3)-Gal (Ga1)
NeuGc (Sg1)
SimGlycan ID
Quantitation + ID
UNIFI processed MSMS spectrum
CFG nomenclature
Oxford nomenclature
likely assignments
FA2G2Ga1Sg1
Tag
mass200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600
528.186
366.137
204.086
673.226
1733.671
835.276 1368.5621206.501882.4011571.626
1895.747
2567.927
2260.845 2421.880
Tag
(M +H)+
Tag
Tag
Tag
TagTagTag
Tag
Tag
Fucose
GlcNAc
Manoses
Galactose
NeuGc
MSMS
