Upload
others
View
6
Download
0
Embed Size (px)
Citation preview
©2015 Waters Corporation 3
Rapid or
Traditional
LC or LCMS
Quantitative or Qualitative
Cost/ROI
Training Requirements
The Road to Glycan Analysis Without Compromise
RELEASED GLYCAN ANALYSIS
©2015 Waters Corporation 4
Conventional Workflow
16 hrs
Conventional
3 hrs
0.1 hrs
(Pre-Labeling Clean-Up) (Drying) 1-3 hrs
(Drying) 2 hrs
>1 work day
©2015 Waters Corporation 5
Streamlined and Simplified Workflow
16 hrs
Conventional
3 hrs
0.1 hrs
(Pre-Labeling Clean-Up) (Drying) 1-3 hrs
(Drying) 2 hrs
>1 work day
<15 min
5 min
30 min
GlycoWorks RapiFluor-MS N-Glycan Kit
10 min
©2015 Waters Corporation 7
Rapid Deglycosylation RapiGest SF Assisted
1% RapiGest SF Surfactant
2 min ≥90˚C
GlycoWorks Rapid Buffer
5 min 50˚C
GlycoWorks Rapid PNGase F
Enzymatic Deglycosylation
Customized Buffer specific for RapiFluor-MS™
©2015 Waters Corporation 8
mass140000 145000 150000 155000 160000
%
0
100
VicamIgG_PNGaseFnoDTT_7p5ug_100414 638 (10.803) M1 [Ev-612128,It15] (Gs,1.500,1994:5000,1.00,L30,R30); Cm (633:642)1.71e4145345
mass140000 145000 150000 155000 160000
%
0
100
VicamIgG_PNGaseFnoDTT_7p5ug_100414 654 (11.074) M1 [Ev-629947,It13] (Gs,1.500,1955:4983,1.00,L30,R30); Cm (650:658)3.49e3146786
146952
mass140000 145000 150000 155000 160000
%
0
100
VicamIgG_PNGaseFnoDTT_7p5ug_100414 671 (11.361) M1 [Ev-562807,It10] (Gs,1.500,1993:5000,1.00,L30,R30); Cm (666:679)352148401
148240
148560148.4 kDa
145.3 kDa
146.8 kDa
2 Step
2 min Heat Denaturation
5 min 50˚C
5 min 50˚C
No PNGase F (control)
Rapid Deglycosylation Completeness measured by Intact Mass Analysis
©2015 Waters Corporation 9
Rapid Deglycosylation Completeness measured by gel shift assays
(-) Negative Control (no enzyme) (+) Positive Control (SDS/DTT + PNGase F)
(R) Rapid Deglycosylation (RapiGest, Rapid PNGase F, 10 min)
Positive control: denaturation with DTT-SDS (incompatible with MS applications), followed by PNGase F treatment is a well established method for complete N-glycan removal. • Complete deglycosylation is achieved with the 2 step rapid method
across a wide range of samples. • Compatible with rapid labeling and MS analysis.
©2015 Waters Corporation 11
N-Glycan(Glycosylamine)
N-Glycan( Acyclic Free Reducing End)
Mild Acid
Hydrolysis
PNGase F
+
ProteinN-Glycan
Released N-Glycan
(Glycosylamine)
Protein
+
HOAc
DMSO
N-Glycan(Free Reducing End) 2-AB
N-Glycan(2-AB Labeled)
Reduction
NaBH3CN
SPE
Reductive Amination (conventional) • Anhydrous sample • Numerous chemical
conversions • Laborious • Heterogenous reaction
products
Rapid Tagging Glycosylamine labeling circumvents these issues
Out with Conventions Reductive Amination is Laborious
©2015 Waters Corporation 12
RapiFluor-MSTM Reagent Built Upon Our Expertise
From Waters’ expertise in rapid, fluorescence labeling of amino acids Enhanced chemical properties for glycan analysis: • Rapid Tagging • Efficient Fluorescence • Enhanced Ionization Efficiency
AccQ·Fluor
Rapid Fluorescence
Patent Pending
RapiFluor-MS
©2015 Waters Corporation 13
+
+
H2O
+ + CO2
rapiFluor-MS
Monoisotopic mass shift (from glycosylamine)312.1586 Da
Glycosylamine+C17H20N4O2
RapiFluor-MS Reagent Rapid Reaction Kinetics
NHS Carbamate Rapid Tagging Group
Tertiary Amine MS-Active Charge Tag
Reaction Time = Seconds Procedure Time:
Quinoline Fluorophore
Glycan
Glycan
Highly stable urea linkage
ΔMass
312 Da
©2015 Waters Corporation 14
Unparalleled FLR and MS Sensitivity
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Fluorescence MS (BPI)
Instant AB Labeled
RapiFluor-MS Labeled
2-AB Labeled
Rela
tive P
erf
orm
ance (
%)
52.5
7.0
0.1 0.6
Procainamide Labeled
0
10
20
30
40
50
60
70
80
90
100
30.0*
7.0*
RapiFluor-MS vs Instant: 2x ↑ in FLR and 1000x ↑ in MS RapiFluor-MS vs Traditional: 14x ↑ in FLR and 160x ↑ in MS RapiFluor-MS vs PCA: 14x ↑ in FLR and 3x ↑ in MS
©2015 Waters Corporation 16
Labeled Glycan Labeled Deglycosylated Protein
Byproducts
Colle
ct
Labeled Glycan
Reaction Byproducts
Robust HILIC SPE
©2015 Waters Corporation 17
0.0E+0
1.2E+6
10 15 20 25 30 35
Positive Control
0.0E+0
1.2E+6
10 15 20 25 30 35
2x SPE
FA2
FA2G2S1
A3S1G3S3
1x SPE
0
5
10
15
20
25
30
Rela
tive A
bundance (
%)
Positive
Control
SPE
Processed
A3G3S3
1x SPE
2x SPE
min
5.7% 6.1%
GlycoWorks HILIC SPE of RapiFluor-MS N-glycans is quantitative No significant deviation in the glycan profile when comparing neutrals to tetrasialyated upon SPE processing
FLR
hIgG and fetuin N-glycans
Robustness Quantitative Extraction
FLR
©2015 Waters Corporation 18
FA2 Rep #1
1.6 pmol Rep #2
1.7 pmol
100% Theoretical Yield = 2.3 pmol
1.5x107 pg IgG1 pmol
150,000 pg
2 pmol glycan
1 pmol IgG0.45 pmol FA2
1 pmol total
glycan pool
10 μL injection
400 μL
sample
prepared
X X X X = 2.3 pmol
Step Yield Testing to confirm minimal bias
Deglycosylation Complete Intact mass analysis Gel shift assays Subunit LC-MS
Labeling >95% Released glycan profile vs subunit derived glycan information
SPE ~74% Recovery measurements
Glycan profile before vs after SPE
Entire Workflow (experimentally
determined) ~73% Yield
Robustness High Yield and Minimal Bias
0E+0
2E+6
3 4 5 6 7 8 9 10 11 12 13
©2015 Waters Corporation 19
Case Study #1: Glycan Characterization
ACQUITY UPLC H-Class Bio System
ACQUITY UPLC Column Manager
ACQUITY UPLC FLR Detector
Xevo G2-XS QTof MS
UNIFI Glycan Application Solution
or MassLynx Informatics
GlycoWorks RapiFluor-MS N-Glycan Kit
ACQUITY UPLC Glycan BEH Amide Column
©2015 Waters Corporation 20
FA2BG2S2
How RapiFluor-MS™ Helps Enhanced Electrospray Ionization
5.3e2
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
RapiFluorMS_hIgG_14pmol_18Nov14_04 283 (5.065)3.47e6
2AB_hIgG_halfload_20Nov14_10 235 (4.234)2.89e4
2AB_hIgG_halfload_20Nov14_10 495 (8.689)529
RapiFluorMS_hIgG_14pmol_18Nov14_04 507 (8.892)1.15e5
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
RapiFluorMS_hIgG_14pmol_18Nov14_04 283 (5.065)3.47e6
2AB_hIgG_halfload_20Nov14_10 235 (4.234)2.89e4
2AB_hIgG_halfload_20Nov14_10 495 (8.689)529
RapiFluorMS_hIgG_14pmol_18Nov14_04 507 (8.892)1.15e5
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
RapiFluorMS_hIgG_14pmol_18Nov14_04 283 (5.065)3.47e6
2AB_hIgG_halfload_20Nov14_10 235 (4.234)2.89e4
2AB_hIgG_halfload_20Nov14_10 495 (8.689)529
RapiFluorMS_hIgG_14pmol_18Nov14_04 507 (8.892)1.15e5
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
RapiFluorMS_hIgG_14pmol_18Nov14_04 283 (5.065)3.47e6
2AB_hIgG_halfload_20Nov14_10 235 (4.234)2.89e4
2AB_hIgG_halfload_20Nov14_10 495 (8.689)529
RapiFluorMS_hIgG_14pmol_18Nov14_04 507 (8.892)1.15e5
500 2000 m/z
500 2000 m/z
500 2000 m/z
500 2000 m/z
2.9e4
1.2e5
3.5e6 2+
3+
2+ 2+
1+
FA2
2-AB RapiFluor-MS
Example courtesy of M.Lauber
©2015 Waters Corporation 21
Time (minutes)
5.50 10.00 15.00 20.00 25.00
1: TOFMSES+BPI
6.93e6
1: TOFMSES+BPI
6.93e6
Conventional 2-AB Glycan from 10 µg of Protein 1.5 Day Sample Prep
RapiFluor-MS Glycan from 0.1 µg Protein <1 hr Sample Prep 100x
Sensitivity
Example courtesy of Y. Yu
10 20 min
0.3 ugp/ul Refi ma
m/z800 1000 1200 1400 1600
%
0
100
RapiFluor-MS-NIST Refimab_yqy_11212014_01 443 (7.810) Sm (SG, 100x5.00); Cm (442:446)2.07e5
800 1600 m/z
1130.921, 2+
Characterization
High Resolution LC-MS(/MS)
Direct Sensitivity Comparison using UPLC/FLR/Xevo G2-XS
FA2G2Ga1
©2015 Waters Corporation 22
50 µg of a Dextran Ladder
Time5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50
EU
x 1
0e
4
50000.004
100000.008
150000.016
200000.016
250000.016
300000.031
*It is recommended to use of cubic spline fitting
Time4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00
%
0
GU Monoisotopic
Mass (Da)
m/z
(monoisotopic)
[M+H]+
m/z
(monoisotopic)
[M+2H]2+
2 699.333 700.340 350.674
3 861.391 862.398 431.703
4 1023.449 1024.456 512.732
5 1185.507 1186.514 593.761
6 1347.565 1348.572 674.790
7 1509.623 1510.630 755.819
8 1671.681 1672.688 836.848
9 1833.739 1834.746 917.877
10 1995.797 1996.804 998.906
11 2157.855 2158.862 1079.935
12 2319.913 2320.920 1160.964
13 2481.971 2482.978 1241.993
14 2644.029 2645.036 1323.022
15 2806.087 2807.094 1404.051
16 2968.145 2969.152 1485.080
17 3130.203 3131.210 1566.109
18 3292.261 3293.268 1647.138
3
8 13
9 10 11 12
7 6
5 4
Patent Pending
GlycoWorks RapiFluor-MS Standards RapiFluor-MS™ Dextran Calibration Ladder
FLR
MS (BPI)
©2015 Waters Corporation 23
HILIC FLR GU + Accurate Mass
RapiFluor-Dextran Ladder Retention Time Calibration Curve
Assign GU values to N-glycans
BPI MS for accurate mass confirmation
GU
Method Robustness and Transferability, Confident Assignments
RFMS labeled Dextran and System Performance Standard (hIgG) are now available to support this GU workflow
©2015 Waters Corporation 25
Enhanced MSMS with RapiFluor-MS Labeling RFMS Label MS performance extends to MS/MS fragmentation data
©2015 Waters Corporation 26
Case Study #2: Glycan Monitoring
ACQUITY UPLC H-Class Bio System
ACQUITY UPLC Column Manager
ACQUITY UPLC FLR Detector
ACQUITY QDa Mass Detector
Empower or MassLynx Informatics
GlycoWorks RapiFluor-MS N-Glycan Kit
ACQUITY UPLC Glycan BEH Amide Column
Simple sample preparation with simple mass detection
©2015 Waters Corporation 27
FA2BG2S2
How RapiFluor-MS™ Helps Higher Charge State for QDa Compatibility
5.3e2
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
RapiFluorMS_hIgG_14pmol_18Nov14_04 283 (5.065)3.47e6
2AB_hIgG_halfload_20Nov14_10 235 (4.234)2.89e4
2AB_hIgG_halfload_20Nov14_10 495 (8.689)529
RapiFluorMS_hIgG_14pmol_18Nov14_04 507 (8.892)1.15e5
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
RapiFluorMS_hIgG_14pmol_18Nov14_04 283 (5.065)3.47e6
2AB_hIgG_halfload_20Nov14_10 235 (4.234)2.89e4
2AB_hIgG_halfload_20Nov14_10 495 (8.689)529
RapiFluorMS_hIgG_14pmol_18Nov14_04 507 (8.892)1.15e5
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
RapiFluorMS_hIgG_14pmol_18Nov14_04 283 (5.065)3.47e6
2AB_hIgG_halfload_20Nov14_10 235 (4.234)2.89e4
2AB_hIgG_halfload_20Nov14_10 495 (8.689)529
RapiFluorMS_hIgG_14pmol_18Nov14_04 507 (8.892)1.15e5
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
m/z500 1000 1500 2000
%
0
100
RapiFluorMS_hIgG_14pmol_18Nov14_04 283 (5.065)3.47e6
2AB_hIgG_halfload_20Nov14_10 235 (4.234)2.89e4
2AB_hIgG_halfload_20Nov14_10 495 (8.689)529
RapiFluorMS_hIgG_14pmol_18Nov14_04 507 (8.892)1.15e5
500 2000 m/z
500 2000 m/z
500 2000 m/z
500 2000 m/z
2.9e4
1.2e5
3.5e6 2+
3+
2+ 2+
1+
FA2
2-AB RapiFluor-MS
Example courtesy of M.Lauber
©2015 Waters Corporation 28
Routine N-Glycan Detection with Equivalent FLR and MS response
Minutes 5 10 15 20 25 30 35 40
Minutes
5 10 15 20 25 30 35 40
Detection across a broad range of glycoforms:
IgG Simple bi-antennery structures
RNase B High mannose structures
Fetuin Large, complex structures
MS
FLR
©2015 Waters Corporation 29
IgG Glycan Profile and Structure Confirmation Using the QDa
Key take away: Large dynamic range – can clearly see most abundant and least abundant glycoforms.
0.00
240.00
480.00
In
ten
sit
y
0.0
1.6x10 6
3.2x10 6
Minutes 10.00 15.00 20.00 25.00 30.00
896.0
Inte
nsity
0.0
12000.0
24000.0
m/z 600 1200
A2G1b 0.5% RPA
888.2
Inte
nsity
0
300000
600000
m/z 600 1200
FA2 20.5% RPA
EU
©2015 Waters Corporation 30
Intuitive GMP compliant Reporting
Empower integration enables annotation of peaks with names and m/z
A2 -
815.1
FA2 -
888.2
FA2B -
989.6
A2G
1a -
896.2
A2G
1b -
896.0
FA2G
1a -
969.3
FA2G
1b -
969.2
FA2BG
1a -
1070.7
FA2BG
1b -
1070.6
A2G
2 -
977.1
FA2G
2 -
1050.3
FA2BG
2 -
1151.8
FA2G
1S1 -
1114.6
FA2G
2S1 -
1196.0
FA2BG
2S1 -
865.3
FA2G
2S2 -
894.7
FA2BG
2S2 -
962.2
0.00
240.00
480.00
Minutes
10.00 15.00 20.00 25.00 30.00
EU
©2015 Waters Corporation 31
Developing a Rapid Method for Glycan Analysis
EU
0
10
20
30
40
Minutes 2 3 4 5
10 minute
Method
EU
0
10
20
Minutes 5 10 15 20
55 minute
Method
©2015 Waters Corporation 32
Released N-Glycan UPLC Analysis Workflows
SAMPLE PREP SEPARATION DETECTION & INFORMATICS
GlycoWorks Kits RapiFluor-MS N-Glycan Kit ACQUITY FLR/QDa
Detectors and Empower 3
Software
FLR/Xevo G2-XS QTof MS and UNIFI Scientific Information System
ACQUITY UPLC Glycan BEH Amide
Column
Deglycosylation, Labeling and Clean-up in 30 min
Unmatched sensitivity
for FLR and MS detection
FLR Quantification GU Retention
MS Confirmation
FLR Quantification GU Retention
Accurate Mass Confirmation MS/MS Fragmentation
FLR
TIC
MS/MS
©2015 Waters Corporation 33
30.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
24.00 28.00 32.00 36.00 40.00 44.00 48.00 52.00 56.00 60.00 64.00
Alliance HPLC XBridge Glycan BEH Amide, 130Å, 2.5µm 3.0 X 225 mm total length (150 mm + 75 mm) Flow Rate: 0.56 mL/minute
H-Class BIO UPLC ACQUITY UPLC Glycan BEH Amide, 130Å, 1.7µm 2.1 X 150 mm Flow Rate: 0.40 mL/minute
Total Run Time = 55 minutes
Total Run Time = 121.3 minutes
Flu
ore
scence (
rela
tive)
Time (minutes)
Comparable sensitivity and resolution – 3x sample load / 2x increase in time
Transfer between labs with different LC equipment capabilities
Case Study #3: Transferability between UPLC and HPLC
©2015 Waters Corporation 34
Summary GlycoWorks RapiFluor-MS N-Glycan Kit
Simple, streamlined protocol
Fast and complete deglycosylation
Rapid and efficient labeling
Unbiased and robust SPE for neutral to
tetrasialylated N-glycans
Unprecedented FLR and MS sensitivity
GlycoWorks RapiFluor-MS N-Glycan Kit
RapiFluor-MS
Glycoprotein
Analysis-Ready N-glycans
30 min