introducing introducing glycoworks glycoworks ... - waters.com€¦ · recorded version of...

Introducing Introducing GlycoWorksGlycoWorks RapiFluorRapiFluor--MS: MS: Label Label NN--GlycansGlycans Simply and Rapidly Without Simply and Rapidly Without

Sacrificing SensitivitySacrificing Sensitivity

©2015 Waters Corporation 1

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� Reference materials

Today’s Presenters

� Matthew Lauber, Ph.D - Senior Research Chemist, Waters Corporation

� Matthew Lauber is a Senior Applications Chemist in the Chemistry Applied Technology group. He has an academic background in protein chemistry and proteomic methods for the structural characterization of biomolecular complexes. Most recently Matt has worked extensively on chemistry products for bioseparations, with a specific focus on the new


bioseparations, with a specific focus on the new GlycoWorks – RapiFluor MS kit we will discuss today.

� Paula Magnelli, Ph.D – New England Biolabs

� Dr. Paula Magnelli is a Scientist at NEB GlycobiologyGroup. Paula is responsible for developing and characterizing novel glycosidases, creating methods for glycomics and glycoproteomics research with an emphasis in therapeutic glycoproteins workflows and glycansequencing.


Introducing GlycoWorks RapiFluorIntroducing GlycoWorks RapiFluor--MS: MS: Label NLabel N--Glycans Simply and Rapidly Glycans Simply and Rapidly

Without Sacrificing SensitivityWithout Sacrificing Sensitivity

Matthew A. Lauber, Ph.D.Paula Magnelli, Ph.D.

Glycosylation of Biotherapeutics


N-glycolylneuraminic acid

Fucose

GlcNAc

Mannose

Galactose

N-acetylneuraminic acid

Immunogenic (αGal / N-glycolylneuraminic acid)

Low Half Life(high mannose)

Anti-Inflammatory(sialylation)

Effector Functions (ADCC/CDC)(fucosylation/galactosylation)

Overall profile sensitive to manufacturing conditions

� N-glycosylation is a quality attribute of biotherapeutics

� N-glycan profiles are frequently characterized in detail and routinely monitored

Anal Chem 2013, 85 (2), 715-36.

Glycoprotein CharacterizationMultiple Strategies – Complementary Information


Released Glycan AnalysisHILIC Profiling

Conventional


5 Hoursto

2 Days

FLR

Conventional2-AB 5.3e2

500 2000 m/z

2+

Released Glycan AnalysisRecent Developments

Procainamide

Klapoetke, S.; Zhang, J.; Becht, S.; Gu, X.; Ding, X., J Pharm Biomed Anal 2010, 53 (3), 315-24.

Rapid Tagging for MS

Rapid


Rapid Tagging for MS

Rapid Tagging for FLR

FLR

MSCook, K. S.; Bullock, K.; Sullivan, T., Biologicals 2012, 40 (2), 109-17.

Gong, B.; Hoyt, E.; Lynaugh, H.; Burnina, I.; Moore, R.; Thompson, A.; Li, H., Anal BioanalChem 2013, 405 (17), 5825-31.

Part I

Development of the GlycoWorks


Development of the GlycoWorks RapiFluor-MS N-Glycan Kit

Mild AcidHydrolysis

PNGase F

+

ProteinN-Glycan

Released N-Glycan

(Glycosylamine)

Protein

Reductive Amination (conventional)

• Relies on numerous chemical conversions

• Laborious• Time consuming • Often produces

Rapid Tagging

Glycosylamine labeling with rapid tagging reactive groups circumvents these issues

Glycan Labeling Techniques


N-Glycan(Glycosylamine)

N-Glycan( Acyclic Free Reducing End)

+

HOAcDMSO

N-Glycan(Free Reducing End) 2-AB

N-Glycan(2-AB Labeled)

Reduction

NaBH3CN

SPE

• Often produces heterogenous reaction products

these issues

RapiFluor-MSTM ReagentBuilt 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

RapiFluor-MS

AccQ·Fluor

RapidFluorescence


Patent PendingExclusive Licensing

RapiFluor-MS

Mechanism for Rapid Tagging of Glycosylamines with RapiFluor-MS

RapiFluor-MS ReagentRapid Labeling Kinetics

+

H O

rapiFluor-MS

∆mass

NHS Carbamate Rapid Tagging Group

Quinoline Fluorophore

Tertiary AmineMS-Active Charge Tag


+

H2O

+ + CO2

Monoisotopic mass shift (from glycosylamine)312.1586 Da

Glycosylamine+C17H20N4O2

∆time = seconds

∆mass (glycosylamine) = 312.16 Da(reducing end) = 311.18 Da

Quinoline Fluorophore

Revolutionizing N-Glycan Sample PrepSensitivity Comparison – Instant ABTM

0.0E+0

2.6E+6

4.5 5 5.5 6 6.5 71.7E+6

FLR

MS(BPI)

342.8

233.7

300

400FA2

FA2

FLR

MS(BPI)

Res

ponse

Fac

tors

(FA2 P

eak

Are

a per

Sam

ple

of

N-G

lyca

ns

µg o

f In

tact

mAb M

ass

Chec

k Std

/ 1000)

RapiFluor-MS Labeled N-Glycans


0.0E+0

2.6E+6

0.0E+0

1.7E+6

4.5 5 5.5 6 6.5 7

300x zoom

0.0E+0

4.5 5 5.5 6 6.5 7

FLR

MS(BPI)

179.9

0.30

100

200

FA2

FA2

RapiFluor-MSLabeled

Instant ABLabeled

Res

ponse

Fac

tors

(FA2 P

eak

Are

a per

Sam

ple

of

Nfr

om 1

µg o

f In

tact

mAb M

ass

Chec

k Std

/ 1000)

Instant AB Labeled N-Glycans

min

min

Revolutionizing N-Glycan Sample PrepQuantitative Analysis to Compare to Convention

DifferentMechanisms

H O

rapiFluor-MSReductive Amination

2-ABRapid TaggingRapiFluor-MS

3.3E+6

RapiFluor-MS Labeled Human IgG N-Glycans

FA2 (2.2 pmol)(Area = 229.6 x 103)

FLR

y = 113033x - 11366 Quantity (pmol)

hIgG FA2 232105 227079 229592 2.2


0.0E+0

4.0E+6

3 4 5 6 7 8 9 10

0.0E+0

3 4 5 6 7 8 9 10

FA2(Area = 275.5 x 103)

FLR

MS(BPI)

RapiFluor-MS Label(pmol)

y = 113033x - 11366R² = 1

0E+0

2E+5

4E+5

6E+5

8E+5

1E+6

0 5 10

Peak A

rea

(Flu

ore

scence

-Ex 2

65 n

m /

Em

425 n

m)

3.0E+5

FA2 (2.6 pmol)(Area = 19.3 x 103)

FLR

y = 7721.8x-834.17 Quantity (pmol)

hIgG FA2 19665 18955 19310 2.6

Revolutionizing N-Glycan Sample PrepQuantitative Analysis to Compare to Convention

DifferentMechanisms

H O

rapiFluor-MSReductive Amination

2-ABRapid TaggingRapiFluor-MS

2-ABLabeled Human IgG N-Glycans


0.0E+0

3.0E+4

3 4 5 6 7 8 9 10

0.0E+0

3 4 5 6 7 8 9 10

FA2(Area = 2.0 x 103)

FLR

MS(BPI)

2-AB Label(pmol)

y = 7721.8x - 834.17R² = 1

0E+0

1E+4

2E+4

3E+4

4E+4

5E+4

6E+4

7E+4

0 5 10

Peak A

rea

(Flu

ore

scence

-Ex 3

30 n

m /

Em

420 n

m)

Revolutionizing N-Glycan Sample PrepSensitivity Comparison – 2-AB

4.0E+6

0.0E+0

3.3E+6

3 4 5 6 7 8 9

FA2(2.2 pmol)

FA2

105.8

126.9

100

150

FLR

MS(BPI)

RapiFluor-MS Labeled N-Glycans from Pooled Human IgG

FLR

MS(BPI)

Res

ponse

Fac

tors

of L

abel

ed F

A2 G

lyca

n/

1000)


0.0E+0

4.0E+6

3 4 5 6 7 8 9

4 5 6 7 8 9

0.0E+0

3 4 5 6 7 8 9

100x zoom

0.0E+0

3.3E+6

3 4 5 6 7 8 9

FA2 (2.6 pmol)

FA2 7.4

0.8

0

50

RapiFluor-MSLabeled

FLR

MS(BPI)

2-ABLabeled

Res

ponse

Fac

tors

(Pea

k Are

a per

pm

olof

Lab

eled

FA2 G

lyca

n

2-AB Labeled N-Glycans from Pooled Human IgG

50

60

70

80

90

100

50

60

70

80

90

100

Fluorescence MS (BPI)

Instant AB Labeled

RapiFluor-MS LabeledRel

ativ

e Pe

rform

ance

(%

)

52.5

Revolutionizing N-Glycan Sample PrepSensitivity Comparison


0

10

20

30

40

50

0

10

20

30

40

50

2-AB Labeled

Rel

ativ

e Pe

rform

ance

(%

)

7.0

0.1 0.6

Procainamide Labeled

30.0*

7.0*

(*) Comparative result extrapolated from a published comparison of N-glycans, wherein it was found that procainamide provided comparable fluorescence and up to 50 fold greater ESI-MS sensitivity when compared to 2-AB(Klapoetke et al. 2010).

Simplified Workflow

Conventional5 Hours

to 2 Days

Direct Analysis


10 min 5 min 10 min

30 min

Patent Pending

Total Sample Prep Time

GlycoWorks RapiFluor-MS N-Glycan Kit

Direct Analysis(Organic Solvent Dilution)

Powered by New England Biolabs®

:: Global leader in the production and supply of reagents for the life sciences

:: Innovator of technologies from basic molecular biology through to next generation

sequencing and high throughput genomics

:: Provider of best-in-class products with unparalleled technical support

:: Manufacturer of reagents for glycan analysis for over 20 years


2014

Rapid PNGase F

released

2007

K. lactis

glycoengineered

strains

1994

Chitin

research

begins

1974

NEB founded

1980 2000 2010

1992

PNGase F, Endo H &

first exos released

1992

Maltose Binding

Protein Expression

Kit Released

2014

Human GPI

glycoproteome

published

1990

1988

Dwek coins term

“glycobiology”

2005

Cellulase

research

begins

1990

NEB glycobiology

program formed

2011

Heparinases

released

2008

Novel

O-glycosidase

released

2000

α-N-Acetyl-

Galactosaminidase

released

2012

Endo S

released

Rapid DeglycosylationRapiGest SF and Glycoworks Rapid PNGase F

RapiGest SFSurfactant

GlycoWorks Rapid PNGase F

GlycoWorks Enzymatic


2 min80˚C

GlycoWorks Rapid Buffer

5 min50˚C

EnzymaticDeglycosylation

Revolutionizing N-Glycan Sample PrepRapid Deglycosylation


(-) 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.

�Rapid deglycosylation: complete unbiased N-glycan removal in 10 minutes, compatible with rapid labeling and mass spectrometry analysis.

Revolutionizing N-Glycan Sample PrepSpeed: Fast Deglycosylation


(-) Negative Control (no enzyme)

(R) Rapid Deglycosylation (RapiGest, Rapid PNGase F, 10 min)

(RT) Rapid Deglycosylation with 4mM TCEP

(+) Positive Control (SDS/DTT + PNGase F)

Revolutionizing N-Glycan Sample PrepSpeed: Fast Deglycosylation


5 min50˚C

+Rapid PNGase F

2 min80˚C

Glycoprotein +

Rapid Buffer+

Rapigest SF

Labeled GlycanLabeled Deglycosylated ProteinReaction Byproducts

Revolutionizing N-Glycan Sample PrepRobustness: Quantitative Extraction


Byproducts

Collect

Labeled Glycan


Mixture of hIgG and bovine fetuin N-glycans

2.3E+6 No SPE Crude Reaction Mixture

FA2FA2G2S1

A3S1G3S3

A3G3S3

FLR

RapiFluor-MS FA2


0.0E+0

1.9E+6

0 5 10 15

0.0E+0

0 5 10 15

After SPE (1x SPE)

FA2

SPE Recovery

~74%

0E+0

1E+8

2E+8

3E+8

4E+8

5E+8

6E+8

0 30 60 90 120 150 180

RapiFluor-MS FA2 Fluorescence Peak Area

SPE Elution Volume (µL)

Specified elution volume

0.0E+0

1.2E+6

FA2

FA2G2S1

A3S1G3S3

1x SPE

15

20

25

30

Rela

tive

Abundance

(%

)

Positive Control

SPE Processed

A3G3S3

1x SPE

2x SPE


FLR


10 15 20 25 30 35

Positive Control

0.0E+0

1.2E+6

10 15 20 25 30 35

2x SPE

0

5

10

Rela

tive

Abundance

(%

)

min

5.7%6.1%

� GlycoWorks HILIC SPE of RapiFluor-MS N-Glycans is quantitative � No significant deviation in the glycan profile upon SPE processing

Revolutionizing N-Glycan Sample PrepRobustness: Optimized Labeling

� Labeling optimized for high yield and minimization of artifacts

Glycoprotein Concentration0.36 mg/mL

RapiFluor-MSConcentration 1.6E+5

G0F

Fluore

scen

ce P

eak

Are

aFA

2 F

luore

scen

ce P

eak

Are

a

Maximized yield

18 mM

36 mM


0.0E+00 50 100

G0F

Fluore

scen

ce P

eak

Are

aFA

2 F

luore

scen

ce P

eak

Are

a

RapiFluor-MS Reagent Concentration (mM)

54 mM

72 mM

108 mM

4 9 min

Patent Pending

Revolutionizing N-Glycan Sample PrepRobustness: Optimized Labeling

� Labeling optimized for high yield and minimization of artifacts

BPI

10x zoom

FA2+2 Labels

FA2G1+2 Labels

Overlabeled~1% 0.3

0.4

(FA2 +

1 L

abel

/ F

A2 +

2 L

abel

s)

% Overlabeled

Minimization of artifacts+

Maximized yield

Not OptimizedHigh Ionic StrengthpH 8.8180 mM Reagent


0

0.1

0.2

0 50 100

(FA2 +

1 L

abel

/ F

A2 +

2 L

abel

s)

10x zoom

Overlabeled≤0.1%

Optimized50 mM HEPESpH 7.936 mM Reagent

BPI

A2

RapiFluor-MS Reagent Concentration (mM)

Patent Pending

Revolutionizing N-Glycan Sample PrepRobustness: High Yield

2E+6

FA2

N-Glycan Yield

(entire workflow)

~73%

y = 113033x - 11366R² = 11E+6

Ex 2

65 n

m /

Em

425 n

m)


0E+0

3 4 5 6 7 8 9 10 11 12 13

FA2Rep #1

1.6 pmolRep #2

1.7 pmol

100% Theoretical Yield = 2.3 pmol

0E+0

2E+5

4E+5

6E+5

8E+5

0 5 10

Peak A

rea

(Flu

ore

scence

-Ex 2

65 n

m /

Em

425 n

m)

RapiFluor-MS Label

(pmol)

1.5x107 pg IgG1 pmol

150,000 pg

2 pmol glycan1 pmol IgG

0.45 pmol FA21 pmol total

glycan pool

10 µL injection400 µL

sample

prepared

X X X X = 2.3 pmol

Revolutionizing N-Glycan Sample PrepRobustness: High Yield with Minimal Bias

Step Yield Testing to Confirm Minimal BiasIntact mass analysis / Subunit LC-MS


FA2Rep #1

1.6 pmolRep #2

1.7 pmol

100% Theoretical Yield = 2.3 pmol

Deglycosylation Complete � Intact mass analysis / Subunit LC-MS� Gel shift assays

Labeling >95% � Released glycan profile vs subunit derived glycan information

SPE ~74% � Recovery measurements� Glycan profile before vs after SPE


~73% Yield

GlykoPrep® Rapid N-Glycan Preparation

with 2-AB~35% Yield

1

2

3

GlykoPrep is a copyright of Prozyme. These values may not be representative of all applications.

Revolutionizing N-Glycan Sample PrepRobustness: Stable Glycan Derivatives

80000

100000

120000

140000

160000

180000

200000

0 Hours

72 Hours

Fluorescence

Fluore

scen

ce P

eak

Are

as

Initial

Intial

3 Days10x zoom


� No significant change in the glycan FLR signal even after 3 days at 10˚C� Stable glycan derivatives

0

20000

40000

60000

80000

Fluore

scen

ce P

eak

Are

as

A2

FA2

FA2G

1

FA2G

1(i

so)

FA2G

2

FA2G

2Sg1

FA2G

2G

a2

10 30 min

10 30 min

10x zoom

After 3 days10˚C

SummaryPart I� Preparation of labeled N-glycans (from

glycoprotein to analysis ready sample) in 30 minutes

� Unprecedented sensitivity for labeled N-

glycans

� Complete deglycosylation producing unbiased results

� Simple, streamlined protocol

RapiFluor-MS


� Simple, streamlined protocol

� Accurate profiling based on robust SPE for neutral to tetrasialylated N-glycans


Part II




@ Work

Conventional 2-ABGlycan from 10 µg of Protein1.5 Day Sample Prep

Glycan Characterization Using UPLC/FLR/Xevo G2-XS QTof


RapiFluor-MSGlycan from 0.1 µg Protein<1 hr Sample Prep 100x

Sensitivity

10 20 min

1: TOF MS ES+BPI

6.93e6

Example courtesy of Y. Yu

CharacterizationCharacterization

High Resolution LC-MS(/MS)

Conventional 2-ABGlycan from 10 µg of Protein1.5 Day Sample Prep

1130.921, 2+

Glycan Characterization Using UPLC/FLR/Xevo G2-XS

FA2G2Ga1


1: TOF MS ES+BPI

6.93e6

RapiFluor-MSGlycan from 0.1 µg Protein<1 hr Sample Prep 100x

Sensitivity

Example courtesy of Y. Yu

10 20 min

800 1000 1200 1400 1600800 1600 m/z

CharacterizationCharacterization

High Resolution LC-MS(/MS)

Glycan CharacterizationEnhanced Electrospray Ionization

3.47e6 2.89e42.9e43.5e62+ 1+

FA2

2-ABRapiFluor-MS


FA2BG2S25.3e2

500 1000 1500 2000

500 1000 1500 2000

1.15e5

500 1000 1500 2000500 2000 m/z

500 2000 m/z

500 2000 m/z

500 2000 m/z

1.2e5

3+

2+ 2+

FA2QTofCollision Induced Dissociation

Glycan CharacterizationInformation-Rich MS/MS Spectra


� Contiguous series of glycosydic bond fragments(Near exclusively containing label)

� MS/MS spectra additionally contain some cross ring fragmentation

Intact mAb Mass Check Standardanti-citrinin murine monoclonal IgG1FLR

MS

Glycan CharacterizationDetailing the N-Glycan Profile of a mAb

Rapid Preparation

DeglycosylationLabeling

SPE

30 min

2.1E+6

0.0E+6

8.4E+6


0.0E+6

Peak No.

Glycan NameObs Mass Mass Error Approximate

(M+H)+ (ppm) Relative%


MS



F: FucoseA: Antennary G: GalactoseB: BisectingGa: Alpha-galactose

Sg: N-glycolylneuraminic acid

iso: Structural isomer

1 A1 1425.5837 -3.8 0.09

2 FA1 1571.6446 -1.5 0.17

3 A2 1628.6676 -0.5 1.21

4 FA2 1774.7256 -0.4 46.02

5 A2G1 1790.7194 -1.1 0.66

FA1G1 1733.6976 -1.3 <0.04

6 A2G1(iso) 1790.7194 -1.1 0.30

7 FA2G1 1936.7772 -1.0 18.59

8 FA2G1(iso) 1936.7772 -1.0 21.51

9 FA2G1B 2139.8558 -1.3 0.04

10 FA2G1B (iso) 2139.8558 -1.3 0.21

11 FA2G2 2098.828 -1.9 8.03

12 FA2G1Ga1 2098.828 -1.9 0.67

13 FA2G2Ga1 2260.877 -3.4 1.08

14 FA2G2Sg1 2405.9174 -2.0 0.18

15 FA2G2Sg1(iso) 2405.9174 -2.0 0.06

16 FA2G2Ga2 2422.9322 -2.2 0.87

17 FA2G2GaSg1 2567.9694 -2.2 0.20

18 FA2G2Ga1Sg1(iso) 2567.9694 -2.2 0.11


MS FA2G2?



Zoom 10x

?


Relative Abundance8.0%


Prominent 528 m/z ion and loss of GlcNAc

Relative Abundance0.7%



MS FA2G2?FA2G1Ga1


Zoom 10x

?FA2G1Ga1

30.0010.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

H-Class BIO UPLCACQUITY UPLC Glycan BEH Amide, 130Å, 1.7µm2.1 X 150 mmFlow Rate: 0.40 mL/minute

Total Run Time = 55 minutes

Fluore

scen

ce (

rela

tive

)

Glycan MonitoringTransferability – UPLC / HPLC


30.00

24.00 28.00 32.00 36.00 40.00 44.00 48.00 52.00 56.00 60.00 64.00

Alliance HPLCXBridge Glycan BEH Amide, 130Å, 2.5µm 3.0 X 225 mm total length (150 mm + 75 mm)Flow Rate: 0.56 mL/minute

Total Run Time = 121.3 minutes

Fluore

scen

ce (

rela

tive

)

Time (minutes)

� Comparable sensitivity and resolution – 3x sample load / 2x increase in time

� Transfer between labs with different LC equipment capabilities

Glycan MonitoringUPLC/FLR/ACQUITY QDa Mass Detector

Detection across a broad range of glycan species:

IgGSimple bi-antennerystructures

RNase BHigh mannose structures

Rountine Analysis with Mass Detection

FLR


5 10 15 20 25 30 35

Minutes5 10 15 20 25 30 35

High mannose structures

FetuinLarge, complex structures

Example courtesy of S. McCarthy

MonitoringMonitoring

Intermediate Technology UPLC-FLR

UPLC-Mass Detection

3x

3x

QDa

Glycan MonitoringUPLC/FLR/ACQUITY QDa Mass Detector

FLR


QDa

2

6

7

11

14

Labeled Glycans of IgG Pooled from Human Serum 400 pmol

FLR

Peak GlycanApprox

RT (min)

Approx Rel

Abund (%)

1 A2 11.8 0.7

2 FA2 12.8 20.9

3 FA2B 13.9 4.3

4 A2G1 14.2 1.0

5 A2G1 14.5 0.4

6 FA2G1 15.1 19.4

7 FA2G1 15.5 10.0

8 FA2BG1 15.9 5.0

9 FA2BG1 16.3 0.5

10 A2G2 16.8 0.9

11 FA2G2 17.7 14.8

12 FA2BG2 18.2 1.7

FA2BG2S2

FA2

GlycoWorks RapiFluor-MS StandardsRapiFluor-MS Glycan Performance Test Standard


1

3

45

8

91012

13 15 1617

MS(BPI)

5

12 FA2BG2 18.2 1.7

13 FA2G1S1 18.9 2.8

14 FA2G2S1 20.8 9.6

15 FA2BG2S1 21.5 2.4

16 FA2G2S2 23.6 2.2

17 FA2BG2S2 24.0 2.6

� System suitability� Method familiarization� Benchmarking� Troubleshooting

LC Calibration with Labeled Dextran

12

14

16

18

20

6

7

8910 11 12 13

14151617

n

Lo

g M

ol W

t

4.8

4.9

5.0

5.1

5.2

4

5

6

7

8

9

10

Cubic Spline Fitting

Calibration Curve

GU

Valu

e


EU

0

2

4

6

8

10

Retention Time (min)

5 10 15 20 25

3 45 L

og

Mo

l W

t

4.4

4.5

4.6

4.7


5 10 15 20

0

1

2

3

5 6 7 8 9 10 11 12



Lo

g M

ol W

t

4.8

4.9

5.0

5.1

5.2

12

14

16

18

20

4.44 min

4

5

6

7

8

9

10


Calibration Curve

GU

Valu

e



Lo

g M

ol W

t

4.4

4.5

4.6

4.7


5 10 15 20

EU

0

2

4

6

8

10

12


5 10 15 20 25

5.89GU

0

1

2

3

5 6 7 8 9 10 11 12



5.85GU

5.95GU

25

30

35

Multiple sites reporting onone glycan profile

Significant deviations in RTs

8

9

10

11

12

Cubic Spline Fit Type

Ret

ention T

ime

(min

)

GU

Val

ue



10

15

20

0 5 10 15 20 25 30

Glycan Profile Component

RSDmax = 8.505

6

7

0 5 10 15 20 25 30

RSDmax = 0.49

Ret

ention T

ime

(min

)

GU

Val

ue

Glycan Profile Component Glycan Profile Component

� GU values improve the robustness of data reporting


� Dextran – no amine to modified with rapid tagging reagents

� Reductive amination with analogs is disadvantageous- different physicochemical properties due to different linkage- FLR wavelengths / basicity / pKas / polarity / HILIC retention

Reductive AminationWith an Analog

RapiFluor-MS Labeled N-Glycans

Dextran

Novel Dextran Ladder

DextranGlycan


235 nm

240 nm

245 nm

250 nm

255 nm

260 nm

265 nm

270 nm

275 nm

280 nm

285 nm

290 nm

295 nm

λExcitation,Max=265 nm λEmission,Max=425 nm Ex 265 nm

Em 425 nm

400 500 nm 400 500 nm 400 500 nm

x x265 nm

325 nm

370 nm

x

400 450 500

265 nm

315 nm

365 nm

Ex 265 nmEm 425 nm

xEx 370 nmEm 480 nm

DextranGlycan

50 µg of a Novel Dextran Ladder

GlycoWorks RapiFluor-MS StandardsRapiFluor-MS Dextran Calibration Ladder

FLR 813

9 1011 12

76

5

Ethanolamino Urea Linkage

RapiFluor-MS LabelDextran

� FLR properties match RapiFluor-MS labeled glycans � Tuned for HILIC Retention

1415

1617


4.00 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

MS(BPI)

GU 3

54

Patent Pending

1617 18

4 38 min

GlycoWorks Mobile Phase Concentrate

� High purity mobile phases are critical to successful glycan LC-MS

� LC-MS grade mobile phase concentrate– Eliminate concerns

LC-MS Grade


– Eliminate concerns on NH4 HCOOH purity and glass-ware cleanliness

– 10 mL concentrate simply poured into a 1L bottle of LC-MS water

Adduct Formation• Na• K• KCl / NaCl

Not LC-MS Grade

SummaryPart II

� N-glycan characterization facilitated by combining RapiFluor-MS labeling and high sensitivity QTof MS

� RapiFluor-MS enhances ESI of glycans, producing high charge state, protonated ions

� RapiFluor-MS enables the possibility of QDa mass detection during routine


RapiFluor-MS


QDa mass detection during routine analyses

� In addition to the GlycoWorks RapiFluor-MS N-glycan kit, new glycan standards and reagents have been developed to support analytical workflows, such as GU value assignment

RapiFluor-MS N-Glycan Kit

Ethanolamino Urea Linkage

RapiFluor-MS LabelDextran

Calibration Ladder

� “Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Novel Fluorescence and MS-Active Labeling Reagent” Waters Application Note 720005275EN. January 2015.

� “Robustness of RapiFluor-MS N-Glycan Sample Preparation and HILIC Analysis” Waters

Application Note in preparation. March 2015.

� Manuscript in preparation

Useful Literature

Posters

Application Notes / Publications


WCBP 2015 / P-206-W“Routine Monitoring of N-Glycans Using a Novel Labeling Reagent with Fluorescence and Mass Detection”

WCBP 2015 / P-216-W“Rapid Preparation of N-Glycans Using a Novel Fluorescence and MS Active Labeling Reagent”

WCBP 2015 / P-115-T“Developing a Scientific Library for UPLC/FLR/MS Analysis of Released N-glycans Labeled with a Novel

www.waters.com/Glycans www.waters.com/RapiFluor-MS

Posters

Thank You for Attending!

� Post-Event Home Page: http://www.waters.com/Feb24

� 30% Introductory Offer On GlycoWorks RapiFluor-MS

– Full Webinar Recording of Today’s Session w/PDF Slide Deck

– Compilation of TODAY’S KEY Literature, Brochures etc…

� For Questions and to Submit your Ideas for our Next Topic


� For Questions and to Submit your Ideas for our Next Topic

– Please eMail - [email protected]

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