TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2017 Waters Corporation

ENHANCING GLYCOPEPTIDE MS PERFORMANCE THROUGH DERIVATIZATION WITH A NOVEL RAPID LABELLING REAGENT William R. Alley, Jr.

1, Brad J. Williams

2, Ying Qing Yu

1, Mark Hilliard

3, Ciara McManus

3, Pauline Rudd

3

1. Scientific Operations, Waters Corporation, Milford, MA 01757; 2. Waters Corporation, Beverly, MA 01915; 3. National Institute for Bioprocessing Research and Training, Dublin, IR

Experimental

The analysis of intact glycopeptides provides a wealth of data, including determining the site of glyco-

sylation, amino acid sequence, and site-specific glycan information. However, glycopeptides are

challenging to characterize by routine LC-MS/MS approaches. Their collision-induced dissociation

(CID) patterns show extensive glycan fragmentation, while the peptide backbone undergoes minimal

fragmentation. However, electron-transfer dissociation (ETD) can be used for glycopeptide backbone

fragmentation, when derivatized to allow higher charge states to be generated, thus shifting their m/z

values towards the most efficient m/z region for ETD. Here, we utilize a rapid tagging molecule

(RapiFluor-MS or RFMS), originally developed to improve fluorescence (FLR) and mass spectrometry

(MS) performance for glycans, to derivatize the N-termini of IgG glycopeptides. Following the conju-

gation with RFMS, labelled glycopeptides were detected with increased charge states and improved

ETD fragmentation. To further enhance the overall UPLC-MS analysis of glycopeptides, we utilized

HILIC chromatography, which requires the use of TFA that can lower the overall charge state of gly-

copeptides. Therefore, to maximize the possibility for efficient ETD fragmentation, we optimized the

HILIC mobile phase conditions to strike a balance between MS and chromatographic performance.

Sample Preparation Workflow

UPLC Conditions

UPLC System: ACQUITY UPLC H-Class Bio

UPLC Column: ACQUITY UPLC Glycoprotein

BEH Amide, 2.1 x 100 mm, 300 Å pore size,

1.7 μm particle size

Column Temp.: 45o C

Sample Temp.: 10o C

FLR Detection: Ex 265 nm/Em 425 nm

Gradient Conditions

Mobile Phase A: Water, 0.01% TFA/0.1% FA

Mobile Phase B: ACN, 0.01% TFA/0.1% FA

MS Conditions

QTof MS Systems: XEVO G2-XS, SYNAPT

G2-Si

Analyzer Mode: ESI+, sensitivity mode

Capillary Voltage: 3.0 kV

Cone Voltage: 60 V

m/z Range: 500-2500

Scan Rate: 2 Hz

Instrument Control: UNIFI 1.8.2 (XEVO G2-

XS), MassLynx (SYNAPT G2-Si)

Sample

Time (min)

Flow (mL/min)

%A %B

0 0.4 25 75

2 0.4 25 75

27 0.4 50 50

32 0.2 80 20

41 0.2 80 20

46 0.2 25 75

48 0.4 25 75

60 0.4 25 75

Instrumental Conditions

UPLC Gradient

RapiFluor-MS

Resuspend/dilute glycoproteins in PBS (pH 7.5), 0.1% RapiGest-SF, 10 mM TCEP

Incubate for 1 hr at 60o C.

Alkylate with 25 mM iodoacetamide.

Digest with trypsin, 1:25 ratio, for 18 hours at 37o C.

Derivatize with RapiFluor-MS for 5 min at room temperature.

HILIC-UPLC-MS/MS analysis of glycopeptides. (Non-glycopeptides are minimally retained.)

Practical Considerations: Balancing Ion Charge, MS Signal Intensity, and Chromatographic Performance

TFA is a critical mobile phase additive for HILIC separations as it acts as an ion-pairing agent with charged amino acids of the peptide backbone and allows the separation to be driven more by the glycan. Including TFA also improves chromatographic peak shape and width. However, TFA lowers the overall MS signal and can reduce the charge state of ESI-generated ions. To maximize the possibility for a successful ETD experiment, the minimum concentration of TFA must be used to maintain good chromatographic perfor-mance, the highest possible MS signal, and the highest possible ion charge states.

The tertiary amino group of RFMS, a molecule orig-inally designed to improve both FLR and MS per-formance of glycans,

1 is estimated to have a pKa of

~11. This is higher than that of the N-terminus, ~9. Therefore, derivatizing the N-terminus with RFMS should enhance the MS performance of glycopep-tides and should result in more highly charged ions.

With optimum HILIC mobile phase conditions of 0.01% TFA / 0.1% FA in both A and B, an ac-

ceptable balance between charge state, MS intensity, and chromatographic performance can be

achieved and is important for efficient ETD fragmentation of intact glycopeptides.

Derivatization with RFMS is complete and results in a shift to higher charge states.

ETD of RFMS-derivatized glycopeptides produced extensive fragmentation, while minimal frag-

mentation was observed for native analytes.

1. Lauber, M. A., et al. Rapid Preparation of Re-leased N-Glycans for HILIC Analysis Using a Label-ing Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection. Anal. Chem., 2015, 87, 5401–5409.

Derivatization with RFMS Significantly Improves the Efficiency of

ETD Fragmentation for Intact Glycopeptides Optimization of HILIC Mobile Phase Conditions Introduction

References

The Influence of TFA Concentration on Chromatographic Performance

The Influence of TFA Concentration on Charge State Increasing the TFA concentration lowered the charge state for all of the glycopeptides included in this study. In all cases, abundance of the highest charged ions were observed for 0.01% TFA (as a reference, 0% is also included). As an example, the 4+ ion of the IgG1:F(6)A2G2S1 glycopeptide (bottom right) was observed at about 15% relative abundance with 0.01% TFA and about 5% with 0.02% TFA. Further in-creasing the concentration to 0.05% resulted in only 2% abun-dance and none was observed with 0.1%. Therefore, to maxim-ize the potential for a successful ETD fragmentation, 0.01% TFA seems optimal.

0

10

20

30

40

50

60

70

80

90

100

0% 0.01% 0.02% 0.05% 0.10%

[TFA] (%)

Rel

ativ

e %

Inte

nsit

y

3+ 2+

0

10

20

30

40

50

60

70

80

90

100

0% 0.01% 0.02% 0.05% 0.10%

[TFA] (%)

Re

lati

ve %

Inte

nsi

ty

3+ 2+

0

10

20

30

40

50

60

70

80

90

100

0% 0.01% 0.02% 0.05% 0.10%

[TFA] (%)

Rel

ativ

e %

Inte

nsit

y

3+ 2+4+

0

10

20

30

40

50

60

70

80

90

100

0% 0.01% 0.02% 0.05% 0.10%

[TFA] (%)

Rel

ativ

e %

Inte

nsit

y

3+ 2+

IgG3:F(6)A2 IgG3:F(6)A2[6]G1

IgG1:F(6)A2G2S1IgG3:F(6)A2G2

0

1

2

3

4

5

6

7

8

0 0.01 0.02 0.05 0.1

[TFA] (%)

Peak

Wid

th (

sec,

FW

HM

)

IgG3:F(6)A2

0

1

2

3

4

5

6

7

0 0.01 0.02 0.05 0.1

IgG1:F(6)A2G2S1

Pe

ak

Wid

th (

sec,

FW

HM

)

[TFA] (%)

The primary goal of this project was to evaluate the effect of derivatization of glycopeptides with RFMS to improve their MS performance and, in particular, their ETD fragmentation efficiency. As demonstrated above, this derivatization significantly improves the ETD perfor-mance. For the underivatized species, few fragments were detected, even with employing 5, 10, or 15 volts of supplemental CID activa-tion. Conversely, complete sets of both c and z ions were detected following derivatization and allows for the entire amino acid se-quence to be determined, along with the site of glycosylation. For the IgG3:F(6)A2 glycopeptide, both sets of ions were detected with-out the supplemental activation, though their intensities were seemingly improved with 5V, even though 2 CID-type oxonium ions were detected. The sialylated IgG1 glycopeptide showed a greater benefit to supplemental CID activation, as 2 z ions and 1 c ion were not observed with 0 V of supplemental activation and all were detected with 5V. Thus, there is a clear benefit to derivatizing glycopeptides with RFMS for ETD analyses of mAb-like glycopeptides.

Conclusions

The Influence of TFA Concentration on MS Signal Intensity These bar graphs repre-sent the sum of all isotope peak areas for a given gly-copeptide across all ob-served charge states and demonstrate the influence of the TFA concentration on the MS signal. Formic acid-only in both A and B resulted in the highest MS signals and adding TFA resulted in a loss of signal. As a general trend, the MS signal did not change sig-nificantly between 0.01-0.05% TFA but there was a noticeable decrease in sig-nal for 0.1% TFA. It is still believed that 0.01% TFA is optimal based on the high-er charge states observed at this concentration.

Glycopeptide derivatization with RFMS results in the generation of more highly charged ions, as shown by these two figures. For derivatized IgG3:F(6)A2, the predominant ion was the 3+ and significant levels of 4+ were observed and only observed for the derivatized sample. For IgG1:F(6)A2G2S1, nearly equal levels of the 3+ and 4+ ions were generated for the RFMS-labelled pep-tide, while for the native species, the 3+ was most abundant ions and much lower levels of the 4+ ion detected.

More Highly Charged Ions are Generated for RFMS

-derivatized Intact Glycopeptides

Here, we show the need for TFA as a mobile phase addi-tive. Including TFA improves both chro-matographic resolu-tion and peak shape while reducing peak width. In this work, we found that 0.1% FA was needed when using lower concentrations of TFA to maintain ac-ceptable chromato-graphic performance.

F(6)A2[6]G1

F(6)A2[3]G1

F(6)A2[6]G1 F(6)A2[3]G1

F(6)A2[3]G1

F(6)A2[3]G1

F(6)A2[3]G1

F(6)A2[6]G1

F(6)A2[6]G1

F(6)A2[6]G1

0% TFA/0.1% FA

0.01% TFA/0.1% FA

0.02% TFA/0.1% FA

0.05% TFA/0.1% FA

0.1% TFA/0.1% FA

Rs: 0.7

Rs: 1.05

Rs: 1.2

Rs: 1.25

Rs: 1.35

(EEQFNSTFR:F(6)A2) (EEQFNSTFR:F(6)A2[6]G1)

(EEQFNSTFR:F(6)A2G2) (EEQYNSTYR:F(6)A2G2S1)

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

9000000

10000000

0 0.01 0.02 0.05 0.1

[TFA] (%)

To

tal X

IC P

ea

k A

rea

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

0 0.01 0.02 0.05 0.1

[TFA] (%)

Tota

l XIC

Pea

k A

rea

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

0 0.01 0.02 0.05 0.1

[TFA] (%)

To

tal X

IC P

ea

k A

rea

0

200000

400000

600000

800000

1000000

1200000

1400000

0 0.01 0.02 0.05 0.1

[TFA] (%)

Tota

l XIC

Pea

k A

rea

IgG3:F(6)A2 IgG3:F(6)A2[6]G1

IgG1:F(6)A2G2S1IgG3:F(6)A2G2

EEQFNSTFR:F(6)A2 EEQFNSTFR:F(6)A2[6]G1

EEQFNSTFR:F(6)A2G2 EEQYNSTYR:F(6)A2G2S1

Sequence for both peptides: EEQFNSTFR EEQFNSTFR:F(6)A2

EEQYNSTYR:F(6)A2G2S1

Labelling with RFMS Enables FLR Detection

As with RFMS-labelled glycans, gly-copeptides derivatized with RFMS can be detected by FLR. Presented here is an FLR HILIC chromatogram annotated with a number of IgG gly-copeptides from pooled human se-rum IgG that were verified by MS techniques. With HILIC, nonglyco-peptides are minimally retained and elute much earlier than glycopep-tides.

* Minor peaks from overlabelled glycopeptides

IgG1:EEQYNSTYR IgG3:EEQFNSTFR

EEQFNSTFR:F(6)A2 EEQYNSTYR:F(6)A2G2S1

Labelling Efficiency Studies To assess the labelling efficiency of glycopeptides with RFMS, two differ-ent glycopeptide backbones from pooled human serum IgG were cho-sen: IgG3 and IgG1. The derivatized XICs for a glycopeptide with each backbone are shown in the blue trac-es of each panel and the XICs corre-sponding to the underivatized glyco-peptides are shown as the black trac-es, which show complete labelling. As rapid labelling molecules featuring NHS carbamates can react with hy-droxyls, the overlabelling (red traces) was investigated. The IgG3 back-bone, did not show any overlabelling However, the IgG1 backbone, with two tyrosines did appear to be overla-belled. Two chromatographic peaks were observed and could be associ-ated with overlabelling of one tyrosine or the other. Additionally, the orange trace of the IgG1 panel presents the XIC for a doubly-overlabelled ana-logue, where presumably both tyro-sines were labelled.

IgG1:EEQYNSTYR:F(6)A2G2S1

IgG3:EEQFNSTFR:F(6)A2

Derivatized

Underivatized

Overderivatized

Doubly Overderivatized

RFMS-EEQYNSTYR

EEQYNSTYR

RFMS-EEQYNSTYR

RFMS

RFMS-EEQYNSTYR

RFMS-EEQYNSTYR

RFMS

RFMS RFMS

|

|

| |

250x zoom

Derivatized

Underivatized

Overderivatized

RFMS-EEQFNSTFR

EEQFNSTFR

RFMS-EEQFNSTFR

RFMS