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MSE: An Alternate Scanning Methodology for Acquiring Peptide and Fragmentation Data

OVERVIEW

An integrated workflow featuring high-resolution UPLC® separation, multiplexed MS data acquisition (MSE), and automatic batch processing of data by BiopharmaLynx™, is developed for fast mapping of protein disulfide bonds

Highly reproducible, fully annotated peptide maps for therapeutic

proteins can be routinely acquired using the workflow The workflow enables automatic assignment of disulfide bonded

peptides in the same run as peptide profiling

EXPERIMENTAL METHODS

Samples: RapiGest SF™ aided LysC

digested IgG1 Instrumentation: Liquid Chromatography,

ACQUITY UPLC® Mass spectrometers Xevo® G2 QTof or SYNAPT® G2 Columns: Peptide Separation Technology,

ACQUITY UPLC® C4 BEH300, 1.7µm, 2.1×150 mm

Informatics: BiopharmaLynx™ v. 1.2

application manager

TOWARDS FAST MAPPING OF PROTEIN DISULFIDE BONDS: AN INTEGRATED WORKFLOW FOR AUTOMATIC ASSIGNMENT OF DISULFIDE PAIRING

Hongwei Xie, Asish B. Chakraborty and Weibin Chen Biopharmaceutical Sciences, Waters Corporation, 34 Maple Street, Milford, MA 01757

Figure 1. Waters biopharmaceutical LC/MS system: ACQUITY UPLC LC, Xevo G2 QTof, and BiopharmaLynx Informatics

CONCLUSIONS

Fast mapping of disulfide linked peptides has been

achieved using LC-MSE methodology coupled with

BiopharmaLynx informatics.

Assignment of disulfide bonded peptides is automated,

based on accurate MS measurement and confirmed by

high-energy MSE fragmentation data.

Effective sample preparation in combination with robust

analytical workflow facilitates routine analysis of disulfide

bonded peptides within a non-reduced peptide map.

Extension of this work for automatic detection of

scrambled disulfide bonded peptides is in progress.

Global analysis Minimize bias/selection of ions

(Improved Reproducibility) Qualitative and quantitative

data from one analysis

Collision Energy Alternates: MS: Low (5ev) MSE: Elevated (15 45eV)

+9

+8

+7

+6

+10

+11

1185.15

5.9129 4996.4247M+H

1000 2000 3000 4000

Inte

nsity

(cou

nts)

0

61860

Control: THTCPPCPAPELLGGPSVFLFPPK=THTCPPCPAPELLGGPSVFLFPPK

TH TCPPCPAPELLGGPSVFLFPPK=THTCPPCPAPELLGGPSVFLFPPK bMaxK F L F L E AP CPPCT =KPPFLFVSPGGLLEPAPCPPCTHT

1145.63771/y11

1031.59481/y9

341.21951/y3

748.44122/y6

3974.90331/y24-2/b15

488.28931/y4 3747.7788

1/y24-2/b122603.14651/b12-2/b13

1597.90101/y15

3634.68851/y24-2/b11

4665.27151/y24-2/b21

244.16681/y2 4405.1382

1/y24-2/b192263.96561/b9-2/b13 3240.5327

1/y24-2/b7

8.7311 7360.281M+H

1000 2000 3000 4000 5000 6000 7000

Inte

nsity

(cou

nts)

0

65775

Control: DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK=STSGGTAALGCLVK

DY V S ... TSGVHTFPA LQ YSLSSVVTVPSSSLGTQTYICNVNHK=STSGGTAALGCLVK bMaxKV L AATGGSTS =KH NVNCIY TGP S T...

3110.52912/y17-1/y14

3409.70782/y14-1/y20

4216.13092/y28-1/y14

2760.33571/b26

2335.17432/y10-1/b13251.1040

1/a2 1048.50021/b9 7112.5469

2/y56-1/y144631.31932/y32-1/y14

6543.28082/y14-1/y515722.8638

2/y45-1/b131975.96462/y6-1/y14

0 7848.0396M+H

1000 2000 3000 4000 5000 6000 7000

Inte

nsity

(cou

nts)

0

54302

Control: DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK=STSGGTAALGCLVK

DY F...V WN LTS GVHTFPAVS Y SSVVTVPS SSLGTQTYICNVNHKPSNTK=STSGGTAALGCLVK bMaxKVLCG ATGGSTSNVNCIY TGL VTVVSSLSYLGSSQLVAPFTH VGSTLA...

3637.79202/y22-1/y14

3936.97052/y14-1/y25

4210.10792/y28-1/y14

4573.28812/y14-1/y312760.3296

1/b26251.1039

1/b8 3088.49681/b3

7266.64112/y58-1/y141048.4985

1/b9 5426.73542/y41-1/b13

2218.08792/y7-1/y13

2:K14-4:K14

2:K7-2:K8

2:K7-2:K8-9

Control: 20100806_UCA_064_mab_LysC_04.raw

3.4368 90.1586Retent ion Time (mins)

10 20 30 40 50 60 70 80

% (

max

= 2

9040

00.0

Cou

nts)

0

25

50

75

100

2:K16-2 :K2146 .36

1:K7-1:K1348 .71

2:K7-2:K876 .73

2:K7-2:K8-974 .18

2:K27-2 :K3140 .81

1:K1-1:K470 .59

2:K1-2:K566 .93

53 .27

50 .15

51 .53

37 .65

30 .31

71 .04

34 .6317 .70

27 .36

T

Min Intensity Threshold : 0 counts

Xevo G2 QTof data processed by BiopharmaLynx displaying MS and MS/MS (MSE) information. Fragment ions (b/y ions) are identified and listed in the Peak Match Data Table.

Fragment ions (b/y ions) are identified and displayed in BiopharmaLynx to validate MS peak assignment of disulfide pairing.

Identify and Confirm Disulfide Pairing using LC-MSE Data: Peptide Table Showing the Assignment of S-S Linked Peptides

The combination of high resolution TOF MS with wide dynamic range enables the detection of larger disulfide bonded peptides

Automatic Annotation of the Fragmentation Peaks (MSE) for Fast Confirmation of Disulfide Bond Linked Peptides

Influences of Sample Preparation on the Identities of Disulfide Bonded Peptides

LC‐MS TIC

LC‐MSE TIC

Lys‐C Digest

C12

Analyte: 20100806_UCA_064_mab_LysC_04.raw

3.2261 88.6448Retention Time (mins)

20 40 60 80

% (m

ax =

482

1000

.0 C

ount

s)

0

25

50

75

100

2:K16-2:K2146.36

2:K7-2:K876.73

2:K2953.18

2:K7-2:K8-974.18

2:K2630.29

2:K27-2:K3140.81

1:K1037.72

1:K1-1:K470.59

1:K917.662:K4

14.322:K2320.94

2:K1-2:K566.94

T

Min Intens ity Threshold : 0 counts

Control: 20100803_UCA_064_mab_LysC_07.raw

3.2261 88.6448Retention Time (mins)

20 40 60 80

% (m

ax =

482

1000

.0 C

ount

s)

0

25

50

75

100

2:K7-2:K8-973.81

1:K7-1:K1348.05

2:K27-2:K3139.99

1:K1-1:K470.07

2:K2629.66

2:K7-2:K876.57

2:K634.26

2:K3217.46

2:K1-2-2:K565.082:K23

20.58

1:K10-H2O36.98

68.71

70.49

67.55

T

Min Intens ity Threshold : 0 counts

Analyte: 20100806_UCA_064_mab_LysC_04.raw

64.5031 77.6213Retention Time (mins)

66 68 70 72 74 76

% (m

ax =

482

1000

.0 C

ount

s)

0

25

50

75

100

2:K14-4:K1475.93

2:K7-2:K876.73

2:K7-2:K8-974.18

1:K1-1:K470.59

1:K1-1:K4*71.13

2:K1-2:K566.94

2:K7-2:K8-9*75.26

1:K1-2-1:K4*69.98

T

Min Intensity Threshold : 0 counts

Control: 20100803_UCA_064_mab_LysC_07.raw

64.5031 77.6213Retention Time (mins)

66 68 70 72 74 76

% (m

ax =

482

1000

.0 C

ount

s)

0

25

50

75

100

2:K7-2:K8-973.81

1:K1-1:K470.07

2:K14-4:K14-1572.24

2:K7-2:K876.57

1:K1-2-1:K468.74

2:K1-2-2:K5-6*67.58

2:K1-2-2:K565.08

2:K27-2:K31-32*66.54

2:K26-27-2:K30-31*74.90

69.14 70.29

74.7469.36

75.30

71.30 73.37

T

Min Intensity Threshold : 0 counts

15 hrs Digestion

8 hrs Digestion

Decrease of mis-cleavage peptides in longer digestion time