A Method for Detecting Post- Translational Modifications ... · Waters ©2002 Waters Corporation Separation of Oxidized Methionine 100 0 100 % 0 100 % 0 100 % 012202_a_casein_ox_MSC18_LpH_2

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©2002 Waters Corporation©2002 Waters Corporation©2002 Waters Corporation

A Method for Detecting Post-Translational Modifications in Intact

Proteins and Peptides

Reb J. Russell II, Paul Rainville, Claude Mallet, Jeff Mazzeo

Waters Corporation. Milford, MA

WA20699

Waters Carbamylation, Oxidation, Deamidation

Occur • In Vivo• In Sample Preparation

– Upstream and Downstream Processing• In Storage over time

• Markers of Disease State• Affect Efficacy of Proteins and Peptides• Affect Identification and Characterization of

proteins and peptides

©2002 Waters Corporation

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Methodological Approachfor Characterization of

• Carbamylation, Oxidation, Deamidation

• LC/UV Proteins• LC/MS Proteins

• LC/UV Peptides• LC/MS Peptides• LC/MS/MS

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Instrument Methodologyfor Proteins and Peptides

• Waters ZQ™ 4000 or a Micromass Q-ToF 2™ MS• Waters Alliance 2790 or 2690 LC• Positive electrospray ionization• Capillary Voltage = 3.3 kV• Extraction Cone Voltage between 20-25 V Protein / 25-35

Peptide• Source Temperature = 150°C, • Desolvation Temperature = 400°C• Cone Gas Flow = 50 L/Hr, Desolvation Gas Flow = 500 L/Hr. • MS/MS collision engergy between 30-35• MassLynx™ v3.5 Control

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Protein Separations MethodologyDelta-Pak™ or Symmetry® 300 columns

C18; 4.6 x 50mm; 5 micron200Å or 300Å Pores

Gradient from 15% B to 80% B in 15 min A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrileFlow rate of 0.5 mL/min Split

0.2 mL/min into the mass spectrometer 0.3mL/min being diverted into the 996 PDA detector.

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LCLC

UVUV

MSMS

ColumnColumn

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Peptide Separations MethodologyRapiGest™ Digestion AidAtlantis™ columns

C18; 4.6 x 50mm; 3.5 micron100Å Pores

Gradient from 0% B to 40% B in 45 min A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrileFlow rate of 0.75 mL/min Split

0.2 mL/min into the mass spectrometer 0.55mL/min being diverted into the 996 PDA detector.

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Carbamylation ∆43 MassIf you use urea,

you mostly like have carbamylations!

SN2 RxN withNucleophilic Amino Acids

-N-Term Amine-Lysine -Arginine-Cysteine

HNOR'

NH2

HNOR'

NH

NH2

HN C O

H2N NH2O

O

∆

RR

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©2002 Waters Corporation©2002 Waters Corporation©2002 Waters Corporation2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.000

100

%

6.86

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.000

100

%

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.000

100

%

TOF MS ES+TIC

1.30e4

TOF MS ES+TIC

1.30e4TIC

1.30e4

6.86

16000 16250 16500 16750 17000 17250 17500 17750mass0

100

%

HHM 365 (6.859) TOF MS ES+1.21e4

16946

17002

16000 16250 16500 16750 17000 17250 17500 17750mass0

100

%

HHM 365 (6.859) TOF MS ES+1.21e4

16000 16250 16500 16750 17000 17250 17500 17750mass0

100

%

HHM 365 (6.859) TOF MS ES+1.21e4

16946

17002

Horse Heart Myoglobin

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Horse Heart Myoglobin Carbamylated

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.000

100

%

2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.000

100

%

7.407.40

TOF MS ES+TIC

5.43e4

TOF MS ES+TIC

5.43e4

16000 16500 17000 17500 18000 18500mass0

100

%

HHM_Test 394 (7.404) TOF MS ES+3.44e4

16000 16500 17000 17500 18000 18500mass0

100

%

HHM_Test 394 (7.404) TOF MS ES+3.44e4

17161

17118

17075

17032

16989

16946

17247

17290

17333

17376

17419

17462

17505

2 Carbamylations

17204

17161

17118

17075

17032

16989

16946

17247

17290

17333

17376

17419

17462

17505

6 Carbamylations

2 Carbamylations

13 Carbamylations

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A B C D

Overlays of LC/MS of Carbamylated Cyto C

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Varying Amounts of Carbamylation

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Cyto C Peptides LC/MS

StandardT15+1 779 m/z

CarbamylatedT15+1 779 m/z T15+1 822 m/z

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y Ion Series b Ion Series

Ion m/zUnmodified

∆ m/z m/zmodified

Ion m/zUnmodi

fied

∆ m/z m/zmodifie

d

y1 147 ∆ 0 147 b1 - - -

y2 260 ∆ 0 260 b2 287 ∆ 43 245

y3 317 ∆ 0 317 b3 435 ∆ 43 392

y4 388 ∆ 0 388 b4 506 ∆ 43 -

y5 535 ∆ 0 535 b5 563 ∆ 43 520

y6 648 ∆ 0 648 b6 - - 633

yMax 822 ∆ 43 779 bMax - - -

M I F A G I Ky6 y5 y4 y3 y2 y1

CarbamylatedN-Term M.

b1 b6b5b4b3b2

Not C-Term KM I F A G I Ky6 y5 y4 y3 y2 y1

CarbamylatedN-Term M.

b1 b6b5b4b3b2

Not C-Term K

MSMS Ion Comparison T15 (Carbamylation)

Waters


Met Oxidation ∆16 & 32 Mass

Met Sulfoxide Sulfone16 mass 32 mass

HNOR'

S

HNOR'

SO

HNOR'

SO O

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Retention Time Shift for Oxidized β Casein

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00Time0

100

%

0

100

%

022002_b_casein_ox_3 TOF MS ES+ TIC

9.17e45.33

0.85

18.6415.9714.42

022002_b_casein_3 TOF MS ES+ TIC

1.05e55.76

0.85

18.6316.22

Oxidized

Unmodified

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©2002 Waters Corporation©2002 Waters Corporation©2002 Waters Corporation10000 10500 11000 11500 12000 12500 13000 13500 14000 14500

mass0

100

%

RNase_Standard 5.74e613689

13707

10000 11000 12000 13000 14000mass0

100

%

RNAse_OX 4.81e613752

13736

13850

13866

13881

RNAse Standard13689 AMU∆16 / Oxidation

3 Oxidations

4 Oxidations

11 Oxidations

12 Oxidations

14 Oxidations

RNAse Protein MS Standard and Oxidized

Waters

©2002 Waters Corporation©2002 Waters Corporation©2002 Waters Corporation5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00Time0

100

%

0

100

%

1.05

4.57

1.34

1.604.04

8.57

6.73

5.17

7.4736.92

35.14

9.27 13.8510.48

26.8826.3717.51 23.0320.17 27.40 32.98

40.55

1.06

4.733.741.38

1.71

2.20

3.47

8.81

7.066.17 7.87

37.43

35.5317.93

10.9017.5111.82

27.3026.75

20.6619.19 22.77 24.35

27.80 29.9833.42 42.4440.37

Alpha Caesin Oxidized

631 m/z T17 Oxidized

615 m/z T15 Not Detected

Alpha Caesin Standard

615 m/z T17

LC/MS: Peptide Unmodified and Oxidized

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Separation of Oxidized Methionine

100

0

100

%

0

100

%

0

100

%

012202_a_casein_ox_MSC18_LpH_2 1: Scan ES+ 780

2.69e623.26

39.1124.42 26.40 41.71


9.36e522.92

20.8717.3816.90 18.5431.12

26.3325.10 27.56 29.07 42.3331.46 41.5736.0433.51 38.5737.34


1.19e628.18

26.5425.9224.7623.6722.5121.3519.0217.3831.4629.07 41.7135.8334.7432.14 40.5538.77

42.39

012202_a_casein_ox_MSC18_LpH_2 1: Scan ES+ TIC

9 74 735.70

34 47 38.70 41 51

methionine

Methionine sulfoxide

Methionine sulfone

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Met Sulfoxide and Sulphone

615 620 625 630 635 640 645 650 655m/z0

100

%

0

100

%

0

100

%

010402_A_CASEIN_NA_3%_1 145 (2.963) Cm (69:219) TOF MS ES+ 728634.49

619.43617.44611.43

647.43

635.50

636.52645.47643.39

648.44

649.46654.45653.45

010402_A_CASEIN_31%_1 161 (3.298) Cm (1:167) TOF MS ES+ 2.25e4634.44

631.41

619.41

634.93

635.45

654.41635.94651.40648.43645.43

010302_A_CASEIN_NA_1 101 (2.073) Cm (9:204) TOF MS ES+ 1.87e4634.47615.43

616.45

631.43617.44619.44 629.47

634.96

635.49

654.45635.98 647.43646.46643.43 648.49

Met Sulfoxide

Met

Met Sulfone

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MSMS Ion Comparison T17 (Oxidation)

L H S M K Alpha Casein T17 Fragment

b ion Series y ion Series

Ion m/z standard

∆ m/z m/z Ox.

Ion m/z standard

∆ m/z m/z Ox.

b1 - - - y1 147 ∆0 147b2 251 ∆0 251 y2 278 ∆16 294b3 338 ∆0 338 y3 365 ∆16 381

b4 467 - - y4 502 - - bmax 597 ∆16 613 ymax 615 ∆16 631

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MSMS Ion Comparison T21 (Oxidation)


Ion m/z standard

∆ m/z m/z Ox.

Ion m/z standard ∆ m/z m/z Ox.

b1 102 ∆0 102 y1 205 ∆0 205 b2 203 ∆0 203 y2 318 ∆0 318

b3 334 ∆16 350 y3 415 ∆0 415 b4 431 ∆16 447 y4 546 ∆16 562 b5 544 ∆16 560 y5 - - - bmax 730 ∆16 746 ymax 748.5 ∆16 764.5

T T M P L W Alpha Casein T21 Fragment

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Immonium Ions

NH

HN

O

R

+H2N CHR

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T21m/z 748

60 80 100 120m/z0

100

%

0

100

%

302129

110

8684

101104 111

10129

110

86

T17m/z 615

50 60 70 80 90 100 110 120 130m/z0

100

%

0

100

%

2.15e4120

8670 116112

125

129

486129

110

86

84 10189

120112

130

T21m/z 764

T17m/z 631

Immonium Ions Indicative of MetOx

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Asp Deamidation ∆1 m/z

NH

CH

NH2

O

RHN

O

R' -NH3

NH

CHR HN R'

C

O

Asparagine Succinimide

NH

CH

OH

O

RHN

O

R'

OH

O

NH

CHR N

O

R'

O

IsoAspartic AcidAspartic Acid

H2OH2O

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Retention Shift: Deamidated ACTH Peptides

Waters

©2002 Waters Corporation©2002 Waters Corporation©2002 Waters Corporation0

100

%

620 m/z MSMS 1: TOF MS ES+

1.00e4

620

621

VYPNGA

616 618 620 622 624 626 628 630m/z

616 618 620 622 624 626 628 630m/z0

100

%

621 m/z MSMS 1: TOF MS ES+1.00e4621

622

623

VYPDGA

Isotope Patterns of Deamidation

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MSMS Ion Comparison ACTH Peptides


Ion m/z standard

∆ m/z m/z Ox.

Ion m/z stan-dard

∆ m/z m/z Ox.

b1 - - - y1 - ∆0 - b2 263 ∆0 263 y2 147 ∆0 147 b3 - - 360 y3 261 ∆1 262 b4 - - - y4 258 ∆1 359 b5 - - - y5 521 ∆1 522

bmax - - - ymax 620 ∆1 621

V Y P N/D G A ACTH Peptide

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Summary

• Carbamylation, Oxidation, Deamidation

• LC/UV Proteins• LC/MS Proteins

• LC/UV Peptides from Proteins• LC/MS Peptides from Proteins• LC/MS/MS Peptides from Proteins

A Simple, Universal Methodological Approach for the Characterization of

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Modification Effects

88*1IncreasePeptideDeamidation

16Protein120 Actual16/32DecreasePeptide

Oxidation43IncreaseProtein

144,172,119*43IncreasePeptideCarbamylation

Signature MSMS ions

∆Change

Retention Time

Sample

*Theoretical

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In Progress

Same methods described here we have done or are doing for;

• Phosphorylation• Formylation • Acylation

Future• Glycosylation

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ACS Poster; Wednesday 6-9 • 248 Identification and Characterization of Post

Translational Modifications of Proteins by LC/MS

• 249 Detecting Carbamylation of Intact Proteins by LC/MS

• 265 Evaluation of Reversed Phase Columns for Protein Separation using a Single LC/UV/MS Methodology

• 284 Significantly improved in-solution tryptic digestion of proteins

Documents

A Method for Detecting Post- Translational Modifications ... · Waters ©2002 Waters Corporation Separation of Oxidized Methionine 100 0 100 % 0 100 % 0 100 % 012202_a_casein_ox_MSC18_LpH_2