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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
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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)
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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
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©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
012202_a_casein_ox_MSC18_LpH_2 1: Scan ES+ 764
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
012202_a_casein_ox_MSC18_LpH_2 1: Scan ES+ 748
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)
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 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
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©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
b ion Series y ion Series
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