An Alternative Approach for Characterization of Impurities … · 2009-10-01 · LT11 127 142...
1
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2009 Waters Corporation INTRODUCTION Sequence variants and posttranslational modifications (PTMs) such as glycosylation, deamidation, and oxidation are common in recombinant protein pharmaceuticals. They potentially affect the safety, activity and stability of protein drugs. Effective monitoring of such impurities and PTMs requires sensitive and reproducible methods. In this study, we have applied an Ultra Performance Liquid Chromatography–Data Independent Acquisition Mass Spectrometry (UPLC/MS E ) approach for characterization and quantification of impurity proteins and site-specific PTMs in protein drugs. The method was evaluated using tryptic digests of yeast enolase, alcohol dehydrogenase (ADH), and a humanized monoclonal antibody (IgG1). Synthetic peptides were used to 1) further confirm the deamidations in the IgG1 “PENNY” peptide, and distinguish aspartyl and isoaspartyl isoforms; 2) exclude potential Met oxidation artifacts produced in the MS ion source; and 3) test ionization efficiency of modified peptides. An Alternative Approach for Characterization of Impurities and Site-specific Modifications in Protein Drugs Hongwei Xie, Martin Gilar, John C. Gebler, and St John Skilton Waters Corporation, 34 Maple Street, Milford, MA 01757 ADVANTAGES OF LC/MS E ● Unbiased data acquisition ● Unbiased sampling of low-abundance peptides ● Improved detection sensitivity ● Suited for quantification via MS signal intensities ● Excellent reproducibility ● Improved analytical speed and efficiency CONCLUSIONS 1. 1. 1. 1. UPLC/MS E analysis of protein tryptic digests yielded qualitative and quantitative information about protein impurities and the presence of protein modifications. 2. 2. 2. 2. Unbiased data acquisition with UPLC/MS E provides high sequence coverage, especially for peptides with low-stoichiometry modfications (<0.5%). 3. 3. 3. 3. Reproducible high resolution UPLC peptide separations ensured confident identification of isobaric modifications with sub Da mass differences (e.g., single and double Asn-deamidated peptides) 4. 4. 4. 4. Synthetic peptides were helpful for distinguishing modified peptides isoforms, and evaluating the ionization efficiency of peptides and their modified forms. Relative concentration of impurity proteins identified by UPLC/MS E analysis and Hi3 quantitation of Enolase and ADH (Normalized to Eno1p or ADH1p) Ionization efficiency (by LC/MS E analysis) of Synthetic HT37(PENNYK) and LT1 and Peptides 1) UPLC/UV (214 nm) and UPLC/MS(TIC) Chromatograms. Peak A - DIQMoxTQSPSSLSAVGDR, Peak B- DIQMTQSPSSLSAVGDR, Peak C - GFPSDIAVEWESNGQPENNYK, Peak D - GFPSDIAVEWESDGQPENNYK UPLC/MS E Identification of “PENNYK” peptide (HT37) modifications in a Humanized IgG1 map (Kept at 4 ̊C for one year). (A) Elution Pattern, (B) MS Spectra, (c) MS E Spectra, (D) RT of Synthetic Peptides y ions in red clearly prove the related modifications UPLC/MS E Separation and identification of an ADH peptide and it’s modified form (Met oxidized) Peptides identified by UPLC/MS E covered 97% of Enolase protein sequence. MS E data also identified peaks corresponding to protein impurities , partial/Non-tryptic peptides, and modified peptides. Retention T i m e (min) 2 4 2 8 3 2 3 6 4 0 0 4 8 12 16 2 0 T1 / T17 / T33 T48 T28 T7 T47 T24 / T46 T2 T3 T12 T19 T10 T5 T9 T40 T23 T18 T22 T50 T41-42 T4 T32 T43 T42 T51 T6 T51-52 T48-49 T11* T11 T44 T38 T16 T16* T14 T30 dimer T45 T35 T37 T27 E1P1 E1P2 S1P1 S1 S2 E1 S3 E2 E1P3 S1P2 S4 Tp1 S5 E1P4 S6 P2 E3 E4 E6 E5 P3 E1P6 E1N1* E1N1 E1P11* E7 E1P7 E1P7* E1N2* E1P7** P4 E9 E8 E1P9 Tp3 P5 P6 E1P10 Synthetic Peptides¹ RT (min) Elution Order A) GFYPSDIAVEWESNGQPENNYK 59.76 3 GFYPSDIAVEWESisoDGQPENNYK 59.36 1 GFYPSDIAVEWESDGQPENNYK 60.57 7 GFYPSDIAVEWESNGQPEisoDNYK 59.66 2 GFYPSDIAVEWESNGQPEDNYK 60.31 5 GFYPSDIAVEWESNGQPENDYK 60.14 4 GFYPSDIAVEWESNGEPENNYK 60.31 5 GFYPSDIAVEWESDGQPEDNYK 61.06 8 GFYPSDIAVEWESDGQPEDDYK 61.55 9 B) DIQMTQSPSSLSASVGDR 42.85 2 DIQMoxTQSPSSLSASVGDR 36.07 1 ¹ A) N/Q-deamidation, "PENNY" peptide T37 of heavy chain; B) M-oxidation, T1 of light chain; isoD - isoaspartic acid; Mox - oxidized M. 100% 13.40% 13.30% 3.10% 1.40% 100% 5.00% 0.70% 0% 50% 100% Percentage Injection 1 -UV (214 nm) Injection 1 -MS (TIC) Injection 2 -UV (214 nm) Injection 2 -MS (TIC) Injection 3 -UV (214 nm) Injection 3 -MS (TIC) (A) (B) (C)(D) % 100 % 0 100 % 0 100 % 0 100 % 0 100 % 0 100 1273.19 1272.69 1273.70 1274.21 1274.70 1275.22 1273.69 1273.18 1274.20 1274.70 1275.21 1275.71 1273.70 1273.19 1274.20 1274.70 1275.22 1276.18 1273.70 1273.20 1274.20 1274.70 1275.17 1275.69 1274.70 1274.20 1273.69 1275.19 1264.68 1264.21 1265.18 1265.66 1266.18 1266.68 58 60 62 RT (min) 1 2 3 4 5 6 Peak #1 Peak #2 Peak #3 Peak #4 Peak #5 Peak #6 0 1262 1266 1270 1274 1278 m/z 1 - GFYPSDIAVEWESNGQPENNYK [MH]+ Mass Difference 2 - GFYPSDIAVEWES iso D GQPENNYK ( isoD - isoaspartic acid ) + 0.98 3 - GFYPSDIAVEWESNGQPE D NYK + 0.98 & GFYPSDIAVEWESNG E PENNYK + 0.98 4 - GFYPSDIAVEWES D GQPENNYK + 0.98 5 - GFYPSDIAVEWES D GQPE D NYK + 1.96 6 - GFYPSDIAVEWES S uc GQPENNYK ( Suc - Succinimide intermediate ) - 16.99 Double charged ion +0.49 Da shift +0.49 Da shift +0.49 Da shift +0.98 Da shift -8.50 Da shift 60.75 61.75 61.25 5 6 (A) Elution Pattern (B) MS Spectra (c) MSE Spectra (D) Synthetic peptides were used to confirm RT of HT37 deamidations, and confirm in-sample LT1 Met-oxidation 2) Ionization Efficiency of Tested Modified Peptides (IE-modified/unmodified) Calculated by IE-modified/unmodified = (MS(modified)/MS(unmodified))/(UV(modified)/UV(unmodified)) * 100% Injection MS(A)* MS(B) MS(C ) MS(D) UV(A) UV(B) UV(C) UV(D) MS(A)/MS(B) UV(A)/UV(B) IE-oxidized/Unmodified** MS(D)/MS(C) UV(D)/UV(C) IE-deamidated/Unmodified 1 1251.11 780.32 612.18 762.96 4594.39 3050.48 7209.42 8960.59 1.6033 1.5061 106.45 1.2463 1.2429 100.27 2 1256.41 789.56 604.16 755.21 4571.44 3073.76 7245.65 9041.04 1.5913 1.4872 106.99 1.2500 1.2478 100.18 3 1283.00 817.76 615.69 760.86 4580.78 3110.66 7175.03 8902.43 1.5689 1.4726 106.54 1.2358 1.2408 99.60 Aveage ± SD 106.66 ± 0.29 100.02 ± 0.36 * MS(X) - peak area of MS total ion chromatographic peak X; UV(X) - peak area of UV (214 nm) chromatographic peak X. Protein Peptide Start End Modification Type Sequence & Modification Site MH+ RT (min) SC (%) 1 Heavy-Chain HT6 51 59 Deamidation N55 IYPTNGYTR 1085.53 27.69 5.5 HT6 51 59 Deamidation N55 IYPTNGYTR 1085.53 28.8 46.2 HT6 51 59 No Modification IYPTNGYTR 1084.55 26.96 48.3 HT10 77 87 Deamidation N84 NTAYLQMNSLR 1311.64 42.47 65.1 HT10 77 87 Deamidation N84 NTAYLQMNSLR 1311.64 45.36 13.4 HT10 77 87 No Modification NTAYLQMNSLR 1310.66 43.7 21.5 HT23 278 291 Deamidation N289 FNWYVDGVEVHNAK 1678.79 51.13 5.1 HT23 278 291 Deamidation N289 FNWYVDGVEVHNAK 1678.79 51.78 9.3 HT23 278 291 No Modification FNWYVDGVEVHNAK 1677.81 50.07 85.6 HT26 305 320 Deamidation N318 VVSVLTVLHQDWLNGK 1809.01 65.9 21 HT26 305 320 Deamidation N318 VVSVLTVLHQDWLNGK 1809.01 67 8.5 HT26 305 320 No Modification VVSVLTVLHQDWLNGK 1808.01 66.1 70.5 HT36 364 373 Deamidation N364 NQVSLTC*LVK 2 1162.61 42.85 1.6 HT36 364 373 Deamidation N364 NQVSLTC*LVK 1162.61 49.12 2.1 HT36 364 373 No Modification NQVSLTC*LVK 1161.63 47.06 96.3 HT37 374 395 Deamidation N387 GFYPSDIAVEWESNGQPENNYK 2545.12 59.36 39.2 HT37 374 395 Deamidation N387 GFYPSDIAVEWESNGQPENNYK 2545.12 60.57 9.4 HT37 374 395 Deamidation N392 & GFYPSDIAVEWESNGQPENNYK & 2545.12 60.31 3.2 Deamidation N389 GFYPSDIAVEWESNGQPENNYK HT37 374 395 Deamidation N387 + N392 GFYPSDIAVEWESNGQPENNYK 2546.1 61.06 0.4 HT37 374 395 Succinimide Intermidate N387 GFYPSDIAVEWESNGQPENNYK 2527.1 61.38 1.3 HT37 374 395 No Modification GFYPSDIAVEWESNGQPENNYK 2544.14 59.76 46.4 HT21 252 258 Oxidation M255 DTLMISR 851.43 28.23 4.7 HT21 252 258 No Modification DTLMISR 835.43 32.76 95.3 Light-Chain LT1 1 18 Oxidation M4 DIQMTQSPSSLSASVGDR 1894.88 43.41 in-source LT1 1 18 No Modification DIQMTQSPSSLSASVGDR 1878.88 43.41 LT3 25 42 Deamidation N30 ASQDVNTAVAWYQQKPGK 1291.98 38.6 1.9 LT3 25 42 Deamidation N30 ASQDVNTAVAWYQQKPGK 1291.98 41.69 32.6 LT3 25 42 No Modification ASQDVNTAVAWYQQKPGK 1291 40.34 65.5 LT11 127 142 Deamidation N137 SGTASVVC*LLNNFYPR 1798.88 67.01 2.51 LT11 127 142 Deamidation N137 SGTASVVC*LLNNFYPR 1798.88 74.61 3.5 LT11 127 142 No Modification SGTASVVC*LLNNFYPR 1797.9 72.05 93.99 LT14 150 169 Deamidation N158 VDNALQSGNSQESVTEQDSK 2136.95 26.88 3.8 LT14 150 169 Deamidation N158 VDNALQSGNSQESVTEQDSK 2136.95 28.11 3.2 LT14 150 169 No Modification VDNALQSGNSQESVTEQDSK 2135.97 27.1 93 1 SC - Stoichiometry in percentage, detected in freshly prepared sample; 2 C* - Carbamidomethyl C Modification Type, Site and Stoichiometry of Modified Peptides Identified from the Humanized IgG1 UPLC/MS E Characterization of Enolase Peptide Map of Tryptic Digestion Target Protein – Enolase 1 (Eno1p - Txx, trytic peptides; E1Px/E1Nx, partially/Non-tryptic peptides, *- Peptides with deamidation) Impurity Proteins – Enolase 2 (Eno2p – Ex); Cu-Zn Superoxide dismutase (Sod1p – Sx, S1Px) ; Glucose-6-phosphate isomerase (Pgi1p – Px); Triosephosphate isomerase (Tpi1p – Tpx) LC System: Waters Acquity™ UPLC ® MS System: Waters SYNAPT™ MS Column : Acquity™ PST C18, BEH300, 2.1 x 100 mm, 1.7 μm Mobile Phase A: 0.1% FA in water, B: 0.1% FA in acetonitrile Gradient: 0-50% B in 60 min, Flow Rate: 0.2 ml/min Column Temp: 40 C, Detection: MS E 424.26 y3 y4 y5 y6 y9 b9/y8 y10 [(M+2H)/2]2+ [(M+2H)/2]2+ [(M+2H)/2]2+ [(M+2H)/2]2+ y3 y3 y3 y4 y4 y 4 y5 y5 y6 y6 y 6 b9/y8 b9/y8 b9/y 8 1063.62 y 9 y 9 y 10 y 10 y 10 Peak #1 - GFYPSDIAVEWESNGQPENNYK Peak #2 - GFYPSDIAVEWESiso D GQPENNYK (PeaK #4 has identical MSE spectrum ) Peak #5 - GFYPSDIAVEWESD GQPED NYK Peak #6 - GFYPSDIAVEWESS uc GQPENNYK m/z 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 % 0 100 % 0 100 % 0 100 % 0 100 764.36 538.28 484.23 424.22 583.32 667.30 1273.06 950.46 1150.54 764.36 538.28 484.25 424.21 583.31 667.31 1273.54 950.44 1151.49 1064.49 997.51 765.35 712.34 583.29 539.24 898.44 950.45 1110.58 1152.48 1273.55 764.36 538.27 424.22 484.25 583.32 667.29 1264.54 1133.49 950.45 851.38 1046.48 m / z 200 600 1000 1400 0 100 0 100 936.52 679.39 608.35 381.22 509.28 879.51 792.48 1351.22 1163.66 1067.53 1251.66 936.54 679.39 608.36 381.22 879.51 792.47 1163.67 1083.51 1234.67 1449.83 65.0 67.0 69.0 71.0 % % 218.15 A B y1 218.15 y2 y1 y2 b2 y3 y3 y4-H20 y4 y4-H20 y4 y5 y5 y6 y6 y7 b2 b8 y7 y8 y8 y9 y9 b9 y10 y10 b10 b10 y11 y11 y12 y12 b11 b11 b12 y13 y13 1267.65 b12 b13 y15 y15 b14 b14-H20 y16 b15 y16 Retenon Time (min) 1) Eluon Paern of Pepde T12 with and without M-oxidaon Peak A - T12 without M-oxidaon Peak B - T12 with M-oxidaon 2) MSE Spectrum of Pepde T12 (Peak A) Sequence: SANLMAGHWVAISGAAGGLGSLAVQYAK 3) MSE Spectrum of T12 with M-oxidaon (Peak B) Sequence: SANL M AGHWVAISGAAGGLGSLAVQYAK M - oxidized M
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Transcript of An Alternative Approach for Characterization of Impurities … · 2009-10-01 · LT11 127 142...
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2009 Waters Corporation
INTRODUCTION
Sequence variants and posttranslational modifications
(PTMs) such as glycosylation, deamidation, and oxidation
are common in recombinant protein pharmaceuticals.
They potentially affect the safety, activity and stability of
protein drugs. Effective monitoring of such impurities and
PTMs requires sensitive and reproducible methods.
In this study, we have applied an Ultra Performance
Liquid Chromatography–Data Independent Acquisition
Mass Spectrometry (UPLC/MSE) approach for
characterization and quantification of impurity proteins
and site-specific PTMs in protein drugs. The method was
evaluated using tryptic digests of yeast enolase, alcohol
dehydrogenase (ADH), and a humanized monoclonal
antibody (IgG1). Synthetic peptides were used to 1)
further confirm the deamidations in the IgG1 “PENNY”
peptide, and distinguish aspartyl and isoaspartyl
isoforms; 2) exclude potential Met oxidation artifacts
produced in the MS ion source; and 3) test ionization
efficiency of modified peptides.
An Alternative Approach for Characterization of Impurities and Site-specific Modifications in Protein Drugs
Hongwei Xie, Martin Gilar, John C. Gebler, and St John Skilton
Waters Corporation, 34 Maple Street, Milford, MA 01757
ADVANTAGES OF LC/MSE
● Unbiased data acquisition
● Unbiased sampling of low-abundance peptides
● Improved detection sensitivity
● Suited for quantification via MS signal intensities
● Excellent reproducibility
● Improved analytical speed and efficiency
CONCLUSIONS
1.1.1.1. UPLC/MSE analysis of protein tryptic digests yielded qualitative and
quantitative information about protein impurities and the presence of protein
modifications.
2.2.2.2. Unbiased data acquisition with UPLC/MSE provides high sequence coverage,
especially for peptides with low-stoichiometry modfications (<0.5%).
3.3.3.3. Reproducible high resolution UPLC peptide separations ensured confident
identification of isobaric modifications with sub Da mass differences (e.g.,
single and double Asn-deamidated peptides)
4.4.4.4. Synthetic peptides were helpful for distinguishing modified peptides isoforms,
and evaluating the ionization efficiency of peptides and their modified forms.
Relative concentration of impurity proteins identified by
UPLC/MSE analysis and Hi3 quantitation of Enolase and ADH
(Normalized to Eno1p or ADH1p)
Ionization efficiency (by LC/MSE analysis) of Synthetic HT37(PENNYK) and LT1 and Peptides
1) UPLC/UV (214 nm) and UPLC/MS(TIC) Chromatograms. Peak A - DIQMoxTQSPSSLSAVGDR, Peak B-
DIQMTQSPSSLSAVGDR, Peak C - GFPSDIAVEWESNGQPENNYK, Peak D - GFPSDIAVEWESDGQPENNYK
UPLC/MSE Identification of “PENNYK” peptide (HT37) modifications in a Humanized IgG1 map
(Kept at 4 ̊C for one year). (A) Elution Pattern, (B) MS Spectra, (c) MSE Spectra, (D) RT of Synthetic Peptides
y ions in red clearly prove the related modifications
UPLC/MSE Separation and identification of an ADH peptide and it’s modified form (Met oxidized)
Peptides identified by UPLC/MSE covered 97% of Enolase protein sequence. MSE data also identified
peaks corresponding to protein impurities , partial/Non-tryptic peptides, and modified peptides.
Retention Time (min)
24 28 32 36 40
0 4 8 12 16 20
T1 / T17 / T33
T48 T28T7 T47
T24 / T46T2
T3T12
T19
T10T5
T9
T40
T23
T18T22T50
T41-42
T4
T32
T43
T42
T51
T6
T51-52
T48-49T11*
T11
T44
T38
T16
T16*
T14
T30 dimer T45
T35
T37
T27
E1P1
E1P2S1P1
S1 S2 E1 S3 E2
E1P3
S1P2
S4Tp1
S5 E1P4 S6P2
E3E4
E6
E5
P3
E1P6
E1N1*
E1N1
E1P11*
E7
E1P7
E1P7*
E1N2*
E1P7**
P4E9E8
E1P9
Tp3 P5 P6
E1P10
Synthetic Peptides¹ RT (min) Elution Order
A)
GFYPSDIAVEWESNGQPENNYK 59.76 3
GFYPSDIAVEWESisoDGQPENNYK 59.36 1
GFYPSDIAVEWESDGQPENNYK 60.57 7
GFYPSDIAVEWESNGQPEisoDNYK 59.66 2
GFYPSDIAVEWESNGQPEDNYK 60.31 5
GFYPSDIAVEWESNGQPENDYK 60.14 4
GFYPSDIAVEWESNGEPENNYK 60.31 5
GFYPSDIAVEWESDGQPEDNYK 61.06 8
GFYPSDIAVEWESDGQPEDDYK 61.55 9
B)
DIQMTQSPSSLSASVGDR 42.85 2
DIQMoxTQSPSSLSASVGDR 36.07 1¹ A) N/Q-deamidation, "PENNY" peptide T37 of heavy chain; B) M-oxidation, T1 of light chain;
isoD - isoaspartic acid; Mox - oxidized M.
100%
13.40%13.30%
3.10% 1.40%
100%
5.00%0.70%
0%
50%
100%
Percentage
Injection 1
-UV (214 nm)
Injection 1
-MS (TIC)
Injection 2
-UV (214 nm)
Injection 2
-MS (TIC)
Injection 3
-UV (214 nm)
Injection 3
-MS (TIC)
(A) (B)(C) (D)
%
100
%
0
100
%
0
100
%
0
100
%
0
100
%
0
100 1273.19
1272.69 1273.70
1274.21
1274.70 1275.22
1273.69
1273.18 1274.20
1274.70
1275.21 1275.71
1273.70
1273.19 1274.20
1274.70 1275.22
1276.18
1273.70
1273.201274.20
1274.701275.17 1275.69
1274.701274.20
1273.69
1275.19
1264.68
1264.211265.18
1265.66
1266.18 1266.68
58 60 62RT (min)
1
2
34
5 6
Peak #1
Peak #2
Peak #3
Peak #4
Peak #5
Peak #6
0
1262 1266 1270 1274 1278m/z
1 - GFYPSDIAVEWESNGQPENNYK [MH]+ Mass Difference
2 - GFYPSDIAVEWESisoDGQPENNYK (isoD - isoaspartic acid) + 0.98
3 - GFYPSDIAVEWESNGQPEDNYK + 0.98
& GFYPSDIAVEWESNGEPENNYK + 0.98
4 - GFYPSDIAVEWESDGQPENNYK + 0.98
5 - GFYPSDIAVEWESDGQPEDNYK + 1.96
6 - GFYPSDIAVEWESSucGQPENNYK (Suc - Succinimide intermediate) - 16.99
Double charged ion
+0.49 Da shift
+0.49 Da shift
+0.49 Da shift
+0.98 Da shift
-8.50 Da shift
60.75 61.7561.25
5
6
(A) Elution Pattern
(B) MS Spectra
(c) MSE Spectra
(D) Synthetic peptides were used to confirm RT of HT37 deamidations, and confirm in-sample LT1 Met-oxidation
2) Ionization Efficiency of Tested Modified Peptides (IE-modified/unmodified) Calculated by
IE-modified/unmodified = (MS(modified)/MS(unmodified))/(UV(modified)/UV(unmodified)) * 100% Injection MS(A)* MS(B) MS(C ) MS(D) UV(A) UV(B) UV(C) UV(D) MS(A)/MS(B) UV(A)/UV(B) IE-oxidized/Unmodified** MS(D)/MS(C) UV(D)/UV(C) IE-deamidated/Unmodified
1 1251.11 780.32 612.18 762.96 4594.39 3050.48 7209.42 8960.59 1.6033 1.5061 106.45 1.2463 1.2429 100.27
2 1256.41 789.56 604.16 755.21 4571.44 3073.76 7245.65 9041.04 1.5913 1.4872 106.99 1.2500 1.2478 100.18
3 1283.00 817.76 615.69 760.86 4580.78 3110.66 7175.03 8902.43 1.5689 1.4726 106.54 1.2358 1.2408 99.60
Aveage ± SD 106.66 ± 0.29 100.02 ± 0.36
* MS(X) - peak area of MS total ion chromatographic peak X; UV(X) - peak area of UV (214 nm) chromatographic peak X.
Protein Peptide Start End Modification Type Sequence & Modification Site MH+ RT (min) SC (%)1
Heavy-Chain HT6 51 59 Deamidation N55 IYPTNGYTR 1085.53 27.69 5.5
HT6 51 59 Deamidation N55 IYPTNGYTR 1085.53 28.8 46.2
HT6 51 59 No Modification IYPTNGYTR 1084.55 26.96 48.3
HT10 77 87 Deamidation N84 NTAYLQMNSLR 1311.64 42.47 65.1
HT10 77 87 Deamidation N84 NTAYLQMNSLR 1311.64 45.36 13.4
HT10 77 87 No Modification NTAYLQMNSLR 1310.66 43.7 21.5
HT23 278 291 Deamidation N289 FNWYVDGVEVHNAK 1678.79 51.13 5.1
HT23 278 291 Deamidation N289 FNWYVDGVEVHNAK 1678.79 51.78 9.3
HT23 278 291 No Modification FNWYVDGVEVHNAK 1677.81 50.07 85.6
HT26 305 320 Deamidation N318 VVSVLTVLHQDWLNGK 1809.01 65.9 21
HT26 305 320 Deamidation N318 VVSVLTVLHQDWLNGK 1809.01 67 8.5
HT26 305 320 No Modification VVSVLTVLHQDWLNGK 1808.01 66.1 70.5
HT36 364 373 Deamidation N364 NQVSLTC*LVK2 1162.61 42.85 1.6
HT36 364 373 Deamidation N364 NQVSLTC*LVK 1162.61 49.12 2.1
HT36 364 373 No Modification NQVSLTC*LVK 1161.63 47.06 96.3
HT37 374 395 Deamidation N387 GFYPSDIAVEWESNGQPENNYK 2545.12 59.36 39.2
HT37 374 395 Deamidation N387 GFYPSDIAVEWESNGQPENNYK 2545.12 60.57 9.4
HT37 374 395 Deamidation N392 & GFYPSDIAVEWESNGQPENNYK & 2545.12 60.31 3.2
Deamidation N389 GFYPSDIAVEWESNGQPENNYKHT37 374 395 Deamidation N387 + N392 GFYPSDIAVEWESNGQPENNYK 2546.1 61.06 0.4
HT37 374 395 Succinimide Intermidate N387 GFYPSDIAVEWESNGQPENNYK 2527.1 61.38 1.3
HT37 374 395 No Modification GFYPSDIAVEWESNGQPENNYK 2544.14 59.76 46.4
HT21 252 258 Oxidation M255 DTLMISR 851.43 28.23 4.7
HT21 252 258 No Modification DTLMISR 835.43 32.76 95.3
Light-Chain LT1 1 18 Oxidation M4 DIQMTQSPSSLSASVGDR 1894.88 43.41 in-source
LT1 1 18 No Modification DIQMTQSPSSLSASVGDR 1878.88 43.41
LT3 25 42 Deamidation N30 ASQDVNTAVAWYQQKPGK 1291.98 38.6 1.9
LT3 25 42 Deamidation N30 ASQDVNTAVAWYQQKPGK 1291.98 41.69 32.6
LT3 25 42 No Modification ASQDVNTAVAWYQQKPGK 1291 40.34 65.5
LT11 127 142 Deamidation N137 SGTASVVC*LLNNFYPR 1798.88 67.01 2.51
LT11 127 142 Deamidation N137 SGTASVVC*LLNNFYPR 1798.88 74.61 3.5
LT11 127 142 No Modification SGTASVVC*LLNNFYPR 1797.9 72.05 93.99
LT14 150 169 Deamidation N158 VDNALQSGNSQESVTEQDSK 2136.95 26.88 3.8
LT14 150 169 Deamidation N158 VDNALQSGNSQESVTEQDSK 2136.95 28.11 3.2
LT14 150 169 No Modification VDNALQSGNSQESVTEQDSK 2135.97 27.1 931 SC - Stoichiometry in percentage, detected in freshly prepared sample;
2 C* - Carbamidomethyl C
Modification Type, Site and Stoichiometry of Modified Peptides Identified from the Humanized IgG1
UPLC/MSE Characterization of Enolase Peptide Map of Tryptic Digestion
Target Protein – Enolase 1 (Eno1p - Txx, trytic peptides; E1Px/E1Nx, partially/Non-tryptic peptides, *- Peptides with deamidation)
Impurity Proteins – Enolase 2 (Eno2p – Ex); Cu-Zn Superoxide dismutase (Sod1p – Sx, S1Px);
Glucose-6-phosphate isomerase (Pgi1p – Px); Triosephosphate isomerase (Tpi1p – Tpx)
LC System: Waters Acquity™ UPLC® MS System: Waters SYNAPT™ MS
Column : Acquity™ PST C18, BEH300, 2.1 x 100 mm, 1.7 µm
Mobile Phase A: 0.1% FA in water, B: 0.1% FA in acetonitrile
Gradient: 0-50% B in 60 min, Flow Rate: 0.2 ml/min
Column Temp: 40 °C, Detection: MSE
424.26
y3y4
y5
y6
y9b9/y8
y10
[(M+2H)/2]2+
[(M+2H)/2]2+
[(M+2H)/2]2+
[(M+2H)/2]2+
y3
y3
y3
y4
y4
y4
y5
y5
y6
y6
y6
b9/y8
b9/y8
b9/y8
1063.62
y9
y9
y10
y10
y10
Peak #1 - GFYPSDIAVEWESNGQPENNYK
Peak #2 - GFYPSDIAVEWESisoDGQPENNYK
(PeaK #4 has identical MSE spectrum)
Peak #5 - GFYPSDIAVEWESDGQPEDNYK
Peak #6 - GFYPSDIAVEWESSucGQPENNYK
m/z450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250
%
0
100
%
0
100
%
0
100
%
0
100764.36
538.28484.23424.22
583.32 667.30
1273.06
950.461150.54
764.36
538.28484.25424.21 583.31 667.31
1273.54
950.441151.491064.49
997.51
765.35
712.34
583.29
539.24
898.44
950.45
1110.58
1152.481273.55
764.36
538.27424.22484.25 583.32
667.29
1264.54
1133.49950.45
851.38 1046.48
m/z
200 600 1000 14000
100
0
100 936.52
679.39
608.35381.22 509.28 879.51
792.48
1351.22
1163.66
1067.53 1251.66
936.54
679.39
608.36381.22
879.51
792.47
1163.671083.51
1234.671449.83
65.0 67.0 69.0 71.0
%
%
218.15
AB
y1
218.15
y2
y1
y2b2 y3
y3
y4-H20
y4
y4-H20
y4
y5
y5
y6
y6
y7
b2
b8
y7
y8
y8
y9
y9
b9 y10
y10
b10
b10
y11
y11
y12
y12
b11
b11
b12
y13
y13
1267.65b12
b13
y15
y15
b14
b14-H20
y16
b15
y16
Reten on Time (min)
1) Elu on Pa"ern of Pep de T12 with and without M-oxida on
Peak A - T12 without M-oxida on
Peak B - T12 with M-oxida on
2) MSE Spectrum of Pep de T12 (Peak A)
Sequence: SANLMAGHWVAISGAAGGLGSLAVQYAK
3) MSE Spectrum of T12 with M-oxida on (Peak B)
Sequence: SANLMAGHWVAISGAAGGLGSLAVQYAK
M - oxidized M
