ELECTROCHEMICAL-ASSISTANCE FOR ELECTRON TRANSFER … · 2013. 10. 3. · ETD reagent. Approximately...
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TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2013 Waters Corporation INTRODUCTION Characterization of intact proteins using tandem mass spectrometry approaches can be challenged by the presence of disulfide bonds that maintain and stabilize three-dimensional structures. Moreover, the presence of multiple disulfide bonds significantly reduces fragmentation efficiency. Electrochemistry- assisted intact protein tandem MS was recently reported for the characterization of multiple disulfide bond containing proteins using DESI- ECD 1 . Following electrochemical reduction and on-line ECD-MS 2 , significant improvements in backbone cleavages were reported compared to the ECD of the oxidized forms of the proteins. This quick reduction of disulfide bonds is an appropriate alternative for disulfide bond reduction using dithiothreitol or other chemical reductants. ESI-ETD-ion mobility-MS was used to investigate the fragmentation efficiency of a number of disulfide bond containing peptides and proteins in their oxidized and reduced forms. ELECTROCHEMICAL-ASSISTANCE FOR ELECTRON TRANSFER DISSOCIATION ION MOBILITY MS OF PEPTIDES AND PROTEINS Jonathan P. Williams, Christopher J. Hughes, Robert Tonge, Jeffery M. Brown Waters Corporation, Manchester, UK METHODS Mass Spectrometry MS: Synapt G2 or G2-S (Fig. 1) Mode: ESI (+) Capillary voltage: 2-3 kV Cone: 25-50V Source temperature: 100 °C Desolvation temperature: 200 °C ETD-MS Reagent: 4-nitrotoluene (4-NT) or 1,4-dicyanobenzene (1,4-DCB) Glow discharge current: 70-90 μA Trap wave height: 0.2-0.25 V Trap RF: 450-500 V Chromatography UPLC: Acquity I-Class Analytical column: BEH300 C4, 1.7μm, 2.1 x 100mm Mobile phase A: H 2 O + 0.1% formic acid Mobile phase B: CH 3 CN + 0.1% formic acid Flow rate: 50μL/min Gradient: 10%B to 35%B in 12minutes Electrochemistry Electrochemical (EC) cell: Roxy (Antec) Reducing solvent: 50% aq. CH 3 CN / 1% formic acid Analyte concentration: ~1 μM Mode: pulse Sample introduction: infusion @ 50 μL/min RESULTS Somatostatin Infusion EC-Ion Mobility MS CONCLUSION Initial studies demonstrate the potential of an Electrochemical-cell for on-line disulfide bond reduction for aiding top-down ETD MS studies. Significant differences were observed between the chemically and electrochemically-reduced forms of the large mAb protein. Further work and method optimization of the pulse profiles for large mAb proteins is currently on-going. References 1. Stokes et al. Top-down protein sequencing by CID and ECD using desorption electrospray ionisation (DESI) and high-field FTICR mass spectrometry. Int J Mass Spectrom289.1 (2010) 54–57 2. Zhang et al. Electrochemistry-Assisted Top-Down Characterization of Disulfide-Containing Proteins. Anal Chem 84.8 (2012): 3838-3842 Figure 1. Synapt G2-S with ETD functionality Bovine Insulin Infusion EC-MS Chicken Lysozyme Infusion EC-MS Figure 3. ETD mass spectra obtained from the ion-ion reaction of the mass-selected [M+3H] 3+ for the (A) oxidized and (B) reduced forms of Somatostatin with the mass-selected anions produced from glow discharge ionization of 4-NT. In (A) low abundance c-/z- type ions were observed by magnification. In (B) abundant c-/z- type ions were directly observed for c 4 -c 13 and z 6 -z 11 and z 12 . Figure 2. Partial mass spectrum highlighting [M+3H] 3+ for (A) oxidized and (B) reduced forms of Somatostatin (Mw 1637, AGCKNFFWKTFTSC) contains a disulfide bond between Cys 3 -Cys 14 . Shown inset are the arrival time distributions (ion mobility) suggesting a more compact and dominant conformation in the Figure 4. ESI mass spectrum of Bovine Insulin in (A) oxidized and (B) reduced form. Low abundance multiply charged ions were observed relative to the abundant [M+4H] 4+ - [M+5H] 5+ for the reduced form of the B-chain. Low abundance multiply charged ions [M+2H] 2+ - [M+4H] 4+ were detected for the reduced form of the A- chain. Bovine Insulin (Mw AVG 5734 Da) has 2 chains containing 2 disulfide bonds and 1 intra-disulfide bond within the A-chain. Figure 7. ESI mass spectrum obtained from Lysozyme in (A) oxidized and (B) reduced form. Multiply charged ions [M+8H] 8+ - [M+12H] 12+ were observed for the oxidized and multiply charged ions [M+8H] 8+ - [M+18H] 18+ were detected for the reduced form. Lysozyme (Mw AVG 14.3 kDa) contains 4 disulfide bonds, Cys6- Cys127, Cys30-Cys115, Cys64-Cys80, and Cys76-Cys94. Figure 8. ETD mass spectrum obtained for the oxidized precursor ion [M+10H] 10+ . Limited sequence information was obtained with only a small number of c-/z-type product ions due to the presence of the disulfide bonds. Figure 9. ETD mass spectrum obtained for the reduced precursor ion [M+15H] 15+ . Extensive sequence coverage is obtained as shown in the mass spectrum. Abundant c– and z-type product ions were observed providing over 50% sequence coverage. Mouse mAb standard LC-EC-MS Synapt G2-S was used for mAb analysis using 1,4-DCB as the ETD reagent. Approximately 30-100μM was injected on-column for EC-ETD analysis depending on the mode selected. Figure 10 shows the individual mAb MS spectra obtained, with com- plementary detail provided in Figures 11 and 12, respectively. Figure 11. LC-ETD mass spectrum obtained for oxidized precursor ion [M+57H] 57+ , m/z 2604. Extensive sequence coverage was obtained for both chains. Shown inset is sequence ions from the mAb heavy chain. Figure 12. LC-EC-ETD mass spectrum of reduced precursor ion [M+27H] 27+ , m/z 897. m/z 544 545 546 547 548 549 550 551 % 0 100 m/z 544 545 546 547 548 549 550 551 % 0 100 546.6 546.9 547.2 547.6 547.9 547.3 546.9 546.6 547.6 547.9 548.3 548.6 (A) (B) EC cell OFF EC cell ON ~ 80% -S—S- bond reduction efficiency 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 % 0 100 4.43 4.64 Time 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 % 0 100 4.64 drift (bins) drift (bins) m/z 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 % 0 100 m/z 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 % 0 100 x16 x10 790.4 546.6 724.4 546.9 547.3 724.9 1622.8 820.4 835.9 1621.8 836.4 1580.8 1448.7 836.9 925.5 1579.7 1623.8 1639.8 1640.8 1640.8 1639.8 820.9 820.4 545.4 c''5 491.3 c''4 377.2 z'6 670.3 547.3 c''6 638.3 547.6 547.9 c''7 785.4 686.3 687.3 1623.8 821.4 c''8 971.5 z'7;856.4 955.5 1622.8 z'9 1150.6 z'8 1003.5 1004.5 z'10 1264.6 1151.6 1408.7 1265.6 1591.8 1641.8 1642.8 1643.8 EC ETD MS EC (cell OFF) ETD MS 2 + charge-reduced 1 + charge-reduced 1 + charge-reduced 2 + charge-reduced (A) (B) precursor ion [M+3H] 3+ precursor ion [M+3H] 3+ Figure 5. ETD mass spectrum of the oxidized precursor ion [M+6H] 6+ showing c-/z- type fragmentation up to the disulfide bonds. m/z 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 % 0 100 x10 2868.7 2868.2 857.4 710.3 400.2 202.1 563.3 2867.7 858.4 2860.7 2859.6 873.4 1071.5 2859.2 1072.5 2846.7 1912.8 1200.5 2440.0 2869.7 2870.2 2870.7 2871.2 5740.0 2871.7 5738.0 4881.0 4879.0 2872.2 4666.7 5028.3 5723.0 5338.9 5742.0 5743.0 5744.0 5745.0 EC (cell OFF) ETD MS singly charged A:B(C20) - A:B(C28) 1 + charge-reduced 3+ charge-reduced 2 + charge-reduced Figure 6. ETD mass spectrum of the reduced precursor ion [M+5H] 5+ from the B-chain showing extensive sequence coverage. m/z 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 % 0 100 1701.4 741.4 z'6 710.4 z'4 400.3 c''3 378.2 c''2 264.2 c''5 643.4 z'5 563.3 c''4 506.3 1700.9 z'7 857.5 742.4 743.4 1700.4 873.5 1141.6 1096.6 874.5 c''9 1003.5 1692.9 1692.4 1142.6 1353.8 1553.9 1482.8 1701.9 3384.9 3383.8 1702.4 1702.9 3382.8 2261.3 1703.4 z'20 2260.3 1849.0 2049.1 2148.2 2661.4 2262.3 2660.4 2278.3 2279.3 3373.9 3356.8 2663.4 3154.7 2758.5 EC ETD MS 1 + charge-reduced 2 + charge-reduced c-ions observed c 1 -c 16 , c 19 , c 21 -c 26 and c 29 z-ions observed z 4 -z 11 and z 14 -z 25 m/z 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 % 0 100 m/z 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 % 0 100 1590.4 1590.3 1590.2 1590.1 1431.6 1431.4 1431.3 1431.2 1301.5 1301.2 1192.9 1301.8 1431.0 1431.7 1431.8 1431.9 1432.0 1432.1 1432.3 1590.7 1590.8 1590.9 1789.2 1788.9 1788.8 1591.2 1591.3 1789.4 1789.6 1789.7 1793.9 1794.2 1193.3 1193.2 1193.1 1101.6 1101.4 1023.0 1022.9 954.8 895.3 1193.4 1301.7 1301.9 1431.7 1302.0 1431.3 1305.2 1431.9 1432.0 1590.7 1590.4 1435.6 1590.9 1594.9 EC cell OFF EC cell ON (B) (A) [M+10H] 10+ [M+9H] 9+ [M+8H] 8+ [M+11H] 11+ [M+12H] 12+ [M+8H] 8+ [M+12H] 12+ [M+18H] 18+ m/z 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 % 0 100 x10 1431.5 1431.4 1431.3 1431.2 1431.1 1431.0 1430.9 1430.8 392.3 245.2 1431.6 1431.7 1431.8 1431.9 1432.0 1590.5 1590.4 1432.1 1590.1 1432.2 1590.0 1589.9 1432.8 1590.6 1590.8 1789.4 1789.1 1789.0 1591.1 1789.5 1789.6 1789.8 2044.9 1789.9 2045.1 EC (cell OFF) ETD MS m/z 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 % 0 100 c''3 392.28 121.06 c''2 245.22 230.20 z'2 z'9 4+ 272.20 433.29 c''11 2+ 582.35 c''13 2+ 711.92 582.84 c''12 2+ 647.87 712.42 c''14 2+ 789.97 1001.58 c''18 2+ 1001.06 z'18 2+ 1058.60 1059.09 1140.63 c''47 4+ 1328.73 1141.13 1428.79 1429.28 1821.99 m/z 1000 1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 1110 1120 % 0 100 1001.58 c''18 2+ 1001.06 993.56 994.06 994.56 z'18 2+ 1058.60 1002.07 c''19 2+ 1058.10 1002.57 c''9 1021.60 1003.07 c''36 4+ z'36 4+ 1013.30 1003.57 1013.79 1022.60 1050.58 1049.89 1049.56 1023.61 z'27 3+ 1049.22 1041.56 1051.08 1059.09 1059.59 c''10 1092.64 1060.09 1060.59 1091.62 1076.62 1093.64 1094.64 c''39 4+ 1106.91 1104.94 1101.58 1107.90 1118.61 EC ETD MS m/z 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 % 0 100 m/z 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 % 0 100 1147.5 1147.3 956.4 956.2 956.0 1147.1 956.5 956.7 956.9 1147.7 1147.9 1148.1 1434.1 1148.3 1433.8 1434.6 1434.8 681.0 680.8 680.6 687.2 850.7 687.4 850.4 858.7 858.9 (A) (B) EC cell ON EC cell OFF [M+3H] 3+ [M+4H] 4+ [M+5H] 5+ [M+6H] 6+ B:[M+4H] 4+ B:[M+5H] 5+ A: [M+3H] 3+ A: [M+4H] 4+ A: [M+2H] 2+ FVNQH LCGSH LVEAL YLVCG ERGFF YTPKA GIVEQ CCASV CSLYQ LENYC N C C C C C C B A Figure 10. LC-MS and LC-EC-MS spectra illustrating analysis of the light and heavy chains (middle and bottom) and the intact form (top) of mouse mAb standard. m/z 500 1000 1500 2000 % 0 100 898.9 z'24 3+ 896.3 895.9 z'15 2+;834.0 z'8 2+ 491.3 899.8 900.8 1345.2 901.3 1343.8 975.1 1325.2 1347.3 z'31 2+ 1817.0 1350.8 1796.0 1818.0 1828.3 EC (cell ON) ETD MS m/z 500 1000 1500 2000 % 0 100 1271.3 1271.0 z'34 3+;1252.9 c''33 3+ 1108.1 z'4 381.2 121.1 1080.1 c''30 3+ 1015.7 1452.6 1452.7 1453.0 1547.1 1694.7 1702.2 c''75 4+ 2004.0 m/z 1950 1975 2000 2025 2050 2075 2100 2125 % 0 100 c''75 4+ 2004.0 1964.4 c''75 4+ 2003.8 1964.6 2003.6 1964.8 1985.6 2004.4 2004.6 2033.4 2007.4 2033.8 z'72 4+ 2055.6 2034.0 2081.6 2081.3 2056.6 2083.9 2109.3 2084.3 EC (cell OFF) ETD MS m/z 1000 2000 3000 % 0 100 1210.82 1153.35 1100.87 1100.78 1053.03 968.96 931.80 1210.96 1274.57 1274.66 1274.72 1345.35 1345.40 1424.30 1614.14 1729.54 1862.52 2699.70 light chain light chain oxidized mAb 10 mM DTT reduction EC cell OFF Electrochemical reduction EC cell ON Intact mAb EC cell OFF m/z 1000 2000 3000 % 0 100 2910.93 2855.06 2749.29 2650.99 2604.57 2559.91 2914.15 3032.80 3092.81 3092.88 3096.43 3160.43 m/z 1000 2000 3000 % 0 100 m/z 1000 2000 3000 % 0 100 1218.99 1139.61 1090.18 1044.83 1350.67 1392.66 1566.77 1665.51 1009.40 969.12 931.91 1101.20 1211.14 1274.71 1424.58 1862.62 2201.06 heavy chain oxidized mAb
Transcript of ELECTROCHEMICAL-ASSISTANCE FOR ELECTRON TRANSFER … · 2013. 10. 3. · ETD reagent. Approximately...
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INTRODUCTION
Characterization of intact proteins using tandem mass spectrometry approaches can be challenged by the presence of disulfide bonds that maintain and stabilize three-dimensional structures. Moreover, the presence of multiple disulfide bonds significantly reduces fragmentation efficiency. Electrochemistry-
assisted intact protein tandem MS was recently reported for the characterization of multiple disulfide bond containing proteins using DESI-ECD1. Following electrochemical reduction and on-line ECD-MS2, significant improvements in backbone cleavages were reported compared to the ECD of the oxidized forms of the proteins. This quick reduction of disulfide bonds is an appropriate alternative for disulfide bond reduction using dithiothreitol or other chemical reductants. ESI-ETD-ion mobility-MS was used to investigate the fragmentation efficiency of a number of disulfide bond containing peptides and proteins in their oxidized and reduced forms.
ELECTROCHEMICAL-ASSISTANCE FOR ELECTRON TRANSFER DISSOCIATION ION MOBILITY MS OF PEPTIDES AND PROTEINS
Jonathan P. Williams, Christopher J. Hughes, Robert Tonge, Jeffery M. Brown
Waters Corporation, Manchester, UK
METHODS
Mass Spectrometry
MS: Synapt G2 or G2-S (Fig. 1) Mode: ESI (+)
Capillary voltage: 2-3 kV Cone: 25-50V
Source temperature: 100 °C Desolvation temperature: 200 °C
ETD-MS
Reagent: 4-nitrotoluene (4-NT) or 1,4-dicyanobenzene (1,4-DCB)
Glow discharge current: 70-90 µA Trap wave height: 0.2-0.25 V
Trap RF: 450-500 V
Chromatography
UPLC: Acquity I-Class
Analytical column: BEH300 C4, 1.7µm, 2.1 x 100mm Mobile phase A: H2O + 0.1% formic acid
Mobile phase B: CH3CN + 0.1% formic acid Flow rate: 50µL/min
Gradient: 10%B to 35%B in 12minutes
Electrochemistry
Electrochemical (EC) cell: Roxy (Antec) Reducing solvent: 50% aq. CH3CN / 1% formic acid
Analyte concentration: ~1 μM
Mode: pulse Sample introduction: infusion @ 50 µL/min
RESULTS
Somatostatin Infusion EC-Ion Mobility MS
CONCLUSION
Initial studies demonstrate the potential of an
Electrochemical-cell for on-line disulfide bond
reduction for aiding top-down ETD MS studies.
Significant differences were observed between the
chemically and electrochemically-reduced forms of the large mAb protein.
Further work and method optimization of the pulse
profiles for large mAb proteins is currently on-going.
References
1. Stokes et al. Top-down protein sequencing by CID and ECD using desorption electrospray ionisation (DESI) and high-field FTICR mass spectrometry. Int J Mass Spectrom289.1 (2010) 54–57
2. Zhang et al. Electrochemistry-Assisted Top-Down Characterization of Disulfide-Containing Proteins. Anal Chem 84.8 (2012): 3838-3842
Figure 1. Synapt G2-S with ETD functionality
Bovine Insulin Infusion EC-MS Chicken Lysozyme Infusion EC-MS
Figure 3. ETD mass spectra obtained from the ion-ion reaction of
the mass-selected [M+3H]3+ for the (A) oxidized and (B) reduced forms of Somatostatin with the mass-selected anions produced from
glow discharge ionization of 4-NT. In (A) low abundance c-/z- type ions were observed by magnification. In (B) abundant c-/z- type ions were directly observed for c4-c13 and z6-z11 and z12.
Figure 2. Partial mass spectrum highlighting [M+3H]3+ for (A)
oxidized and (B) reduced forms of Somatostatin (Mw 1637, AGCKNFFWKTFTSC) contains a disulfide bond between Cys3-Cys14.
Shown inset are the arrival time distributions (ion mobility) suggesting a more compact and dominant conformation in the
Figure 4. ESI mass spectrum of Bovine Insulin in (A) oxidized and
(B) reduced form. Low abundance multiply charged ions were observed relative to the abundant [M+4H]4+- [M+5H]5+ for the
reduced form of the B-chain. Low abundance multiply charged ions [M+2H]2+- [M+4H]4+ were detected for the reduced form of the A-chain. Bovine Insulin (MwAVG 5734 Da) has 2 chains containing 2
disulfide bonds and 1 intra-disulfide bond within the A-chain.
Figure 7. ESI mass spectrum obtained from Lysozyme in (A)
oxidized and (B) reduced form. Multiply charged ions [M+8H]8+- [M+12H]12+ were observed for the oxidized and multiply charged
ions [M+8H]8+- [M+18H]18+ were detected for the reduced form. Lysozyme (MwAVG 14.3 kDa) contains 4 disulfide bonds, Cys6-Cys127, Cys30-Cys115, Cys64-Cys80, and Cys76-Cys94.
Figure 8. ETD mass spectrum obtained for the oxidized precursor
ion [M+10H]10+. Limited sequence information was obtained with only a small number of c-/z-type product ions due to the presence of
the disulfide bonds.
Figure 9. ETD mass spectrum obtained for the reduced precursor
ion [M+15H]15+. Extensive sequence coverage is obtained as shown in the mass spectrum. Abundant c– and z-type product ions were
observed providing over 50% sequence coverage.
Mouse mAb standard LC-EC-MS
Synapt G2-S was used for mAb analysis using 1,4-DCB as the
ETD reagent. Approximately 30-100µM was injected on-column for EC-ETD analysis depending on the mode selected. Figure
10 shows the individual mAb MS spectra obtained, with com-plementary detail provided in Figures 11 and 12, respectively.
Figure 11. LC-ETD mass spectrum obtained for oxidized precursor
ion [M+57H]57+, m/z 2604. Extensive sequence coverage was obtained for both chains. Shown inset is sequence ions from the
mAb heavy chain.
Figure 12. LC-EC-ETD mass spectrum of reduced precursor ion
[M+27H]27+, m/z 897.
m/z544 545 546 547 548 549 550 551
%
0
100
m/z544 545 546 547 548 549 550 551
%
0
100546.6
546.9
547.2
547.6
547.9
547.3
546.9546.6
547.6
547.9
548.3
548.6
(A)
(B)
EC cell OFF
EC cell ON
~ 80% -S—S-
bond reduction efficiency
Time2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
%
0
100
2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
%
0
1004.43
4.64
Time2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
%
0
100
2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00
%
0
1004.43
4.64
drift (bins)
drift (bins)
m/z300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700
%
0
100
m/z300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700
%
0
100x16x10
790.4546.6
724.4546.9
547.3
724.9
1622.8820.4
835.9 1621.8
836.4
1580.8
1448.7836.9925.5 1579.7
1623.8
1639.8
1640.8
1640.8
1639.8
820.9
820.4545.4
c''5491.3c''4
377.2
z'6670.3
547.3 c''6638.3547.6
547.9
c''7785.4686.3
687.3
1623.8
821.4c''8
971.5
z'7;856.4
955.5
1622.8z'9
1150.6z'8
1003.51004.5
z'101264.6
1151.61408.7
1265.61591.8
1641.8
1642.8
1643.8
EC ETD MS
EC (cell OFF) ETD MS
2+
charge-reduced
1+
charge-reduced
1+
charge-reduced
2+
charge-reduced
(A)
(B)
precursor ion
[M+3H]3+
precursor ion
[M+3H]3+
Figure 5. ETD mass spectrum of the oxidized precursor ion [M+6H]6+
showing c-/z- type fragmentation up to the disulfide bonds.
m/z500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500
%
0
100x10
2868.7
2868.2
857.4
710.3
400.2
202.1
563.3
2867.7
858.4
2860.7
2859.6873.4
1071.5
2859.2
1072.5 2846.7
1912.81200.5 2440.0
2869.7
2870.2
2870.7
2871.2
5740.0
2871.75738.0
4881.0
4879.02872.2
4666.7
5028.3 5723.05338.9
5742.0
5743.0
5744.0
5745.0
EC (cell OFF) ETD MS
singly charged
A:B(C20) - A:B(C28)
1+
charge-reduced
3+
charge-reduced
2+
charge-reduced
Figure 6. ETD mass spectrum of the reduced precursor ion [M+5H]5+
from the B-chain showing extensive sequence coverage.
m/z200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200
%
0
1001701.4
741.4
z'6710.4
z'4400.3
c''3378.2
c''2264.2
c''5643.4
z'5563.3
c''4506.3
1700.9
z'7857.5
742.4
743.4
1700.4
873.5
1141.6
1096.6
874.5
c''91003.5
1692.9
1692.4
1142.6
1353.8 1553.9
1482.8
1701.9
3384.9
3383.8
1702.4
1702.9
3382.82261.3
1703.4z'20
2260.31849.0 2049.1
2148.2
2661.42262.3
2660.42278.3
2279.3
3373.9
3356.82663.4
3154.72758.5
EC ETD MS
1+
charge-reduced
2+
charge-reduced
c-ions observed c1-c16, c19, c21-c26 and c29
z-ions observed z4-z11 and z14-z25
m/z200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800
%
0
100
m/z200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800
%
0
1001590.4
1590.3
1590.2
1590.11431.6
1431.4
1431.3
1431.21301.5
1301.2
1192.9
1301.8
1431.0
1431.7
1431.8
1431.9
1432.0
1432.1
1432.3
1590.7
1590.8
1590.91789.2
1788.9
1788.8
1591.2
1591.3
1789.4
1789.6
1789.7
1793.9
1794.2
1193.3
1193.2
1193.1
1101.6
1101.4
1023.0
1022.9
954.8
895.3
1193.4
1301.7
1301.9
1431.7
1302.0
1431.3
1305.2
1431.9
1432.0
1590.7
1590.4
1435.6
1590.9
1594.9
EC cell OFF
EC cell ON (B)
(A)
[M+10H]10+
[M+9H]9+
[M+8H]8+
[M+11H]11+
[M+12H]12+
[M+8H]8+
[M+12H]12+
[M+18H]18+
m/z200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800
%
0
100x10 1431.5
1431.4
1431.3
1431.2
1431.1
1431.0
1430.9
1430.8392.3245.2
1431.6
1431.7
1431.8
1431.9
1432.0
1590.5
1590.4
1432.1
1590.1
1432.2
1590.0
1589.9
1432.8
1590.6
1590.8
1789.41789.1
1789.0
1591.1
1789.5
1789.6
1789.82044.9
1789.92045.1
EC (cell OFF) ETD MS
m/z200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400
%
0
100
c''3392.28
121.06
c''2245.22
230.20
z'2z'9 4+272.20
433.29c''11 2+582.35
c''13 2+711.92
582.84
c''12 2+647.87
712.42
c''14 2+789.97
1001.58
c''18 2+1001.06
z'18 2+1058.60
1059.09
1140.63
c''47 4+1328.73
1141.13 1428.79
1429.28 1821.99
m/z1000 1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 1110 1120
%
0
1001001.58
c''18 2+1001.06
993.56
994.06
994.56
z'18 2+1058.60
1002.07
c''19 2+1058.10
1002.57
c''91021.60
1003.07
c''36 4+z'36 4+1013.30
1003.57
1013.79
1022.60
1050.58
1049.89
1049.561023.61
z'27 3+1049.22
1041.56
1051.08
1059.09
1059.59 c''101092.64
1060.09
1060.59
1091.621076.62
1093.64
1094.64 c''39 4+1106.91
1104.94
1101.58 1107.901118.61
EC ETD MS
m/z600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
%
0
100
m/z600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
%
0
1001147.5
1147.3
956.4
956.2
956.0
1147.1
956.5
956.7
956.9
1147.7
1147.9
1148.1
1434.11148.3 1433.81434.6
1434.8
681.0
680.8
680.6
687.2
850.7687.4
850.4
858.7
858.9
(A)
(B) EC cell ON
EC cell OFF
[M+3H]3+
[M+4H]4+
[M+5H]5+
[M+6H]6+
B:[M+4H]4+
B:[M+5H]5+
A:
[M+3H]3+
A:
[M+4H]4+
A:
[M+2H]2+
Insulin B-Chain
Average Mass = 5733.5763, Monoisotopic Mass = 5729.6008
N-Terminus = H, C-Terminus = OH
1 FVNQH LCGSH LVEAL YLVCG ERGFF YTPKA
1 GIVEQ CCASV CSLYQ LENYC NC C
C C
C C
B
A
Figure 10. LC-MS and LC-EC-MS spectra illustrating analysis of the
light and heavy chains (middle and bottom) and the intact form (top) of mouse mAb standard.
m/z500 1000 1500 2000
%
0
100898.9
z'24 3+896.3
895.9
z'15 2+;834.0
z'8 2+491.3
899.8
900.8
1345.2901.3
1343.8
975.1 1325.2
1347.3 z'31 2+1817.01350.8
1796.0 1818.0
1828.3
EC (cell ON) ETD MS
m/z500 1000 1500 2000
%
0
1001271.3
1271.0
z'34 3+;1252.9
c''33 3+1108.1z'4
381.2
121.1
1080.1
c''30 3+1015.7
1452.6
1452.7
1453.0
1547.1
1694.7
1702.2c''75 4+2004.0
m/z1950 1975 2000 2025 2050 2075 2100 2125
%
0
100
c''75 4+2004.0
1964.4
c''75 4+2003.81964.6
2003.6
1964.8
1985.6
2004.4
2004.6
2033.4
2007.4 2033.8
z'72 4+2055.6
2034.02081.6
2081.3
2056.6 2083.9 2109.3
2084.3
EC (cell OFF) ETD MS
m/z1000 2000 3000
%
0
1001210.82
1153.35
1100.87
1100.78
1053.03
968.96
931.80
1210.96
1274.57
1274.66
1274.72
1345.35
1345.40
1424.30
1614.14
1729.54
1862.522699.70
light chain
light chain
oxidized
mAb
10 mM DTT reduction
EC cell OFF
Electrochemical reduction
EC cell ON
Intact mAb
EC cell OFF
m/z1000 2000 3000
%
0
1002910.93
2855.06
2749.29
2650.99
2604.57
2559.91
2914.15
3032.80
3092.81
3092.88
3096.43
3160.43
m/z1000 2000 3000
%
0
100
m/z1000 2000 3000
%
0
1001218.99
1139.61
1090.18
1044.83
1350.67
1392.66
1566.77
1665.51
1009.40969.12
931.91
1101.20
1211.14
1274.71
1424.58
1862.62
2201.06
heavy chain
oxidized
mAb
