Don t miss a thing on your peptide mapping journey How to get full coverage peptide maps using high resolution accurate mass spectrometry

Don t miss a thing on your peptide mapping journey How to get full coverage peptide maps using high resolution accurate mass spectrometry Kai Scheffler, PhD BioPharma Support Expert,LSMS Europe The world leader in serving science

Outline What is the exact goal of the analysis? sequence elucidation/verification sequence variant analysis, amino acid substitution analysis of modifications: glycosylation, deamidation, sulfation, oxidation disulfide bridge analysis distinguishing Leu/Ile, Asp/isoAsp Mass spectrometry portfolio: which instrument is applicable? Method setup: what is the most suitable method? MS methods: what are the critical parameters? how do they influence the success of the analysis? 2

MS Portfolio for BioPharma Characterization Thermo Scientific Ion Trap LC-MS systems Full MS with dd MS 2 (CID, HCD, ETD) MSn PERFORMANCE Thermo Scientific Exactive Plus Orbitrap Mass Spectrometer Full MS only (HCD fragmentation, no precursor selection) Thermo Scientific Q Exactive hybrid quadrupole- Orbitrap mass spectrometers Full MS only Full MS with dd MS 2 (HCD) Thermo Scientific LTQ Orbitrap XL Hybrid Ion Trap- Orbitrap Mass Spectrometer Full MS only (OT detection) Full MS with dd MS 2 (CID, HCD, ETD) MSn Thermo Scientific Orbitrap Fusion Tribrid Mass Spectrometers Full MS only (OT detection) Full MS with dd MS 2 (CID, HCD, ETD) MSn VALUE 3

MS Portfolio for BioPharma Characterization Analysis type Thermo Scientific Ion Trap LC-MS systems Full MS with dd MS 2 (CID, HCD, ETD) MSn PERFORMANCE Thermo Scientific Exactive Plus Orbitrap Mass Spectrometer Full MS only (HCD fragmentation, no precursor selection) Thermo Scientific Q Exactive hybrid quadrupole- Orbitrap mass spectrometers Full MS only Full MS with dd MS 2 (HCD) Thermo Scientific LTQ Orbitrap XL Hybrid Ion Trap- Orbitrap Mass Spectrometer Full MS only (OT detection) Full MS with dd MS 2 (CID, HCD, ETD) MSn Thermo Scientific Orbitrap Fusion Tribrid Mass Spectrometers Full MS only (OT detection) Full MS with dd MS 2 (CID, HCD, ETD) MSn Peptde mapping/sequence confirmation Sequence variant analysis Analysis of PTMs and other modifications Disulfide bond analysis low res, low mass accuracy Full MS only Confirmation of expected ones only Confirmation of expected ones only Confirmation of expected ones only VALUE 4

MS Portfolio for BioPharma Characterization Analysis type Thermo Scientific Ion Trap LC-MS systems Full MS with dd MS 2 (CID, HCD, ETD) MSn PERFORMANCE Thermo Scientific Exactive Plus Orbitrap Mass Spectrometer Full MS only (HCD fragmentation, no precursor selection) Thermo Scientific Q Exactive hybrid quadrupole- Orbitrap mass spectrometers Full MS only Full MS with dd MS 2 (HCD) Thermo Scientific LTQ Orbitrap XL Hybrid Ion Trap- Orbitrap Mass Spectrometer Full MS only (OT detection) Full MS with dd MS 2 (CID, HCD, ETD) MSn Thermo Scientific Orbitrap Fusion Tribrid Mass Spectrometers Full MS only (OT detection) Full MS with dd MS 2 (CID, HCD, ETD) MSn Peptde mapping/sequence confirmation Sequence variant analysis Analysis of PTMs and other modifications Disulfide bond analysis low res, low mass accuracy ETD enabled systems only Full MS only Confirmation of expected ones only Confirmation of expected ones only Confirmation of expected ones only VALUE ETD enabled systems only 5

Before you setup, refine and start the MS method, you need to ensure you have an optimized LC-MS setup: Method Setup Calibration Source settings: probe position in the source housing (probe depth, x/y position) source voltage gas flows (sheath/aux gas) optimized for LC flow rate S-lens 6

Peptide Mapping Method Setup Method 1: Full MS scans only Method 2: Full MS scans with data dependent MS 2 scans 7

Peptide Mapping Method Setup Method 1: Full MS scans only Method 2: Full MS scans with data dependent MS 2 scans Sequence confirmation based on intact accurate precursor masses Confirmation of known and expected modifications Confirmation of disulfide bridges Critical parameters: Mass range AGC target setting Fill time 8

Peptide Mapping Method Setup Method 1: Full MS scans only Sequence confirmation based on intact accurate precursor masses Confirmation of known and expected modifications Confirmation of disulfide bridges Critical parameters: Mass range AGC target setting Fill time Method 2: Full MS scans with data dependent MS 2 scans Sequence confirmation based on intact accurate precursor masses and MS 2 spectra Confirmation of known and expected modifications and identification of unknowns Confirmation of disulfide bridges and identification of unknowns Critical parameters: Mass range Charge state range AGC target setting MS + MS 2 Maximum injection time MS/MS 2 Fixed first mass (MS 2 ) Intensity threshold TopN Collision energy (NCE) 9

Peptide Mapping Method Setup Method 1: Full MS scans only Sequence confirmation based on intact accurate precursor masses Confirmation of known and expected modifications Confirmation of disulfide bridges Critical parameters: Mass range AGC target setting Fill time For Exactive MS series the MS method setup and data acquisition can be done via Thermo Scientific Xcalibur Software and/or Thermo Scientific Chromeleon Chromatography Data System Software Method 2: Full MS scans with data dependent MS 2 scans Sequence confirmation based on intact accurate precursor masses and MS 2 spectra Confirmation of known and expected modifications and identification of unknowns Confirmation of disulfide bridges and identification of unknowns Critical parameters: Mass range Charge state range AGC target setting MS + MS 2 Maximum injection time MS/MS 2 Fixed first mass (MS 2 ) Intensity threshold TopN Collision energy (NCE) 1

Critical Parameter 1: Mass Range Parameter relates to the mass range for monitoring intact precursor ions (m/z) Suggested to be set to - m/z for peptide mapping Suggested LC-MS setup is running without a trap column and starting the MS method without a delay time 11

Critical Parameters 2 & 3: AGC Target Setting MS + MS 2 / Maximum Inject Time AGC target values depend on the particular instrument used: AGC target Exactive Q Exactive Hybrid OT OT Fusion/Lumos Full MS 1-3e6 3e6 3e6 2e5/4e5 MS 2 OT --- 1e5 1e5 5e4/1e5 MS 2 IT --- --- 1e4 1e4/1e4 Optimal max. inject times need to be in accordance with: TopN set in the method Gradient length Sample amount injected Max. Inject time Exactive Q Exactive Hybrid OT OT Fusion/Lumos Full MS 5- ms 5- ms 5- ms 5- ms MS 2 OT --- 15- ms 15- ms 15- ms MS 2 IT --- --- - ms - ms 14

Critical Parameter 4: TopN with N=? RT: 5.81 -.54 9 7 5 3 1 RT: 1.88-11.15 9 7 5 3 1 11.7 13.55 15.22 12.16 8.9 9.61 13.45 14.45 11.19 13.36 17.94 9.75 7.54 11.1 8.94 14.33 13.31 15.34 18.77 17.22 14.7 16.83 19.86 9.41 6.13 16.64 12.45 19.35 12.53 15.81 18.63 6.22 15.96 6. 8.22 1.4 1.77 17.38.6 6 7 8 9 1 11 12 13 14 15 16 17 18 19 Time (min) ddtop5 Method Full MS + 5 MS 2 scans per scan cycle Resolution settings : 7k/17,5 k Max. inject time ms 14.79 NL: 4.31E8 Base Peak F: ms MS 11.1 NL: 5.86E8 1 9 1.9 1.92 1.94 1.96 1.98 11. 11.2 11.4 11.6 11.8 11.1 11.12 11.14 Time (min).151 min = 9.1 sec 17

Critical Parameter 4: TopN with N=? RT: 5.81 -.54 9 7 5 3 1 RT: 1.88-11.15 9 7 5 3 1 11.7 13.55 15.22 12.16 8.9 9.61 13.45 14.45 11.19 13.36 17.94 9.75 7.54 11.1 8.94 14.33 13.31 15.34 18.77 17.22 14.7 16.83 19.86 9.41 6.13 16.64 12.45 19.35 12.53 15.81 18.63 6.22 15.96 6. 8.22 1.4 1.77 17.38.6 6 7 8 9 1 11 12 13 14 15 16 17 18 19 Time (min) ddtop5 Method Full MS + 5 MS 2 scans per scan cycle Resolution settings : 7k/17,5 k Max. inject time ms 14.79 NL: 4.31E8 Base Peak F: ms MS 11.1 NL: 5.86E8 9 Full MS scans 8x5= MS 2 scans 49 scans total in 9.1 sec with an average MS 2 injection time of 159 ms 1 9 NL: 7.88E6 1.9 1.92 1.94 1.96 1.98 11. 11.2 11.4 11.6 11.8 11.1 11.12 11.14 Time (min).151 min = 9.1 sec 18

Critical Parameter 4: TopN with N=? RT: 5.81 -.54 # 3655-375 RT: 1.95-11.9 AV: 9 NL: 7.1E7 11.7 T: FTMS + p ESI Full lock ms [.-.] 14.79 9 x1 x5 x5 639.2792 13.55 15.22 z=2 12.16 7 8.9 9.61 13.45 14.45 11.19 13.36 17.94 658.257 9.75 5 7.54 11.1 8.94 z=2 14.33 13.31 15.34 Relative Abundance NL: 4.31E8 Base Peak F: ms MS 279.159 3 18.77 17.22 14.7 16.83 19.86 9.41 1277.5472 6.13 574.2987 16.64 12.45 19.35 12.53 18.63 1 6.22 394.7293 439.1697 123.4753 15.81 15.96 6. z=2 z=3 1.4 691.291 865.185 8.22 58.2773 1.77 17.38 z=3 181.9952 1248.9232 1299.5267 1467.36 1579.85 16.31 1835.6348.6 1985.4446 z=2 z=? z=? z=? z=? z=? z=? 6 7 8 9 1 11 12 13 14 15 16 17 18 19 3 5 7 9 Time (min) 1 1 13 1 15 1 17 1 19 RT: 1.88-11.15 m/z 11.1 NL: 5.86E8 NL: 7.88E6 9 7 5 3 1 ddtop5 Method Full MS + 5 MS 2 scans per scan cycle Resolution settings : 7k/17,5 k Max. inject time ms 9 Full MS scans 8x5= MS 2 scans 49 scans total in 9.1 sec with an average MS 2 injection time of 159 ms 1 9 1.9 1.92 1.94 1.96 1.98 11. 11.2 11.4 11.6 11.8 11.1 11.12 11.14 Time (min).151 min = 9.1 sec 19

Estimation of Top N For the decisions on what N exactly shoud needs to be considered the average chromatographic peak width at the base and calculate the time available for each scan cycle to obtain at least ~6 Full MS scans over the peak. How many MSMS-scans can fit in every scan cycle to stay within the calculated max. cycle time will depend on the maximum injection time set. E.g. if the peak width is 7 secs aiming at 6 full MS scans, the resulting the cycle time should not exceed 1.15 sec, and can fit one full MS scan + 5 MSMS scans with ms max. inject time. 2 3 4 5 A ddtop5 method has been proven as a very good standard method for peptide mapping on all Q Exative MS instruments and only requires modification if extended max. injection times are required. 1 1.1 sec 7sec 6

Critical Parameter 5: Intensity Threshold TIC 4.84 4.86 4.88 4.9 4.92 Time (min) 9 7 5 3 1 214.898 276.27 391.2846 468.1968 z=3 672.2928 z=4 682.8185 z=2 Full MS spectrum 71.791 z=2 883.47 z=3 1118.4283 1364.636 1 1 m/z 21

Critical Parameter 5: Intensity Threshold TIC 4.84 4.86 4.88 4.9 4.92 Time (min) 682.8185 z=2 682 684 m/z 9 7 5 3 1 214.898 276.27 391.2846 468.1968 z=3 672.2928 z=4 682.8185 z=2 Full MS spectrum 71.791 z=2 883.47 z=3 1118.4283 1364.636 1 1 m/z 22

Critical Parameter 5: Intensity Threshold TIC 4.84 4.86 4.88 4.9 4.92 Time (min) 682.8185 z=2 682 684 m/z intensity threshold charge state peptide match: on/off/preferred dynamic exclusion 9 7 5 3 1 214.898 276.27 391.2846 468.1968 z=3 672.2928 z=4 682.8185 z=2 Full MS spectrum 71.791 z=2 883.47 z=3 1118.4283 1364.636 1 1 m/z 929.858 z=3 929.7545 z=3 933.9597 z=4 934.2145 z=4 928 93 932 934 m/z 24

Critical Parameter 6: Fixed First Mass C-trap HCD cell Quadrupole 25

Critical Parameter 6: Fixed First Mass C-trap HCD cell Quadrupole Scan range: last mass 15 first mass Leaving the first mass open will automatically adjust the mass range based on m/z of the precursor ensuring that also high mass fragments ions are captured. Setting a fixed first mass is suggested only when certain low mass ions are desired for monitoring, e.g. 4 (oxonium ions) or for denovo sequencing aiming e.g. at the Lys/Arg y 1 ions at m/z 147/175. 26

Critical Parameter 4: Fixed First Mass - Set to 1 m/z 159.9 FTMS + p ESI d Full ms2 333.19@hcd27. [1.-3.] 288.17 y 1 17.6 147.11 289.17 333.19 316.17 27.16 158.6 185.9 298.16 1.1 171.6 188.7 315.18 3.14 317.17 334. 157.1 z=? 239.12 243.15 253.13 z=? 1 15 1 17 1 19 21 2 23 2 25 2 27 2 29 3 31 3 33 3 35 3 m/z y 1 169.1 254.19 278.11 296.12 297.13 339.17 367.16 43.23 412.22 368.16 452.25 517.26 518.27 588.3 657.32 675.33 676.33 FTMS + p ESI d Full ms2 634.33@hcd27. [1.-1315.] 15 25 3 35 45 5 55 65 7 75 85 9 95 15 1 115 1 125 13 m/z y 1 175.12 276.1 6.19 439.16 569.25 57.25 78.27 732.31 1421. 733.32 744.29 831.38 989.45 99.46 991.46 114.48 1233.52 13.55 1322.56 77.41 788.42 789.42 79.42 883.49 91.5 972.54 z=? FTMS + p ESI d Full ms2 1347.59@hcd27. [1.-277.] 1 1 1 1 2 m/z 1422. 1424. z=? 1534.69 1621.72 178.75 171.75 1711.74 1821.84 1824.82 z=? 1949.9 33.91 2195.98 z=? 22.96 27

Critical Parameter 4: Fixed First Mass Set to Auto Adjust 318.15 FTMS + p ESI d Full ms2 563.32@hcd28. [5.-595.] 86.1 84.8 563.32 87.1 5.6 246.18 147.11 284.16 129.1 213.16 256.11 72.8 359.26 185.16 273.13 29.15 319.15 389.22 417.22 435.1 462.28 564.32 66.98 11.11 177.7 228.17 373.19 51.29 519.3 5 15 25 3 35 45 5 55 m/z 328.15 FTMS + p ESI d Full ms2 814.88@hcd28. [112.-16.] 199.11 487.18 12.53 227.1.15 171.11 326.17 427.22 71.27.34 399.22 899.41 111.49 13.54 31.14 586.24 667.3 766.38 998.49 131.61 267.11 354.14 536.74 613.27 73.35 169.51 112.49 132. 143.63 469.17 815.46 9.41 168.51 1184.52 14.54 149.42 1431.65 4.9 3 5 7 9 1 1 13 1 15 1 m/z FTMS + p ESI d Full ms2 1399,5@hcd28. [191.67-2875.] 366.14 1392.59 1538.65 2431.96 1393.59 1539.65 1272.55 2268.91 2432.96 338.18 1919.79 65.83 367.14 1273.55 1595.67 1741.74 5.9 1189.51 1394. 2122.85 528.19 1921.76 2286.9 1759.73 2594.2 664.25 731.28 797.36 1134.5 1723.74 1 1 1 1 2 m/z 28

Reviewing ox ox Identified Modifications ox ox 3

Peptide Mapping with Short and Long Separation Times 2.7 6.58 Thermo Scientific Vanquish UHPLC Platform coupled to a Thermo Scientific Q Exactive HF MS 8.42 1.78 14.64 2.73 1.59 13.22 8.63 19.56 14.52 min gradient.4 ml min -1 bar 3 min gradient.4 ml min -1 bar Heavy Chain Light Chain % Sequence coverage obtained for Rituximab Light and Heavy Chain 1.86 6.97 9.43 5.47 5. 7.44 9.82 13 min gradient.6 ml min -1 85 bar 1.12 3.58 5.55 5.83 8 min gradient 1. ml min -1 115 bar 1.7 3.82 4.2 5 min gradient 1.1 ml min -1 13 bar 5 1 15 25 Time [min] 31

Peptide Mapping with Short and Long Separation Times Thermo Scientific Vanquish UHPLC Platform coupled to a Thermo Scientific Q Exactive HF MS Heavy Chain Light Chain 1.12 1.7 1.86 2.7 3.58 3.82 6.58 8.42 1.78 6.97 9.43 5.47 5. 7.44 9.82 4.2 5.55 5.83 14.64 2.73 1.59 13.22 8.63 19.56 5 min gradient 1.1 ml min -1 13 bar 8 min gradient 1. ml min -1 115 bar 14.52 13 min gradient.6 ml min -1 85 bar min gradient.4 ml min -1 bar 3 min gradient.4 ml min -1 bar 5 1 15 25 Time [min] % Sequence coverage obtained for Rituximab Light and Heavy Chain Gradient Time 3 min min 13 min 8 min 5 min Modification Abundance σ Light Chain Q1+NH3 loss 91.95% 91.17% 89.69% 9.93% 26.57% 28.% Heavy Chain ~Q1+NH3 loss 99.62% 99.67% 99.61% 99.68% 99.69%.4% Heavy Chain N33+Deamidation.52%.51%.58% -.51%.3% Heavy Chain M34+Oxidation 1.64% 1.54% 1.73% 1.42% 1.45%.13% Heavy Chain N31+A1GF 4.32% 4.42% 3.83% 3.52% 3.38%.46% Heavy Chain N31+A1G1F 1.87% 1.91% 1.72% 3.32% 1.46%.73% Heavy Chain N31+A2G 1.9% 1.2% 1.2% -.98%.5% Heavy Chain N31+A2GF 37.88% 37.11% 38.59%.48% 43.12% 2.41% Heavy Chain N31+A2G1F 42.6% 41.89% 43.42% 43.% 43.35%.75% Heavy Chain N31+A2G2F 1.23% 1.17% 9.81% 1.36% 1.5%.21% Heavy Chain N31+A2S1GF.83%.86% - - -.2% Heavy Chain N31+A2S1G1F 2.14% - - - - - Heavy Chain N31+A3Sg1G 1.3% - - - - - Heavy Chain N31+M5 1.61% 1.59% 1.66% 1.87% 1.86%.14% Heavy Chain N31+Unglycos..54%.9%.76%.83%.97%.16% Heavy Chain G45+Lys 3.57% 3.56% 3.92% 3.% 3.15%.28% median.19% 32

Identified Deamidation of Peptide VVSVLTVHQDWLN*GK 5 5 94.587 95.99 R=323 R=336 z=2 z=2 95.511 1.99 ppm R=322 z=2 1.32 ppm 95.1 R=3297 z=2 measured 95.514 R=332 z=2 96.5143 R=2792 z=2 96.4576 R=33 z=2 94 95 96 97 m/z NL: 2.51E6 RT 18.85 min NL: 1.87E6 RT 18.99 min 96.9589 R=3 z=2 Intact peptide precursor 94.569 95.83 95.597 94.9989 95.54 96.11 96.5124 97.136 97.5149 96.17 simulated 96.531 97.44 97.557 95 96 97 m/z NL: 8.26E3 c 83 h 134 n 22 o 23 +H : C 83 H 136 N 22 O 23 p (gss, s /p : ) Chrg 2 R : 3 Res.Pwr. @FWHM NL: 8.26E3 unmodified peptide measured spectrum simulated MS 2 spectrum unmodified peptide c 83 h 133 n 21 o 24 +H : C 83 H 135 N 21 O 24 p (gss, s /p : ) Chrg 2 R : 3 Res.Pwr. @FWHM deamidated peptide 33

Identified Deamidation of Peptide VVSVLTVHQDWLN*GK simulated MS 2 spectrum.6 ppm deamidated peptide measured spectrum.97 ppm 1.5 ppm.14 ppm 34

Identified Deamidation of Peptide VVSVLTVHQDWLN*GK.6 ppm.97 ppm 1.5 ppm deamidated peptide measured spectrum.14 ppm 35

Disulfide Bridge Analysis 36

Sample Analysis by Mass Spectrometry Q Exactive Plus Analysis of reduced (+alkylated) sample Analysis of unreduced sample Method used for both samples: data-dependent Top5 Method: Full MS + top5 MS/MS scans per cycle 9 7 5 3 1 9 7 5 3 1 reduced sample 5 1 15 25 3 35 Time (min) unreduced sample 5 1 15 25 3 35 Time (min) 37

Sample Analysis by Mass Spectrometry Q Exactive Plus Analysis of reduced (+alkylated) sample Analysis of unreduced sample Method used for both samples: data-dependent Top5 Method: Full MS + top5 MS/MS scans per cycle 9 7 5 3 1 9 7 5 3 1 peaks deteced in reduced sample only peaks detected in unreduced sample only reduced sample 5 1 15 25 3 35 Time (min) unreduced sample 5 1 15 25 3 35 Time (min) 38

Disulfide Bridge Analysis - Result Review R e l a t i v e I n t e n s i t y 95 9 85 75 7 65 55 5 45 35 3 25 15 1 5 HC:39-43 HC:338-341 LC:62-66 HC:39-43 Reduced LC:8-211 LC:14-17 HC:-65 LC:14-17 HC:-65 HC:217-221 LC:62-66 HC:338-341 LC:8-211 HC:413-417 Non-Reduced HC:413-417 LC:15-169 HC:68-83 HC:51-59 LC:15-169 HC:81-87 LC:146-149 HC:68-83 LC:19-24 HC:68-76 HC:88-98 LC:189-7 RT (min) HC:245-251 HC:68-76 HC:33-337 HC:33-337 LC:1-18 HC:252-258 LC:25-42 HC:31-38 HC:443-449 HC:44-5 HC:77-87 LC:62-86 HC:364-373 HC:278-291 LC:5-61 HC:259-277 LC:191-7 HC:44-5 HC:278-291 LC:25-42 HC:-3 HC:31-38 HC:51-84 HC:443-449 HC:125-136 HC:125-136 HC:278-288 HC:16-124 LC:17-183 HC:278-288 HC:374-395 LC:46-49 HC:374-395 LC:46-49 LC:18-126 HC:66-76 HC:16-124 HC:1-19 HC:396-412 2 1 3 HC:396-412 HC:-11 LC:127-142 HC:226-251 HC:35-3 HC:99-124 LC:46-53 Cysteine containing peptides HC:396-41 HC:151-213 HC:151-183 Expected/confirmed disulfide bridge linked peptides HC:137-15/HC:151-213 HC:151-183 39

Disulfide Bridge Analysis - Result Review R e l a t i v e I n t e n s i t y 95 9 85 75 7 65 55 5 45 35 3 25 15 1 5 HC:39-43 HC:338-341 LC:62-66 HC:39-43 Reduced LC:8-211 LC:14-17 HC:-65 LC:14-17 HC:-65 HC:217-221 LC:62-66 HC:338-341 LC:8-211 HC:413-417 Non-Reduced HC:413-417 LC:15-169 HC:68-83 HC:51-59 LC:15-169 HC:81-87 LC:146-149 HC:68-83 LC:19-24 HC:68-76 HC:88-98 LC:189-7 RT (min) HC:245-251 HC:68-76 HC:33-337 HC:33-337 LC:1-18 HC:252-258 LC:25-42 HC:31-38 HC:443-449 HC:44-5 HC:77-87 HC:364-373 HC:278-291 HC:125-136 LC:17-183 HC:396-412 LC-HC HC4 HC1 HC2 LC2 LC1 Hinge HC3 LC:62-86 LC:5-61 HC:259-277 LC:191-7 HC:44-5 HC:278-291 LC:25-42 HC:-3 HC:31-38 HC:51-84 HC:443-449 HC:125-136 HC:278-288 HC:16-124 HC:278-288 HC:374-395 LC:46-49 HC:374-395 LC:46-49 LC:18-126 HC:66-76 HC:16-124 HC:1-19 2 1 3 HC:396-412 HC:-11 LC:127-142 HC:226-251 HC:35-3 HC:99-124 LC:46-53 Cysteine containing peptides HC:396-41 HC:151-213 HC:151-183 Expected/confirmed disulfide bridge linked peptides HC:137-15/HC:151-213 HC:151-183

Details for Expected Light Chain S-S-Bond: C134-C194 Light chain amino acid sequence XICs Non-Reduced Precursor isotope pattern Fragment ion coverage maps Reduced No correlating peak present 41

Details for Unexpected Heavy Chain S-S-Bond: C22-C264 Heavy chain amino acid sequence location of scrambled disulfide bridge in the trastuzumab heavy chain sequence XICs Non-Reduced Precursor isotope pattern Fragment ion coverage maps MS pattern = 1.84 ppm 5 638.76 z=5 Reduced No correlating peak present 639 6 m/z 23. 23.5 24. RT (min) 42

Benefits of ETD: Distinguising Leu/Ile Based on Diagnostic ETD-HCD MS 3 ions 43

Benefits of ETD: Distinguising Leu/Ile Based on Diagnostic ETD-HCD MS 3 ions HCD fragmentation ->MS 3 spectrum ETD MS 2 spectrum Leucine Isoleucine 44

What we Learned 45

What we Learned 46

What we Learned 47