Supporting Information to: Disulfide Linkage Characterization of Disulfide ond-containing roteins and eptides y Reducing Electrochemistry and Mass Spectrometry Christian N. Cramer 1,2, Kim F. Haselmann 1, Jesper V. Olsen 2 & eter Kresten Nielsen 1 * 1. rotein Engineering, Novo Nordisk /S, Novo Nordisk ark, 2760 Måløv, Denmark 2. Department of roteomics, The Novo Nordisk Foundation Center for rotein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark * Corresponding author: eter Kresten Nielsen, h.d. rotein Engineering Novo Nordisk /S Novo Nordisk ark 2760 Måløv, Denmark Email: pkrn@novonordisk.com Tel.: (+45) 30 79 03 75 Fax.: (+45) 44 49 05 55 age S-2: Supporting Information Methods age S-3: Figure S-1 CID MS/MS of EC reduced - and -chain age S-4: Figure S-2 IMS separation of intact and partial reduced HI age S-5: Figure S-3 Tertiary structure of HI age S-6: Figure S-4 Identification of disulfide scramled species y CID MS/MS Figure S-5 CID MS/MS of HI:4NEM age S-7: Figure S-6 Chromatogram of partially EC reduced and NEM alkylated lysozyme age S-8: Figure S-7 Tertiary structure and disulfide-containing tryptic fragments of HS Figure S-8 LC-MS and LC-EC-MS chromatogram of non-reduced HS digest age S-9: Figure S-9 nnotation of EC-related titanium oxide species Figure S-10 LC-EC-MS spectra of the disulfide-containing #3 fragment from HS age S-10 Figure S-11 LC-EC-MS spectra of the disulfide-containing #7 fragment from HS S-1
Supporting Information Methods: In addition to the methods descried in the Experimental section in the article, the following methods were used to generate the data in the Supporting Figures: Mass spectrometry: ll MS experiments were carried out as descried in Experimental section using a Waters Synapt HDMS G2-S instrument (Waters Company, UK). Ion moility separation (IMS) was performed with a pressure of 2.87 mar in the IMS cell, with IMS wave height of 40 V and wave velocity of 1600 m/s. Trap and transfer cell parameters were adjusted to wave heights of 2.0 V and 3.0 V and wave velocities of 311 m/s and 250 m/s, respectively to optimize IMS separation. Data analysis: ll MS data were analyzed with the MassLynx 4.1 software (Waters Company, UK). Visualization of the three-dimensional structure and location of disulfide onds of HI was done using ymol v. 1.7.2.3 (Schrodinger LCC). S-2
Supporting Figures: recursor ion :[M+3H] 2 3 4 5 6 7 8 G I V E Q C C T S 910 11 1213 14 1516 17 18 19 20 I C S L Y Q L E N Y C N y8 y7 y1 y 1 iy a 2 2 y 2 [ 13 H 2 O] y 5 YQ y 3 4 3 5 6 20 19 10 y 6 12 16 y y 8 4 13 15 y 9 7 11 8 17 18 y 9 y 10 1 2 14 3 4 5 6 7 8 9 1011 12 13 1415 1617 18 19 20 21 22 23 24 25 26 27 F V N Q H L C G S H L V E L Y L V C G E R G F F Y T 9 8 7 6 54 3 1 0y19 y18 y17 y16y15 y14y13 y12 y11 y7 29 K T y 12 y 13 15 iy [y 3 -y 1 ] 10 11 13 ir a 1 y 5 12 y 14 16 a 2 2 y 2 y 3 y 4 y 11 18 19 y 28 3 4 5 7 8 y 15 17 y 16 9 y 18 y19 y 9 Figure S1. CID MS/MS fragmentation of the EC reduced free - (:[M+3H] / m/z = 795) and -chains (:[M+5H] 5+ / m/z = 687), with trap CE ramps of 15-35 V (-chain) and 20-40 V (-chain), are shown in () and (), respectively. roduct ions providing full sequence coverage of oth chains are oserved, as highlighted y the annotation of all identified fragments in the insets of the - and -chain sequences. Only the -chain N-terminal glycine is not oserved due to data acquisition in the 100-4000 m/z-region. The on-line EC reduction of intact HI to free - and -chains is seen in Figure 2 in the article. S-3
HI + 2H EC cell ON Intact HI IMS of: (968-971) m/z Figure S2. IMS separation of intact and EC partially reduced HI. Comined IMS spectrum of the m/z-region around the [M+6H] 6+ charge state of HI (m/z = 968-971) with the EC cell on (- 1.5V, 1.0V, 1990ms, 1010ms) in direct infusion EC-IMS-MS of 10 µm HI, 1% F, 10% MeCN. IMS parameters were; IMS wave height: 40V and IMS wave velocity: 1600m/s. The inset shows the comined MS spectra at the IMS drift-times indicated y doule-headed arrows; (12.5-13.0 ms) and (14.5-15.0 ms). Evident from the isotope distriutions, the drift-times corresponded to intact HI and HI + 2H, respectively. S-4
30 7 7 1 6 11 19 20 1 Figure S3. Tertiary structure of HI with disulfide onds highlighted. HI monomer in T-state (D code 1MSO, chain and ) displayed with -chain colored in red, -chain in lue and the three disulfide onds in yellow. The structure of HI is in () represented in cartoon structure with disulfide onds represented in sticks. In () the structure in shown in mesh structure with disulfide onds represented as spheres to visualize location and solvent exposure of the disulfide onds. The solvent exposed sulfur-atoms of the 7-7 disulfide ond are indicated y arrows. S-5
I 3 2 G I V 1 F 2 4 5 7 8 9 E Q C C T S I C S L Y Q L E N Y C N 3 4 5 6 7 8 y12 10 9 y11 11 10 12 11 13 y8 12 14 13 y7 14 15 15 16 V N Q H L C G S H L V E L Y L V C G E R G F F Y T K T 16 17 18 y11 ( 7-2H) y1 ( 8-2H) ( 9-2H) ( 10-2H) :(y 11 ) :(y 12 ) II 3 2 G I V 1 F 2 4 5 6 11 E Q C C T S I C S L Y Q L E N Y C N 3 4 5 6 7 8 9 10 11 12 13 12 y8 13 14 y7 14 15 15 16 V N Q H L C G S H L V E L Y L V C G E R G F F Y T K T 16 17 18 y11 ( 6 ) y8 y1 C Main 3 2 G I V 1 F 2 4 5 11 E Q C C T S I C S L Y Q L E N Y C N 3 4 5 6 7 8 9 10 11 12 13 12 y8 13 14 y7 14 15 15 16 V N Q H L C G S H L V E L Y L V C G E R G F F Y T K T 16 17 17 18 y11 y8 y7 y1 Figure S4. Identification of scramled species y characterization of partially reduced species. CID LC-MSMS of the main partially reduced specie (C) (same as article Figure 3C), I () and II () as annotated in article Figure 3, with trap CE ramps of 24-44 V. Only -chain intrachain disulfide ond determining fragments are annotated in the spectra. Full annotation of the main partially reduced specie is shown in article Figure 3C. Sequence coverage and disulfide connectivities of the partially reduced HI species are shown in the HI sequence insets in the spectra, revealing I and II to have scramled -chain intrachain disulfide onds. Retention time: 24.70 recursor ion [M+6H] 6+ HI:4NEM :(y 8 ) ( 3 ) (y 3 ) (y 4 ) ( 5 ) ( 10 ) ( ( 10 ) 11 ) ( 11 ) ( 14 ) (15 ) ( 16 ) :(y 7 ) :(y 10 ) :(y 11 ) Retention time: 25.35 recursor ion [M+6H] 6+ HI:4NEM :(y 8 ) ( 3 ) (y 3 ) (y 4 ) ( 5 ) ( 10 ) ( 11 ) ( ( 10 ) 11 ) ( 14 ) (15 ) ( 16 ) :(y 7 ) :(y 10 ) :(y 11 ) Figure S5. Confirmation of identical fragmentation patterns of the two HI:4NEM peaks in article Figure 4. Comined CID fragmentation spectra of the peaks eluting at 24.70 min and 25.35 min are shown in () and (), respectively, with trap CE ramps of 18-38 V. The identical fragmentation patterns confirmed peak splitting due to introduction of chiral carons in NEM alkylations. S-6
1 disulfide reduced (+2 NEM) (+2 O) 2 disulfides reduced (+4 NEM) (+2 O) 3 disulfides reduced (+6 NEM) (+2 O) II I I (+2 O) (+ O) Intact Lysozyme 8 disulfides reduced (+8 NEM) (+2 O) Figure S6. LC-MS TIC chromatogram of partially EC reduced and NEM alkylated Lysozyme. EC reduction of 10 µm lysozyme was performed in 25% CN and 1 % F with EC potentials of (-1.0V, +0.5, 1000, 500). Sample collection was done with NEM alkylation of free cysteines. LC elution was performed with a gradient of -uffer % from 15 to 30 in 32 min. Elution of intact and partially reduced forms of lysozyme are annotated in the chromatogram, identified according to the intact masses. Intact lysozyme was present unmodified and as singly (+O) and douly (+2O) oxidized forms. ll partially reduced species were present as douly oxidized forms (+2O). The peaks annotated with I and II are partially reduced lysozyme with 1 disulfide reduced (+2 NEM, +2O) and 2 disulfides reduced (+4NEM, +2O), respectively. The appearance of these peaks is due to the chiral caron introduction per NEM alkylation. S-7
C-term ) ) N-term #1 : 7 #2 : #3 : #4 : #5 : #6 : #7 : 9 10 12 14 21 22 28 36 38 40 41 42 49 51 58 61 64 65 52TCVDESENCDK 74LCTVTLR 82ETYGEMDCCK 99NECFLQHK 115LVREVDVMCTFHDNEETFLK 163FTECCQDK 175CLLK 200CSLQK 241VHTECCHGDLLECDDR 263YICENQDSISSK 277ECCEKLLEK 287SHCIEVENDEMDLSLDFVESK 314DVCK 360CCDHECYK 390QNCELFEQLGEYK 437CCK 446MCEDYLSVVLNQLCVLHEK 476CCTESLVNR 485RCFSLEVDETYVK #8 : 66 75 77 501EFNETFTFHDICTLSEK 558CCK 565ETCFEEGK Figure S7. HS. () Tertiary structure of HS (D code: 1O6, chain ) displayed in cyan as cartoon with disulfide onds shown as yellow sticks. N- and C-terminals are indicated. In () the 8 disulfide-containing peptides from a theoretical Trypsin digestion of HS is listed named with #. The numering of residues and the individual peptides linked with disulfide onds are according to the in silico digestion y GMW. #3 LC-MS 0.1 % F #1 #6 #4 #2 #8 #5 #7 #1 #6 #4 #2 #8 #3 #5 #7 LC-EC-MS 0.1 % F C #3 LC-EC-MS 0.5 % F #1 #6 #4 #2 #8 #5 #7 Figure S8. TIC chromatograms of non-reduced tryptic digest of HS under standard LC-MS conditions using 0.1% F solvents () and in a LC-EC-MS setup using either 0.1% F- () or 0.5% F solvents (C) with the EC cell turned off. slight peak-roadening effect of the EC cell dead volume was oserved etween and. In C, the effect of using 0.5% F solvent was found to shift the retention time only slightly for most peptides compared to when using 0.1% F solvents in. Only disulfide-containing peptides are annotated in the figure, named according to an in silico digestion (Figure S7). The LC gradient used was -uffer % from 0 to 33.5 in 75.5 min. S-8
EC cell OFF TiO 2 + CHO + H TiO 2 + CH 3 CN + H CO Ti 2 O 3 + HCOOH + O + H CO CO Ti 2 O 3 + HCOOH + CH 3 CN + O + O + H CO EC cell ON Ti 3 O 4 + HCOOH + CO + CO + O + O + H Figure S9. LC-EC-MS spectra of the low m/z-region with the EC cell turned off () and on (). Different cominations of titanium oxide species and uffer component, generated when the EC cell was turned on, are assigned in the figure. MS spectra represent single scans displayed with linked vertical axes. #3 : 14 21 22 115LVREVDVMCTFHDNEETFLK 163FTECCQDK 175CLLK #3 7+ #3 6+ #3 5+ EC cell OFF - 2 µg #3 7+ 14 21 #3 6+ EC cell ON - 2 µg 0.1% F 22 21-22 #3 8+ #3 5+ 14 C 22 21-22 14 21 22 (y 5 ) #3 6+ 14 #3 5+ EC cell ON - 10 µg 0.5% F Figure S10. LC-EC-MS spectra of the disulfide-containing #3 fragment with the EC cell turned off () and on in 0.1% F- () and 0.5% F LC-solvents (C). When turned on the EC cell potentials were (-1.5V, 1.0V, 1000ms, 500ms). Sample injection amounts were as indicated in the spectra. EC reduction in 0.1% F solvents resulted in a limited reduction efficiency and generation of an increased amount of the partially reduced 21-22 specie. S-9
#7 : 51 58 61 64 65 390QNCELFEQLGEYK 437CCK 446MCEDYLSVVLNQLCVLHEK 476CCTESLVNR 485RCFSLEVDETYVK #7 9+ #7 8+ #7 7+ EC cell OFF - 2 µg #7 6+ 65 64 65 61-64 5+ 61-64 61 #7 9+ #7 8+ #7 7+ EC cell ON - 10 µg 0.1% F C 64 61 + Ti 65 61 EC cell ON - 10 µg 0.5% F Figure S11. LC-EC-MS spectra of the disulfide-containing #3 fragment with the EC cell turned off () and on in 0.1% F- () and 0.5% F LC-solvents (C). When turned on the EC cell potentials were (-1.5V, 1.0V, 1000ms, 500ms). Sample injection amounts were as indicated in the spectra. EC reduction in 0.1% F solvents resulted in a limited reduction efficiency and generation of the partially reduced 61-64 specie. s seen in the spectra, extensive ion suppression is oserved resulting in no oservation of the 51 and 58 peptides. S-10