Ion Mobility-Mass Spectrometry As a Tool for the Structural Characterization of Peptides

Ion mobility-mass spectrometry as a tool for the structural characterization of peptides bearing intra-molecular disulfide bond(s)

Philippe Massonnet(1), Jean R. N. Haler (1), Gregory Upert(2) Michel Degueldre(1) Denis Morsa(1), Nicolas Smargiasso(1), Gilles Mourier (2), Nicolas Gilles(2), Loïc Quinton(1), Edwin De Pauw*(1).

Laboratory of Mass Spectrometry-GIGA-R, University of Liège, Quartier Agora, Allée du six Aout 11, B-4000 Liege, Belgium
Commissariat à l’Energie Atomique, DRF/iBiTec – S/SIMOPRO, CE Saclay, 91191 Gif sur Yvette, France.

AUTHOR EMAIL ADDRESS:

SUPPLEMENTARY INFORMATION

SI 1) Table of the peptides used in this study.

Name / m (Da) / Connectivity / Sequence / Number
of S-S bonds
P1 / 1083,50 / / / CYFQNCPRG / 1
P2 / 1636,80 / / / AGCKNFFWKTFTSC / 1
P3 / 1006,50 / / / CYIQNCPLG / 1
P4 / 3060 / / / SLRRSSCFGGRIDRIGAQSGLGCNSFRY / 1
P5 / 2170,05 / / / C1NC2KAPETALC3ARRC4QQH
(+2Acm on C1 and C2) / 1
P6 / 2170,04 / / / C1NC2KAPETALC3ARRC4QQH
(+2Acm on C2 and C3) / 1
P7 / 3305,60 / / / C1EGWFRFTKTGLEYC2TPGLC3LRWGKLC4* (+2Acm on C2 and C4) / 1
P8 / 2165,04 / / / C1FRFTKYC2C3LRWGKLC4*
(+2 Acm on C3 and C4) / 1
P9 / 3305,60 / / / C1EGWFRFTKTGLEYC2TPGLC3LRWGKLC4* (+2Acm on C2 and C3) / 1
P10 / 3305,60 / / / C1EGWFRFTKTGLEYC2TPGLC3LRWGKLC4* (+2Acm on C3 and C4) / 1
P11 / 2891,48 / / / C1LGWFRFTKTGLVYC2C3LRWGKLC4*
(+2Acm on C3 and C4) / 1
P12 / 2154,15 / 1,4/2,3 / RGGRLCYCRRRFCVCVGR / 2
P13 / 2025,88 / 1,3/2,4 / CNCKAPETALCARRCQQH / 2
P14 / 2505,10 / 1,4/2,3 / CSCADMTDKECLYFCHQDVIW / 2
P15 / 2059,90 / 1,3/2,4 / DCPPHPVPGMHKCVCLKTC / 2
P16 / 1541,68 / 1,3/2,4 / GRCCHPACGKYYSC* / 2
P17 / 1328,52 / 1,3/2,4 / CCHPACGKYYSC* / 2
P18 / 1621,68 / 1,3/2,4 / ECCNPACGRHYSCGK* / 2
P19 / 1436,50 / 1,3/2,4 / ECCNPACGRHYSC* / 2
P20 / 1655,70 / 1,3/2,4 / DGRCCHPACAKHFNC* / 2
P21 / 1454,50 / 1,3/2,4 / YCCHPACGKNFDC* / 2
P22 / 2389,14 / 1,3/2,4 / FNWRCCLIPACRRNHKKFC* / 2
P23 / 1689,69 / 1,3/2,4 / NGRCCHPACARKYNC* / 2
P24 / 1666,57 / 1,3/2,4 / ZSOGCCWNPACVKNRC* / 2
P25 / 1670,60 / 1,3/2,4 / GCCSYPPCFATNSGYC* / 2
P26 / 1725,60 / 1,3/2,4 / GGCCSHPACFASNPDYC* / 2
P27 / 1331,494 / 1,3/2,4 / GCCSDPRCKHEC* / 2
P28 / 1569,76 / 1,3/2,4 / DYCCRROOCTLIC* / 2
P29 / 2091,82 / 1,3/2,4 / RDOCCYHPTCNMSNPQIC* / 2
P30 / 1496,59 / 1,3/2,4 / GRCCHPACGKNYSC* / 2
P31 / 1415,60 / 1,3/2,4 / ECCHPACGKHFSC* / 2
P32 / 1709,84 / 1,3/2,4 / GCCSNPVCHLEHSNLC* / 2
P33 / 1352,60 / 1,3/2,4 / ICCNPACGPKYSC* / 2
P34 / 2909,56 / 1,3/2,4 / FPRPRICNLACRAGIGHKYPFCHCR* / 2
P35 / 1301,50 / 1,4/2,3 / CCHSSWCKHLC / 2
P36 / 1447,66 / 1,3/2,4 / KPCCSIHDSSCCGI / 2
P37 / 1392,60 / 1,4/2,3 / VGVCCGYKLCHOC / 2
P38 / 1356,39 / 1,4/2,3 / DPCCGYRMCVOC* / 2
P39 / 1930,83 / 1,3/2,4 / LWQNTWCCRDHLRCC* / 2
P40 / 1274,54 / 1,3/2,4 / DCCPAKLLCCNP / 2
P41 / 1358,56 / 1,3/2,4 / ZCCITIPECCRI* / 2
P42 / 1236,49 / 1,4/2,3 / VCCGYKLCHOC / 2
P43 / 1266,45 / 1,4/2,3 / GICCGVSFCYOC / 2
P44 / 1311,51 / 1,4/2,3 / RCCGYKMCHOC / 2
P45 / 1248,50 / 1,4/2,3 / GVCCGVSFCYOC / 2
P46 / 2589,42 / 1,3/2,4 / IKCNCKRHVIKPHICRKICGKN* / 2
P47 / 1089,50 / 1,3/2,4 / CCPGKPCCRI* / 2
P48 / 1175,42 / 1,3/2,4 / PCCSIHDNSCC* / 2
P49 / 1596,62 / 1,3/2,4 / ECCEDGWCCTAAPLT / 2
P50 / 3161,48 / 1,2/3,4 / CEGWFRFTKTGLEYCTPGLCLRWGKLC / 2
P51 / 3161,48 / 1,3/2,4 / CEGWFRFTKTGLEYCTPGLCLRWGKLC / 2
P52 / 3161,48 / 1,4/2,3 / CEGWFRFTKTGLEYCTPGLCLRWGKLC / 2
P53 / 2020,96 / 1,2/3,4 / CFRFTKYCCLRWGKLC* / 2
P54 / 2692,29 / 1,2/3,4 / CEGWFRFTKGLEYCCLRWGKLC* / 2
P55 / 2025,95 / 1,4/2,3 / CNCKAPETALCARRCQQH / 2
P56 / 2025,95 / 1,2/3,4 / CNCKAPETALCARRCQQH / 2
P57 / 2747,40 / 1,2/3,4 / CLGWFRFTKTGLVYCCLRWGKLC* / 2
P58 / 4705,20 / 1 - 5 , 2 - 4 , 3 - 6 / AAPCFCSGKPGRGDLWILRGTCPGGYGYTSNCYKWPNICCYPH / 3
P59 / 4773,10 / 1 - 5 , 2 - 4 , 3 - 6 / AAPCFCPGKPDRGDLWILRGTCPGGYGYTSNCYKWPNICCYPH / 3
P60 / 2607,20 / 1 - 4 , 2 - 5 , 3 - 6 / RDCCTOOKKCKDRQCKOQRCCA* / 3
P61 / 2603,25 / 1 - 4 , 2 - 5 , 3 - 6 / ZRLCCGFOKSCRSRQCKOHRCC* / 3
P62 / 2648,22 / 1 - 4 , 2 - 5 , 3 - 6 / GOOCCLYGSCRPFOGCYNALCCRK* / 3
P63 / 2805,36 / 1 - 4 , 2 - 5 , 3 - 6 / LOOCCTOOKKHCOAOACKYKOCCKS / 3
P64 / 2737,32 / 1 - 4 , 2 - 5 , 3 - 6 / CKLKGQSCRKTSYDCCSGSCGRSGKC* / 3
P65 / 3447,71 / 1 - 4 , 2 - 5 , 3 - 6 / VGCAECPMHCKGKMAKPTCENEVCKCNIGKKD / 3
P66 / 5199,55 / 1 - 5 , 2 - 4 , 3 - 6 / GNCKCDDEGPNVRTAPLTGYVDLGYCNEGWEKCASYYSPIAECCRKK / 3
P67 / 3804,90 / 1 - 6 , 2 - 4 , 3 - 5 / GCKDNFSANTCKHVKANNNCGSQKYATNCAKTCGKC / 3
P68 / 3982,04 / 1 - 6 , 2 - 4 , 3 - 5 / ACKDYLPKSECTQFRCRTSMKYKYTNCKKTCGTC / 3
P69 / 4052,16 / 1 - 6 , 2 - 4 , 3 - 5 / RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC / 3
P70 / 1656,66 / 1 - 3 , 2 - 4 / GCCSRPPCIANNPDIC* / 2
P71 / 1711,48 / 1 - 5 , 2 - 4 , 3 - 6 / CCSWDVCDHPSCTCCG / 3
P72 / 1722,58 / 1 - 5 , 2 - 4 , 3 - 6 / VCCPFGGCHELCQCCE / 3
P73 / 1473,56 / 1 - 3 , 2 - 4 / KPCCSIHDNSCCGI* / 2
P74 / 2954,36 / 1 - 4 , 2 - 5 , 3 - 6 / GCNNSCQ(Gla)HSDC(Gla)SHCICTFRGCGAVN* / 3
P75 / 3088,49 / 1 - 4 , 2 - 5 , 3 - 6 / CGGYSTYC(Gla)VDS(Gla)CCSDNCVRSYCTLF* / 3
P76 / 1888,74 / 1 - 4 , 2 - 5 , 3 - 6 / RCCKFPCPDSCRYLCC* / 3
P77 / 1946,76 / 1 - 4 , 2 - 5 , 3 - 6 / RCCKFPCPDSCRYLCCG / 3
P78 / 2150,70 / 1 - 5 , 2 - 4 , 3 - 6 / KFCCDSNWCHISDCECCY* / 3
P79 / 3034,20 / 1 - 4 , 2 - 5 , 3 - 6 / WCKQSGEMCNLLDQNCCDGYCIVLVCT / 3

Acm = Acetamidomethyl :

* designates a terminal amidation

O = hydroxyproline

Z = Pyroglutamic acid

Gla = Gamma carboxylic glutamic acid

SI 2) Collision Cross-Sections of peptides in different solvent conditions:

A)For peptides bearing disulfide bond(s)

Ω (Ȧ2)
100% H20 - 0,1% FA / Ω (Ȧ2)
75% H20 - 25% ACN - 0,1% FA / Ω (Ȧ2)
50% H20 - 50% ACN - 0,1% FA / Ω (Ȧ2)
25% H20 - 75% ACN - 0,1% FA / Ω (Ȧ2)
100% ACN - 0,1% FA
p3 / 2+ / 236 / 236 / 235 / 236 / 236
p13 / 3+ / / / 419 / 422 / 419 / 422
4+ / 475 / 476 / 478 / 476 / 477
5+ / 523 / 523 / 523 / 522 / 523
p14 / 3+ / / / 394 / 394 / 396 / /
4+ / 430 / 435 / 433 / 435 / /

B) For peptides without disulfide bond (tryptic digest)

Ω (Ȧ2) - 90% H20 - 10% ACN - 0,1% FA / Ω (Ȧ2) - 75% H20 - 25% ACN - 0,1% FA / Ω (Ȧ2) - 50% H20 - 50% ACN - 0,1% FA / Ω (Ȧ2) - 25% H20 - 75% ACN - 0,1% FA / Ω (Ȧ2) - 90% ACN - 10% H2O - 0,1% FA
DIPVPKPK
(tryptic digest ADH) / 2+ / 224 / 225 / 224 / 224 / 224
3+ / 268 / 273 / 270 / 273 / 267
EKDIVGAVLK
(tryptic digest ADH) / 3+ / 299 / 302 / 299 / 302 / 301
2+ / 248 / 250 / 248 / 249 / 248
LEYKDPIPVPKPPKANELLINVK
(tryptic digest ADH) / 3+ / 467 / 468 / 469 / 467 / 468
ANGTTVLVGMPAGAK (tryptic digest ADH) / 2+ / 292 / 291 / 292 / 291 / 291
3+ / 343 / 346 / 345 / 345 / 344
LEYK
(tryptic digest ADH) / 2+ / 174 / 178 / 176 / 178 / 177
S(Acetyl)IPETQK
(tryptic digest ADH) / 2+ / 212 / 213 / 213 / 212 / 212
IGDYAGIK
(tryptic digest ADH) / 2+ / 213 / 214 / 214 / 213 / 213
AELDFFAR
(tryptic digest ADH) / 2+ / 233 / 234 / 234 / 233 / 233
ANELLINVK
(tryptic digest ADH) / 2+ / 242 / 243 / 242 / 241 / 241
GVIFYESHGK
(tryptic digest ADH) / 3+ / 292 / 295 / 296 / 298 / 295
AMGYR
(tryptic digest ADH) / 2+ / 182 / 186 / 183 / 186 / 186
VLGIDGGEGK
(tryptic digest ADH) / 2+ / 228 / 228 / 227 / 227 / 227
VLGIDGGEGKEELFR (tryptic digest ADH) / 2+ / 343 / 344 / 341 / 341 / 341
3+ / 369 / 370 / 369 / 369 / 369
EELFR
(tryptic digest ADH) / 2+ / 199 / 201 / 201 / 200 / 200
DIVGAVLK
(tryptic digest ADH) / 2+ / 211 / 213 / 212 / 212 / 213
ATDGGAHGVINVSVSEAAIEASTR
(tryptic digest ADH) / 4+ / 524 / 525 / 518 / 525 / 525
SISIVGSYVGNR
(tryptic digest ADH) / 2+ / 273 / 272 / 272 / 272 / 272
VVGLSTLPEIYEK
(tryptic digest ADH) / 2+ / 301 / 301 / 301 / 300 / 300
GQIVGR
(tryptic digest ADH) / 2+ / 185 / 186 / 188 / 189 / 189

SI 3) Table of ion mobility calibrants used to calibrate the drift times of the travelling wave ion mobility setup.

Molecule / mass / z / m/z / omega
Bradykinine / 1059,62 / 2 / 530,81 / 246
LVTDTDLTK / 788,5 / 2 / 395,25 / 205
GACLLPK + CAM / 757,48 / 2 / 379,74 / 207
ATEEQLK / 817,46 / 2 / 409,73 / 209
LCVLHEK+CAM / 897,52 / 2 / 449,76 / 228
AEFVEVTK / 921,52 / 2 / 461,76 / 219
YLYEIAR / 926,52 / 2 / 464,26 / 237
LVVSTQTALA / 1001,62 / 2 / 501,81 / 234
QTALVELLK / 1013,66 / 2 / 507,83 / 245
LVNELTEFAK / 1162,68 / 2 / 582,34 / 262
HLVDEPQNLIK / 1304,78 / 2 / 653,39 / 288
TVMENFVAFVDK / 1398,72 / 2 / 700,36 / 297
SLHTLFGDELCK + CAM / 1418,76 / 2 / 710,38 / 297
YICDNQDTISSK + CAM / 1442,7 / 2 / 722,35 / 298
LGEYGFQNALIVR / 1478,86 / 2 / 740,43 / 307
DDPHACYSTVFDK + CAM / 1553,72 / 2 / 777,86 / 313
MPCTEDYLSLILNR + CAM / 1723,88 / 2 / 862,94 / 338
YNGVFQECCQAEDK + CAM / 1748,72 / 2 / 875,36 / 325
HLVDEPQNLIK / 1304,76 / 3 / 435,92 / 319
SLHTLFGDELCK + CAM / 1291,65 / 3 / 431,55 / 334
TCVADESHAGCEK+CAM / 1461,75 / 3 / 488,25 / 340
DDPHACYSTVFDK+CAM / 1248,63 / 3 / 417,21 / 323
KVPQVSTPTLVEVSR / 1638,99 / 3 / 547,33 / 338
DAFLGSFLYEYSRR / 1722,75 / 3 / 575,25 / 383
NECFLSHKDDSPDLPK / 1900,96 / 4 / 476,24 / 442
Ubiquitine / 8560,8 / 9 / 952,2 / 1649
Ubiquitine / 8560,9 / 10 / 857,09 / 1732
Ubiquitine / 8561,08 / 11 / 779,28 / 1802
Cytochrome c / 12352,6 / 13 / 951,2 / 2391
Cytochrome c / 12348,7 / 14 / 883,05 / 2473
Cytochrome c / 12354,75 / 15 / 824,65 / 2579
Myo / 16950,6 / 18 / 942,7 / 3489
Myo / 16947,81 / 19 / 892,99 / 3570
Myo / 16948 / 20 / 848,4 / 3682
Myo / 16947 / 21 / 808 / 3792
Myo / 16940 / 22 / 771 / 3815

SI 4) Calibration curve to convert drift times into Collision Cross-Sections.

-Blue linescorrespond to the 95% confidence bands

-Red lines correspond to the 95% prediction bands

SI 5) Plot of Ω(CCS) as a function of the mass-to-charge(m/z)of all the analyzed peptides at different charge states

SI 6) Plots of the CCS (Ω)as a function of the mass (m)of peptides bearing no disulfide bond atdifferent charge states (z=2 to z=6). The linear fits are depicted in black with the equations and theircorresponding R2 values.

SI 7) Plots of the CCS (Ω) as a function of the mass (m) of peptides bearing 0, 2 or 3 disulfide bonds for z=5.A linear fit function has been used to fit the trend ofthe peptides without disulfides. ∆Ω value calculations are presented in green.

SI 8)Plots of the ∆CCS (∆Ω) as a function of the mass (m) for peptides bearing different numbers of disulfide bonds (8)a) and 8)b)plots) or different cysteine connectivities (8)c) and 8)d) plots).

a)Plot of the ∆CCS (∆Ω) as a function of the mass (m) for peptides bearing different numbers of disulfide bonds for z = 4.

b)Plot of the ∆CCS (∆Ω) as a function of the mass (m) for peptides bearing different numbers of disulfide bonds for z = 5.

c) Plot of the ∆CCS (∆Ω) as a function of the mass (m) for peptides bearing different cysteine connectivities (2 intra-molecular disulfide bonds) for z = 4.

d)Plot of the ∆CCS (∆Ω) as a function of the mass (m) for peptides bearing different cysteine connectivities (2 intra-molecular disulfide bonds) for z = 5.

SI - 1