2D FT-ICR MS of Calmodulin: a Top-Down and Bottom-Up Approach

Supporting information

2D FT-ICR MS of Calmodulin: a top-down and bottom-up approach

Floris, F.1; van Agthoven M.1; Chiron, L.2; Soulby, A.J.1; Wootton, C.A.1; Lam P.Y.1; Barrow M.P.1; Delsuc, M-A2,3 ; O'Connor, P.B.1

1University of Warwick, Coventry, UK;2CASC4DE, Illkirch-Graffenstaden, France; 3IGBMC, Illkirch-Graffenstaden, France

Tables with peaks assignments for the top-down (S1) and bottom-up (S2.1-2.6) data. Table of the parameters used for the acquisition of 2D mass spectra (S3). Table with peak assignments for the bottom-up data in 1D MS (S4.1). Cleavage coverage map for One-Dimensional FT-ICR MS analysis of CaM (Fig. S1).

Table S1: Peak assignment for top-down 2D-IRMPD/FT-ICR MS Calmodulin

Fragment ions / Charge / Theoretical m/z / Observed m/z / Error [ppm]
y2 / 1 / 218.149918 / 218.150115 / 0.90
b2 / 1 / 229.081899 / 229.082081 / 0.79
y3 / 1 / 319.197597 / 319.197628 / 0.10
b3 / 1 / 357.140477 / 357.140358 / -0.33
PTEA-H2O / 1 / 381.176900 / 381.176642 / -0.58
y4-H2O / 1 / 432.227517 / 432.227401 / -0.27
y4 / 1 / 450.238082 / 450.237949 / -0.29
b4 / 1 / 470.224541 / 470.224312 / -0.49
b5-H2O / 1 / 553.261655 / 553.260604 / -1.90
y5-H2O / 1 / 563.268002 / 563.267836 / -0.29
b5 / 1 / 571.272220 / 571.271558 / -1.16
y5 / 1 / 581.278567 / 581.278309 / -0.44
EFVQM-H2O / 1 / 617.275200 / 617.274524 / -1.09
PTEAEL / 1 / 641.314100 / 641.313744 / -0.53
b6-H2O / 1 / 682.304249 / 682.302961 / -1.89
y6-NH3 / 1 / 692.310596 / 692.309784 / -1.17
? / 2 / 696.847448
y6 / 1 / 709.337145 / 709.337590 / 0.63
QDMINE-NH3 / 1 / 714.276300 / 714.275576 / -1.04
MIREAD / 1 / 716.339600 / 716.339328 / -0.36
EEFVQM-H2O / 1 / 746.317800 / 746.317438 / -0.47
NPTEAEL / 1 / 755.357000 / 755.357591 / 0.77
? / 2 / 804.884127
y7 / 1 / 808.405559 / 808.406023 / 0.57
EMIREAD-H2O / 1 / 827.371600 / 827.368425 / -3.86
b7 / 1 / 829.357408 / 829.355787 / -1.95
EMIREAD / 1 / 845.382200 / 845.380091 / -2.47
PTEAELQD-H2O / 1 / 866.389000 / 866.388953 / -0.10
b15 / 2 / 873.415060 / 873.415287 / 0.26
y15-H2O / 2 / 878.905293 / 878.904927 / -0.42
? / 2 / 898.900820
? / 1 / 903.915715
? / 1 / 912.920464
y8 / 1 / 955.473973 / 955.473651 / -0.34
y17-H2O / 2 / 964.929497 / 964.930418 / 0.96
? / 2 / 984.442158
? / 6 / 1,002.815433
b37-H2O / 4 / 1,035.758406 / 1,035.757729 / -0.65
? / 1 / 1,044.466336
b9-H2O / 1 / 1,052.489485 / 1,052.488617 / -0.82
b9 / 1 / 1,070.500050 / 1,070.494920 / -4.79
y19-H2O / 2 / 1,078.985001 / 1,078.984842 / -0.15
b39-2H2O / 4 / 1,081.284788 / 1,081.286864 / 1.92
b39-H2O / 4 / 1,085.787430 / 1,085.788762 / 1.23
b39 / 4 / 1,090.290071 / 1,090.289966 / -0.10
b40-H2O / 4 / 1,100.042796 / 1,100.043707 / 0.83
b50-NH3 / 5 / 1,105.531262 / 1,105.533070 / 1.64
b51-NH3 / 5 / 1,131.739359 / 1,131.743737 / 3.87
b41-H2O / 4 / 1,132.057440 / 1,132.056631 / -0.71
b41 / 4 / 1,136.560081 / 1,136.556569 / -3.09
b10 / 1 / 1,141.537164 / 1,141.534267 / -2.54
b52-NH3 / 5 / 1,154.356172 / 1,154.357187 / 0.88
? / 4 / 1,156.564910
b42-NH3 / 4 / 1,160.814176 / 1,160.822771 / 7.40
b42 / 4 / 1,165.070813 / 1,165.072374 / 1.34
MH / 1,199.924136
b45-H2O / 4 / 1,242.353931 / 1,242.357154 / 2.59
b45 / 4 / 1,246.856573 / 1,246.854922 / -1.32
? / 2 / 1,248.638688
? / 2 / 1,257.139891
? / 1 / 1,277.617715
? / 1 / 1,299.609740
b22 / 2 / 1,299.615946 / 1,299.617500 / 1.20
? / 1 / 1,317.554812
? / 2 / 1,361.650768
? / 2 / 1,370.195414
? / 2 / 1,378.707026
b24 / 2 / 1,385.640150 / 1,385.638665 / -1.07
? / 1 / 1,611.810091
y15 / 1 / 1,774.813874 / 1,774.813333 / -0.30
Average Error / -0.18
Standard deviation / 1.85

Table S2.1 Peak assignment for bottom-up 2D-IRMPD/FT-ICR MS Calmodulin

Peptide / Theoretical
m/z / Observed
m/z / Error
[ppm] / Peptide sequence / Charge
31_37 / 403.215464 / 403.215329 / -0.33 / ELGTVMR / 2
76_86 / 451.538131 / 451.537898 / -0.52 / MKDTDSEEEIR / 3
22_30 / 454.221999 / 454.222146 / 0.32 / DGDGTITTK / 2
14_21 / 478.739827 / 478.740266 / 0.92 / EAFSLFDK / 2
75_86 / 494.236452 / 494.236087 / -0.74 / KMKDTDSEEEIR / 2
87_90 / 522.267074 / 522.266960 / -0.22 / EAFR / 1
78_90 / 532.909393 / 532.910201 / 1.52 / DTDSEEEIREAFR / 3
78_86 / 547.235835 / 547.235994 / 0.29 / DTDSEEEIR / 2
91_106 / 585.628449 / 585.628964 / 0.88 / VFDKDGNGYISAAELR / 3
d91_106 / 585.956454 / 585.956483 / 0.05 / VFDKDGNGYISAAELR / 3
14_30 / 615.635270 / 615.636243 / 1.58 / EAFSLFDKDGDGTITTK / 3
76_86 / 676.803559 / 676.804002 / 0.66 / MKDTDSEEEIR / 2
1_13 / 782.380488 / 782.380453 / -0.04 / Ac-ADQLTEEQIAEFK / 2
78_90 / 798.860452 / 798.856303 / -5.19 / DTDSEEEIREAFR / 2
106_125 / 801.062778 / 801.057993 / -5.97 / HVMTNLGEKLTDEEVDEMIR / 3
31_37 / 805.423652 / 805.422482 / -1.45 / ELGTVMR / 1
d91_106 / 878.431044 / 878.429467 / -1.80 / VFDKDGNGYISAAELR / 2
14_30 / 922.949267 / 922.950409 / 1.24 / EAFSLFDKDGDGTITTK / 2
78_86 / 1093.464394 / 1093.461091 / -3.02 / DTDSEEEIR / 1
127_148 / 1245.543609 / 1245.534997 / -6.91 / EADIDGDGQVNYEEFVQMMTAK / 2
Average Error / -0.94
Standard deviation / 2.42

Table S2.2: Bottom-up 2D-IRMPD/FT-ICR MS CaM, peak assignment for ion at 403.215463m/z

Ion: [31_37]2+
Fragment / Theoretical m/z / Observed m/z / Error [ppm]
y / 175.118952 / 175.118877 / -0.43
b2-H2O / 225.123369 / 225.123466 / 0.43
y2 / 306.159437 / 306.159510 / 0.24
b4-2H2O / 365.181946 / 365.182217 / 0.74
b4-H2O / 383.192511 / 383.192650 / 0.36
MH-H2O / 394.210181 / 394.208981 / -3.04
[MH2]2+ / 403.215463 / 403.215860 / 0.98
y3 / 405.227851 / 405.227721 / -0.32
b5-H2O / 482.260925 / 482.260791 / -0.28
y4 / 506.275530 / 506.275810 / 0.55
y5-H2O / 545.286428 / 545.286426 / 0.00
y5 / 563.296993 / 563.297137 / 0.26
b6-2H2O / 595.290845 / 595.290598 / -0.42
b6-H2O / 613.301410 / 613.301637 / 0.37
Average Error / -0.04
Standard deviation / 0.93

Table S2.3: Bottom-up 2D-IRMPD/FT-ICR MS CaM, peak assignment for ion at 547.235832m/z

Ion: [78_86]2+
Fragment / Theoretical m/z / Observed m/z / Error [ppm]
y / 175.118952 / 175.118983 / 0.18
b2 / 217.081898 / 217.081864 / -0.16
y2 / 288.203016 / 288.202904 / -0.39
b3-H2O / 314.098276 / 314.098260 / -0.05
b3 / 332.108841 / 332.108772 / -0.21
y3 / 417.245609 / 417.245397 / -0.51
[MH2-H2O]2+ / 538.230550 / 538.231097 / 1.02
y4 / 546.288202 / 546.287524 / -1.24
[MH2]2+ / 547.235832 / 547.235367 / -0.85
b5 / 548.183462 / 548.183563 / 0.18
b6-2H2O / 641.204926 / 641.203951 / -1.52
b6-H2O / 659.215491 / 659.214806 / -1.04
y5 / 675.330795 / 675.329826 / -1.43
b6 / 677.226056 / 677.225820 / -0.35
y6 / 762.362824 / 762.363086 / 0.34
b7-2H2O / 770.247519 / 770.247237 / -0.37
b7-H2O / 788.258084 / 788.258059 / -0.03
b7 / 806.268649 / 806.267984 / -0.82
y7-H2O / 859.379202 / 859.377517 / -1.96
b8-3H2O / 865.321018 / 865.324568 / 4.10
y7 / 877.389767 / 877.388923 / -0.96
b8-2H2O / 883.331583 / 883.332027 / 0.50
b8-H2O / 901.342148 / 901.342227 / 0.09
b8 / 919.352713 / 919.352639 / -0.08
Average Error / -0.23
Standard deviation / 1.14

Table S2.4: Bottom-up 2D-IRMPD/FT-ICR MS CaM, peak assignment for ion at 585.628447m/z

Ion: [91_106]3+
Fragment / Theoretical m/z / Observed m/z / Error [ppm]
? / 441.767966
? / 443.783189
y4 / 488.282723 / 488.282700 / -0.05
? / 519.799132
[MH3]3+ / 585.628447 / 585.628184 / -0.45
d[MH3]3+ / 585.956452 / 585.955704 / -1.28
y6 / 646.351865 / 646.351900 / 0.05
y142+ / 755.362627 / 755.362600 / -0.04
Average Error / -0.35
Standard deviation / 0.49

Table S2.5: Bottom-up 2D-IRMPD/FT-ICR MS CaM, peak assignment for ion at 615.635268m/z

Ion: [14_30]3+
Fragment / Theoretical m/z / Observed m/z / Error [ppm]
y3 / 349.208161 / 349.208136 / -0.07
y4 / 462.292225 / 462.292275 / 0.11
b5-2H2O / 512.250360 / 512.250999 / 1.25
b5-H2O / 530.260925 / 530.261639 / 1.35
[y15-2H2O]3+ / 536.934989 / 536.936343 / 2.52
[y11-2H2O]2+ / 557.772385 / 557.773139 / 1.35
y5 / 563.339904 / 563.341754 / 3.28
[y11-H2O]2+ / 566.777668 / 566.777777 / 0.19
[MH3-5H2O]3+ / 585.617660 / 585.617477 / -0.31
[MH3-4H2O]3+ / 591.621181 / 591.621636 / 0.77
[MH3-3H2O]3+ / 597.624703 / 597.624851 / 0.25
y6-H2O / 602.350802 / 602.351181 / 0.63
[MH3-2H2O]3+ / 603.628225 / 603.629659 / 2.38
[MH3-H2O]3+ / 609.631746 / 609.631368 / -0.62
[MH]3+ / 615.635268 / 615.635618 / 0.57
y6 / 620.361367 / 620.361259 / -0.17
[y12-3H2O]2+ / 622.301310 / 622.300789 / -0.84
[y12-2H2O]2+ / 631.306592 / 631.306366 / -0.36
[y12-H2O]2+ / 640.311875 / 640.311903 / 0.04
y122+ / 649.317157 / 649.317692 / 0.82
[b13-2H2O]2+ / 674.296224 / 674.296382 / 0.23
[b13-H2O]2+ / 683.301507 / 683.299349 / -3.16
[y13-2H2O]2+ / 687.848624 / 687.847927 / -1.01
[y13-H2O]2+ / 696.853907 / 696.851851 / -2.95
[b14-3H2O]2+ / 721.832974 / 721.832555 / -0.58
[b14-2H2O]2+ / 730.838256 / 730.837826 / -0.59
[y14-H2O]2+ / 740.369921 / 740.370325 / 0.55
Average Error / 0.21
Standard deviation / 1.37

Table S2.6: Bottom-up 2D-IRMPD/FT-ICR MS CaM, peak assignment for ion at782.380486 m/z

Ion: [1_13]2+
Fragment / Theoretical m/z / Observed m/z / Error
[ppm]
y2 / 294.181218 / 294.181670 / 1.54
b3 / 357.140476 / 357.140078 / -1.11
y3-H2O / 405.213246 / 405.213674 / 1.06
y3 / 423.223811 / 423.223816 / 0.01
b4 / 470.224540 / 470.224526 / -0.03
y4 / 494.260925 / 494.260813 / -0.23
b5-H2O / 553.261653 / 553.261102 / -1.00
b5 / 571.272218 / 571.271978 / -0.42
y5 / 607.344989 / 607.346291 / 2.14
TEEQIA-H2O / 654.309334 / 654.309487 / 0.23
TEEQIA / 672.319899 / 672.320422 / 0.78
b6-H2O / 682.304246 / 682.304275 / 0.04
y12(2+) / 725.856646 / 725.855826 / -1.13
[MH2-H2O]2+ / 773.375203 / 773.375467 / 0.34
[MH2]2+ / 782.380486 / 782.380127 / -0.46
LTEEQIA / 785.403963 / 785.403437 / -0.67
b7-H2O / 811.346839 / 811.349151 / 2.85
b7 / 829.357404 / 829.357680 / 0.33
y7-H2O / 846.435595 / 846.435030 / -0.67
b8 / 957.415982 / 957.451126 / 36.71
DQLTEEQIA-H2O / 1010.478920 / 1010.476830 / -2.07
DQLTEEQIA / 1028.489485 / 1028.490346 / 0.84
b9-2H2O / 1034.478916 / 1034.478881 / -0.03
b9-H2O / 1052.489481 / 1052.488447 / -0.98
b9 / 1070.500046 / 1070.501445 / 1.31
y9 / 1094.536431 / 1094.538171 / 1.59
b10-H2O / 1123.526595 / 1123.526826 / 0.21
b10 / 1141.537160 / 1141.534467 / -2.36
DQLTEEQIAE / 1157.532079 / 1157.534665 / 2.23
b11 / 1270.579753 / 1270.580712 / 0.75
b12 / 1417.648167 / 1417.647512 / -0.46
Average Error / 1.33
Standard deviation / 6.56

Table S3: Parameters used for 2D FT-ICR MS spectral acquisition

Parameter / Experiment
Top-down 2D FT-ICR MS / Bottom-up FT-ICR MS
Acquisition mode
Acquisition size [datapoints] / 4 / 512k
Horizontal low mass cut-off [m/z] / 147.41 / 147.41
Horizontal high mass cut-off [m/z] / 3000 / 3000
Transient length, t2 [s] / 1.6777 / 0.2097
Ion accumulation time [s] / 0.1 / 0.1
Ion cooling time [s] / 0.1 / 0.25
Source (nanoESI)
Capillary voltage [V] / 900 / 1300
Capillary exit [V] / 200 / 200
Deflector plate [V] / 220 / 220
Funnel [V] / 150 / 150
Skimmer [V] / 10 / 10
Funnel RF [Vpp] / 200 / 200
Collision cell
Collision voltage [V] / 0 / 0
DC extract bias [V] / 0.6 / 0.6
RF frequency [MHz] / 2 / 2
Collision RF Amplitude [Vpp] / 1400 / 1400
Octopole
Frequency [MHz] / 2 / 2
RF Amplitude [Vpp] / 1400 / 1400
Transfer optics
Time of Flight [s] / 0.0012 / 0.0012
Frequency [MHz] / 4 / 4
RF Amplitude [Vpp] / 350 / 350
ICR-cell
Transfer exit Lens [V] / -15 / -15
Analyser entrance [V] / -8 / -8
Side kick [V] / 12 / 10
Side kick offset [V] / -2.3 / -2.9
Front Trap plate [V] / 0.51 / 0.69
Back Trap plate [V] / 0.55 / 0.74
Sweep Excitation power, P3 [%] / 16 / 16
2D-MS Pulse Program
t1 [s] / 1.2E-06 / 2.4E-06
t1 increment [s] / 1.2E-06 / 2.4E-06
P1 and P2 pulse length [s] / 4.0E-07 / 4.0E-07
P1 and P2 excitation power [Db] / 10.42 / 10.42
Laser Power (25W) [%] / 70 / 70
IR pulse length [s] / 0.3 / 0.3

Table S4.1 Peak assignment for bottom-up 1D IRMPD FT-ICR MS Calmodulin

Peptide / Theoretical
m/z / Observed
m/z / Error
[ppm] / Peptide sequence / Charge
31_37 / 403.215464 / 403.21421 / -3.11 / ELGTVMR / 2
75_77 / 406.2483 / 406.24719 / -2.73 / KMK / 1
76_86 / 451.538131 / 451.53762 / -1.13 / MKDTDSEEEIR / 3
14_21 / 478.739827 / 478.73947 / -0.75 / EAFSLFDK / 2
75_86 / 494.236452 / 494.23584 / -1.24 / KMKDTDSEEEIR / 3
87_90 / 522.267074 / 522.26659 / -0.93 / EAFR / 1
78_90 / 532.909393 / 532.90945 / 0.11 / DTDSEEEIREAFR / 3
78_86 / 547.235835 / 547.23583 / -0.01 / DTDSEEEIR / 2
91_106 / 585.628449 / 585.62840 / -0.08 / VFDKDGNGYISAAELR / 3
d91_106 / 585.956454 / 585.95641 / -0.08 / VFDKDGNGYISAAELR / 3
14_30 / 615.6352697 / 615.63524 / -0.05 / EAFSLFDKDGDGTITTK / 3
76_86 / 676.803559 / 676.80370 / 0.21 / MKDTDSEEEIR / 2
1_13 / 782.380488 / 782.38055 / 0.08 / Ac-ADQLTEEQIAEFK / 2
78_90 / 798.8604515 / 798.86131 / 1.07 / DTDSEEEIREAFR / 2
106_125 / 801.0627777 / 801.06433 / 1.94 / HVMTNLGEKLTDEEVDEMIR / 3
31_37 / 805.423652 / 805.42358 / -0.09 / ELGTVMR / 1
91_106 / 877.9390355 / 877.94069 / 1.88 / VFDKDGNGYISAAELR / 2
d91_106 / 878.431044 / 878.43151 / 0.53 / VFDKDGNGYISAAELR / 2
22_30 / 907.436721 / 907.43861 / 2.08 / DGDGTITTK / 1
14_30 / 922.949267 / 922.94928 / 0.01 / EAFSLFDKDGDGTITTK / 2
14_21 / 956.472377 / 956.47375 / 1.44 / EAFSLFDK / 1
78_86 / 1093.464394 / 1093.46538 / 0.90 / DTDSEEEIR / 1
127_148 / 1245.543609 / 1245.54480 / 0.96 / EADIDGDGQVNYEEFVQMMTAK / 2
1_13 / 1563.7537 / 1563.75544 / 1.11 / Ac-ADQLTEEQIAEFK / 1
Average Error / 0.09
Standard deviation / 1.28

Figure S1 Cleavage coverage map for one-dimensional top-down and bottom-up analysis

Map of the cleavages obtained with the top-down and bottom-up analysis of CaM using IRMPD FT-ICR MS/MS. The total cleavage coverage for the one-dimensional MS analysis is ~52%. For comparison, the total cleavage coverage obtained with the 2D MS analysis is ~40%.

1