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Intact and Top-Down Protein Analysis Guide: Orbitrap Fusion Lumos Seema Sharma October 2016
2
Orbitrap Fusion Lumos
3
Application Dependency: Pressure & Resolution
MS Protein
>50 kDa
Intact Protein Mode IRM @ 2mtorr R @ 120000
880.30 880.35 880.40 880.45 880.50 880.55 880.60 880.65 880.70 880.75 880.80 880.85m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
880.55880.52
880.49
880.61
880.45
880.42
880.64880.39
880.70880.67
880.36
880.73
880.33880.76
880.30
880.79880.82
880.85880.27
Intact mAb Charge States Carbonic Anhydrase Isotopic Resolution
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
0 9
1007.41
592.94
1086.88836.80 881.61
987.56729.94
337.19 1175.14519.04793.12
539.28667.51
627.85
1022.30
1161.12270.11
748.24
355.20 1268.02436.221210.40
311.13
468.28
239.111362.39365.18 1409.97 1548.16
Carbonic Anhydrase HCD MS/MS
CS 33+
89.45 1189.50 1189.55 1189.60 1189.65 1189.70 1189.75 1189.80 1189.85 1189.90 1189.95 1190.00 1190.05 1190.10m/z
Reduced mAb Heavy Chain Isotopic Resolution
CS 42+
Standard Pressure Mode
R @ 15000
Intact Protein Mode IRM @ 2mtorr R @ 120000
Intact Protein Mode IRM @ 1mtorr R @ 240000
MS Protein <40kDa
MS/MS
MS Protein
40-50 kDa
4
• Check all positive calibrations before proceeding to extended positive calibrations.
• Check Negative mode Ion optics & Ion Trap electron multiplier gain calibrations prior to ETD calibration.
General Calibrations
5
Extended Calibrations MS
Protein >50 kDa
Normal Mass Range No Extended
Calibrations Required
High Mass Range Positive Extended: High Mass Range
CalMix & Enfuvirtide
6
Extended Calibrations Contd. MS
Protein <40 kDa
Set IRM Pressure @ 2mtorr
Positive Extended: Intact Protein Mode
MS Protein
40-50 kDa
Set IRM Pressure @ 1mtorr
Positive Extended: Intact Protein Mode
7
IPM Pressure Setting • IPM pressure can be changed in diagnostics, by changing the parameter value under ‘Set IRM pressure (Intact)’ and then click on Start.
• HardwareSystemIRM Set IRM Pressure (Intact) ‘IRM_Pressure’ Parameter Value
• Note: The instrument can only be calibrated for one ‘IRM Pressure (Intact)’ at a given time.
• The intact protein mode calibration (optics) needs to be run at the desired IRM pressure (Intact).
8
Extended Calibrations Contd.
MS/MS Set IRM Pressure @
2mtorr
Positive Extended: Intact Protein Mode
& ETD Extended:
Intact Protein Mode
9
• To improve spectra S/N use either microscans (each is a single transient) or Averager in Tune. S/N improves as square root of number of transients averaged.
• When spectra are averaged in Qual browser, both signal and noise are averaged, the gain in S/N is somewhat lower.
• Whenever possible try to average as many transients as possible to improve spectral S/N.
• Averager updates the scan with every transient added. • Microscans update the scan only when all transients are added. • The end result is exactly the same whether you use n microscans or
average n transients through Averager. The only difference is in the scan update frequency.
• Averager uses rolling average routine. • When using microscans (n) the scan appear after averaging the last n
transients.
FT Signal: Averaged Transients vs. Averaged Scans
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Direct Infusion: Carbonic Anhydrase
11
• P/N C2522, Sigma • Direct Infusion: Flow rate 3ul/min • 1-5 pmol/ul in 50:50 AcN: H2O, 0.1% FA • HESI probe (same source conditions as for calmix)
Carbonic Anhydrase (29 kDa)
(42.0106) SHHWGYGKH NGPEHWHKDF PIANGERQSP VDIDTKAVVQ DPALKPLALV YGEATSRRMV NNGHSFNVEY DDSQDKAVLK DGPLTGTYRL VQFHFHWGSS DDQGSEHTVD RKKYAAELHLVHWNTKYGDF GTAAQQPDGL AVVGVFLKVG DANPALQKVL DALDSIKTKG KSTDFPNFDPGSLLPNVLDY WTYPGSLTTP PLLESVTWIV LKEPISVSSQ QMLKFRTLNF NAEGEPELLM LANWRPAQPL KNRQVRGFPK
N-terminal acetylation
12
p [ ]
660 680 700 720 740 760 780 800 820 840 860 880 900 920 940 960 980 1000 1020 1040 1060 1080 1100 1120 1140 1160 1180 1200 1220m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
908.03880.48854.65830.26
968.46937.19
807.251001.89
1037.60785.43764.79
745.23 1076.03908.65 951.14855.38 969.26726.60 1117.38881.48
CA_1uscan_240K_1_mtorr_2e5_RF30_XT_00001_M_ #2 RT: 2.00 AV: 1 NL: 3.59E7T: FTMS + p ESI Full ms [400.00-1600.00]
2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 22000 24000 26000 28000 30000 32000 34000 36000 38000 40000 42000 44000 46000 48000 50000 52000m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
29006.7218
7597.07292958.65108748.6145
15309.40423798.0566 21392.68395325.5634 19787.7503 29946.928610202.3672 24197.0083 28131.8040 42513.8661 53033.3840
Carbonic Anhydrase Full MS 240K, IPM 1mtorr
AGC 2e5, 20uscans CS 30+
Isotopic Resolution CA_1uscan_240K_1_mtorr_2e5_RF30 #17-35 RT: 0.18-0.37 AV: 19 NL: 1.06E7T: FTMS + p ESI Full ms [400.0000-1600.0000]
968.15 968.20 968.25 968.30 968.35 968.40 968.45 968.50 968.55 968.60 968.65 968.70 968.75 968.80 968.85m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
968.46
968.50968.60968.43
968.53
968.57968.40968.63
968.37
968.33
968.67
968.70968.30968.26
968.23968.73
Monoisotopic Mass
13
Carbonic Anhydrase MS/MS CID
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
951.15
1086.88740.92
1007.41592.94
807.24766.93
881.61902.09
730.46
1021.70
1175.14
826.12
627.86 1134.53 1251.53702.35
539.28337.19
436.22 1362.40
468.28
1449.25355.20
1300.71
311.17 1506.73251.10
ProSight Lite: 10 ppm fragment mass
tolerance
120K, 2mtorr, CID CE 25
AGC 5e5, 200 uscans
14
Carbonic Anhydrase MS/MS HCD
p @ [ ]
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
740.92
1007.41
592.94
1086.88836.80 881.61
987.56729.94
337.19 1175.14519.04793.12
539.28667.51
627.85
1022.30
1161.12270.11
748.24
355.20 1268.02436.221210.40
311.13
468.28
239.111362.39365.18 1409.97 1548.16
120K, 2mtorr, HCD CE 10
AGC 5e5, 200 uscans
15
Carbonic Anhydrase ETD HD
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
534.76
776.46
563.51
1086.88669.97
801.06 951.15588.34 751.01
506.74
486.29 630.14
819.91884.38
1105.59
421.201036.14
973.64
937.39
1243.66
1166.131267.86
375.22
284.14 1360.23 1421.33
1281.70
1447.91332.65 1554.83249.10
120K, 2mtorr, ETD Rxn 10ms
AGC 1e6, Reag AGC 7e5, 200 uscans
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Direct Infusion: Enolase
17
• P/N E6126, Sigma • Direct Infusion: Flow rate 3ul/min • Clean-up using Bio-Rad Micro spin columns (follow manufacturer’s
instructions) • 10 -20 pmol/ul in 50:50 AcN: H2O, 0.1% FA
Enolase (47 kDa)
AVSKVYARSVYDSRGNPTVEVELTTEKGVFRSIVPSGASTGVHEALEMRDGDKSKWMGKGVLHAVKNVNDVIAPAFVKANIDVKDQKAVDDFLISLDGTANKSKLGANAILGVSLAASRAAAAEKNVPLYKHLADLSKSKTSPYVLPVPFLNVLNGGSHAGGALALQEFMIAPTGAKTFAEALRIGSEVYHNLKSLTKKRYGASAGNVGDEGGVAPNIQTAEEALDLIVDAIKAAGHDGKVKIGLDCASSEFFKDGKYDLDFKNPNSDKSKWLTGPQLADLYHSLMKRYPIVSIEDPFAEDDWEAWSHFFKTAGIQIVADDLTVTNPKRIATAIEKKAADALLLKVNQIGTLSESIKAAQDSFAAGWGVMVSHRSGETEDTFIADLVVGLRTGQIKTGAPARSERLAKLNQLLRIEEELGDNAVFAGENFHHGDKL
18
p [ ]
500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relat
ive Ab
unda
nce
953.47
934.36
881.54
865.27 973.25819.75
993.93778.81
1038.16
766.13 1086.34
753.73
741.77
729.93707.81
1112.251167.64
697.021229.17
686.83
1297.32642.02
632.32571.33
Enolase Full MS
Average Mass
15K, 8mtorr
AGC 2e5, 500 uscans
19
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
686.33
870.25
670.35 978.90859.30788.92
1044.10577.27 612.79 824.45
1153.84
970.881101.58
1000.12735.86
940.49472.26645.33
696.86
569.30 1339.701211.43484.50390.15325.19 1371.64
432.25
1249.65
1174.61
921.38
1521.611464.76280.17 1576.83
Enolase 47 kDa, MS/MS CID
Seq Cov 14% 240K, 1mtorr, CID CE 35
AGC 5e5, 200 uscans
20
T: FTMS + p ESI Full ms2 [email protected] [200.0000 1600.0000]
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
686.33
921.38
978.84
670.35 909.84966.58
577.27788.92612.79
1044.10859.35
1000.12 1074.71735.86457.89
824.50 1153.84645.67
696.85
484.50571.32
390.15258.11 325.19 503.76396.191211.53297.16 1339.701275.29 1392.70 1505.79
Enolase MS/MS HCD
Seq Cov 17% 240K, 1mtorr, HCD CE 10
AGC 5e5, 200 uscans
21
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z
0
20
40
60
80
1000
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e Ab
unda
nce
0
20
40
60
80
100 899.48
936.88
828.94
977.56403.27 802.22621.35 1061.55
864.41
729.38553.28 1119.58446.77 1191.62678.46 1371.221283.66 1421.39360.21311.13828.94
898.76403.27621.35 890.97
929.50771.82 1061.55600.52
814.71
736.43 1221.57446.77 502.33 1091.36 1371.221283.66690.37 1421.061499.78360.21257.16
828.94
403.27
621.35898.76 1061.80771.82 914.48600.52446.77 1221.57502.33
814.71
1149.10
845.44
986.341371.221320.69
732.39665.40 1421.06
1499.79257.16 360.21828.94
403.27
621.35892.54 1061.80771.82553.28446.77 1221.57966.51 1091.56 1320.69 1371.22
849.64814.85
732.59665.40 1443.76 1531.81257.16 360.21
A
A
A
A
Rxn 9ms
Enolase ETD HD CS 52+ Rxn 3ms
Rxn 5ms
Rxn 7ms
240K, 1mtorr, Reagent AGC 7e5
AGC 1e6, 200 uscans
22
Enolase CS 52+, ETD HD Sequence Coverage
5ms 3ms
7ms 9ms
3ms
7ms 9ms
23
200 400 600 800 1000 1200 1400 1600m/z
0
50
100
0
50
100
0
50
100
Rela
tive
Abun
danc
e
0
50
100
742.89z=?
600.52z=5
773.47z=?712.99
z=?403.27
z=1 893.46z=?
1048.04z=2
533.96z=3
1221.57z=2
1443.76z=1
1371.72z=2
322.31z=?
743.06z=6
600.52z=5
828.94z=?403.27
z=1 729.38z=11
914.48z=12533.96
z=3985.52
z=?1061.55
z=41221.57
z=2 1356.73z=1
1443.76z=1
360.16z=?
257.16z=?
743.06z=6
828.94z=?600.52
z=5403.27
z=1552.96
z=3
732.39z=?
914.48z=12 985.52
z=?1061.80
z=4 1221.57z=2 1356.72
z=11443.76
z=1257.16
z=1360.16
z=?
462.28z=?
828.94z=2403.27
z=1 600.52z=5
742.89z=6
914.48z=?
502.33z=1
985.52z=7
1061.80z=4
690.37z=?
1221.57z=2 1356.72
z=11443.76
z=1257.16
z=1360.21
z=1
Rxn 9ms
Enolase ETD HD CS 63+ Rxn 3ms
Rxn 5ms
Rxn 7ms
240K, 1mtorr, Reagent AGC 7e5
AGC 1e6, 200 uscans
24
Enolase CS 63+, ETD HD Seq Coverage
5ms 3ms
7ms 9ms
25
Enolase Combined Sequence Coverage 56% Seq Coverage
26
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
670.35
909.78
969.771015.80
830.27
686.32
1170.16
859.29
803.75727.71 1072.60
1124.51
1339.70612.79577.27 1205.11473.27
645.331026.54
1287.73 1390.75 1487.81 1545.48
Enolase MS2 CID MS3 CID
MS2 CID m/z 898, MS3 m/z 870 CID
MS3 Precursor Sequence
27
Enolase MS2 CID MS3 ETD
S p S u s3 898 5000@c d35 00 8 0 000@etd5 00 [ 00 0000 000 0000]
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
868.47
828.94
979.641085.58
621.35
881.131119.731044.23
809.42 1204.72736.43 1320.70934.99
665.40502.33 1219.67 1438.99539.82 1491.83446.77 1567.36 1655.66
607.32
MS2 CID m/z 898, MS3 ETD m/z 870
MS3 Precursor Sequence
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DDA LC-MS Analysis: Bacterial Lysates
29
• LC: Dionex Ultimate 3000RSLCnano
• Flow rate: 12 ul/min
• Column: ProSwift Monolithic RP-4H 200 μm x 25cm
• Trap Column (optional): Prototype C4, 200
μm x 5mm
• Solvents: A: H2O, 0.1% FA B: AcN, 0.1% FA
• Gradient: 20% B to 60% B in 90min
Chromatography
30
• High / High Method
• Full MS @ 120000 resolution
• MS2 @ 120000 resolution
• Medium / High Method
• Full MS @ 15000 resolution
• MS2 @ 120000 resolution
Instrument Method Full MS
31
Instrument Method DDA
32
DDA Parameters
33
E. coli DDA Total Ion Chromatogram RT: 9.93 - 60.29
10 15 20 25 30 35 40 45 50 55 6Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
29.6325.28
34.2050.36
29.39 48.4238.21
48.1933.30 37.71 50.84
37.1922.96 41.2136.88 42.17
38.8120.98
20.75
44.22
51.5517.95 53.9154.33
17.63 56.1115.8710.28
25.3050.62
34.23 48.4422.86
29.42
51.1820.78
31.97
29.1543.0531.84
38.3834.45 39.6223.13 48.1242.7338.24
45.1336.6547.88
51.6519.95
18.21
Full Scan MS
CID MS/MS
120K, 2mtorr
34
Data Processing: ProSight PD
35
Protein Identifications
0
20
40
60
80
100
120
etd hcd cid ethcd combined
# Pr
otei
n G
roup
s
Fragmentation
S. sonnei lysate Precursor 120K
Precursor 15K
0
20
40
60
80
100
120
etd hcd cid ethcd combined
# Pr
otei
n G
roup
s
Fragmentation
E. coli lysate Precursor 120K
Precursor 15K
Higher number of proteins identified using High/High method
36
Identification of Large Proteins Greater number of large proteins (>30kDa) identified using Med/High
0
20
40
60
80
100
120
etd hcd cid ethcd combined
# Pr
otei
n G
roup
s
Fragmentation
S. sonnei lysate <30kDa, Hi/Hi
<30kDa, Med/Hi
>30kDa, Hi/Hi
>30kDa, Med/Hi
0
20
40
60
80
100
120
etd hcd cid ethcd combined
# Pr
otei
n G
roup
s
Fragmentation
E. coli lysate <30kDa, Hi/Hi
<30kDa, Med/Hi
>30kDa, Hi/Hi
>30kDa, Med/Hi
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High Flow LC-MS Analysis: Intact mAb
38 Proprietary & Confidential
Intact IgG: Seven Major Glycosylated Forms
39 Proprietary & Confidential
How Many Components Are In Each Peak?
•Covalent mods can include: SS red/ox, oxidation, deamidation, aa substitutions, Lys variants, sialated forms
•Non-covalent mods can include: metal adducts, phosphate adducts
40 Proprietary & Confidential
Commonly Observed CS Distribution…….s????
2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
Re
lative
Ab
un
da
nce
3537.813158.13
3710.53
3623.99
3092.333029.23
3154.73
2968.673805.58
3905.672910.54
2971.95
3910.22
4011.472854.532800.77
4122.852748.874240.642698.94
4245.002604.37 4365.30 4638.202500.85 4374.79
2326.35
4632.77
49+ 41+
41 Proprietary & Confidential
Evidence For Multiple Overlapping Distributions
_ _ p _ _ _ _ _ _ _ _ _ T: FTMS + p NSI sid=40.00 Full ms [1000.0000-5000.0000]
3016 3018 3020 3022 3024 3026 3028 3030 3032 3034 3036 3038 3040 3042 3044 3046 3048 3050 3052m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
3029.23N=23405.26
3032.62N=23432.303025.99
N=23379.43
3036.02N=23459.39
3039.32N=23485.65
3042.74N=23512.97
3021.79N=23346.01
3045.62N=23535.87
w aters_mAb_2x50perACN_FT_15k_500nl_min_SID40_S60_400C_8mtorr_2 #8 RT: 0.16 AV: 1 NL: 2.28E6T: FTMS + p NSI sid=40.00 Full ms [1000.0000-5000.0000]
3606 3608 3610 3612 3614 3616 3618 3620 3622 3624 3626 3628 3630 3632 3634 3636 3638 3640 3642 3644 3646 3648m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
3620.06N=28113.51
3623.99N=28144.78
3616.19N=28082.65
3628.07N=28177.29
3632.05N=28209.04
3636.25N=28242.463611.15
N=28042.45
148877.7
148873.05
148382.91
148381.2
49+
41+ Varied mass shifts
1-2 Da shifts are not distinguishable from centroiding errors
Larger shifts are indicative of varied abundances of overlapping signals
42 Proprietary & Confidential
41+: Higher Resolution Reveals Multiple Isoforms
15K
30K
60K
Unique to Orbitrap Analyzer
G0F-G0F G0F-G1F
G1F-G1F
G1F-G2F G2F-G2F
43 Proprietary & Confidential
• Sigma mAb (p/n MSQC4)
• Sample Load: 1ug
• LC : Dionex Ultimate 3000 • Column: MabPac RP 2.1 x 10 cm
Chromatography
LC Gradient
44 Proprietary & Confidential
Instrument Method Full MS
45 Proprietary & Confidential
Divert Valve Set-up
46 Proprietary & Confidential
Sigma mAb LCMS: 15K Full MS
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
6.13
6.32
0.06 6.730.49 0.66 6.961.00 1.26 7.30 7.56 7.96 8.16 8.42 8.82 9.08 9.345.85 9.55 11.8910.10 10.87 11.6310.384.604.032.38 3.88 4.403.222.10 5.275.041.72 2.93 5.59
NL:2.93E8TIC MS Sigma_mab_run1
Sigma_mab_run1 #212-225 RT: 6.08-6.24 AV: 14 NL: 3.05E6T: FTMS + p ESI sid=50.00 Full ms [2000.0000-4000.0000]
2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600 3700 3800 3900 4000m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
2824.532879.90
2771.30 2937.48
2997.42
2719.98
3059.822670.55
2622.94 3124.90
3192.772576.832529.68
3263.722489.653415.423334.312331.55
2407.983500.772259.27 3586.082159.98 3671.69 3770.012096.08 3864.98 3966.98
15K, Standard Pressure Mode
AGC 2e5, 10 uscans
47 Proprietary & Confidential
BioPharma Finder Deconvolution Results
G0F/G1F
G0F/G0F G1F/G1F
G1F/G2F G0/G0F
The world leader in serving science Proprietary & Confidential
Targeted LC-MS Analysis: Reduced mAb
49 Proprietary & Confidential
• Sample Load: 3ug
• Sigma mAb (p/n MSQC4) Reduced deglycosylated
• LC : Dionex Ultimate 3000 (High Flow)
• Column: MabPac RP 2.1 x 10 cm
Chromatography:Flow Rate 300 ul/min
LC Gradient
50 Proprietary & Confidential
Divert Valve Set-up
51 Proprietary & Confidential
RT: 0.00 - 10.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
3.793.82
3.88
3.58
4.004.06
4.17 4.30 4.513.25 4.74 9.345.11 5.36 5.61 8.76 8.925.80 8.666.11 6.29 6.52 7.616.79 7.05 7.48 7.75 7.972.912.28 2.792.151.50 1.941.31
600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700m/z
0
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e Ab
unda
nce
998.48956.811093.421043.92918.60 1147.99
1275.551208.37 1434.841350.40883.33 1530.40 1639.62
850.66820.35
792.12765.70 1583.231481.141311.921156.21 1391.331099.28641.82 1083.221217.25
1541.21
718.03 1674.281088.99
1166.68 1195.19 1256.36 1360.941020.93 1324.22960.97907.651139.60 1399.841000.19
1440.99875.281484.61845.11803.62
1531.00 1580.34778.16 1632.90742.79
1689.08700.37 1605.591554.89662.72
Deglycosylated Reduced mAb
Heavy Chain
Light Chain
Heavy Chain
Light Chain
15K, 8mtorr, AGC 2e5
52 Proprietary & Confidential
Heavy Chain: ReSpect Sliding Window Deconvolution
Average Mass
53 Proprietary & Confidential
Light Chain Isotopic Resolution
0 00 0 03
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 1Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
3.73
3.59
3.27 4.70 5.07 9.95.27 9.775.59 5.77 6.03 9.426.26 6.55 8.958.696.72 7.287.16 7.43 7.72 8.457.872.952.542.281.901.651.12 1.43
deglyco_120K_run02 #77-81 RT: 3.53-3.65 AV: 5 NL: 6.66E7T: FTMS + p ESI Full ms [600.0000-1700.0000]
600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
998.41
956.931043.83
1093.44918.69
1148.07
883.361208.49
1275.52850.68
1350.551434.96
820.41 1530.49
1639.59792.12 961.97
765.75 1122.55 1172.71641.82 1236.711079.99740.88 1481.041324.04
1283.64
1583.22684.68
1622.33
1393.62 16
1522.631460.18 1545.721370.06 1659.0
CS 22+ 120K, 2mtorr, AGC 2e5
54 Proprietary & Confidential
Light Chain: Xtract Deconvolution deglyco_120K_run02_XT_00001_M_ #2 RT: 2.00 AV: 1 NL: 2.85E8T: FTMS + p ESI Full ms [600.00-1700.00]
22905 22910 22915 22920 22925 22930 22935 22940 22945 22950 22955 22960 22965m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
22928.1810
22910.258722950.1172
22955.1962
Monoisotopic Mass
55 Proprietary & Confidential
400 600 800 1000 1200 1400 1600 1800 2000m/z
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
1155.61
1245.34
1082.531298.83
996.471660.45
1381.32 1581.80949.45 1454.97
882.48534.79 791.40 1703.131502.97578.31 1765.87
1155.61
1245.081000.74
1298.831334.15
1082.531660.11
1623.541381.651601.18 1703.13
1455.22882.48928.02791.40 1765.87534.79 1996.981897.93
NL: 1.10E7deglyco_etd15_iw400_run07_lc-qb#1 RT: 3.51 AV: 1 T: FTMS + p ESI Full ms2 [email protected] [400.00-2000.00]
NL: 3.97E5deglyco_etd15_run04_lc-qb#1 RT: 3.51 AV: 1 T: FTMS + p ESI Full ms2 [email protected] [400.00-2000.00]
Light Chain: Wide Isolation Window 41% Seq Cov
33% Seq Cov
ETD 15ms
ETD 15ms
ETD MS/MS
56 Proprietary & Confidential
Light Chain: ETD & EThcD 65% Sequence Coverage
• EThcD: 15ms, 25ms • ETD iw 400: 7ms, 15ms
57 Proprietary & Confidential
Heavy Chain: ETD Fragmentation
400 600 800 1000 1200 1400 1600 1800 2000m/z
0
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e Ab
unda
nce
0
20
40
60
80
1001194.29 1492.871327.10957.79
1152.601029.531363.70
1069.551705.841239.63927.48 1461.86
1514.77834.43
1603.80772.43708.361786.91 1916.95577.35487.29
1492.86957.79
1705.841363.701204.621286.66 1403.721069.55
976.80
1605.821549.79
927.47772.43 1752.88708.36 1990.1885.74 1847.94
577.35487.29
957.79
1705.991069.551203.35 1363.70
1990.11436.18 1603.801297.66976.80 1520.771166.58 1787.92
1872.95772.43 916.47708.36586.36
487.29
Rxn 10ms
Rxn 15ms
Rxn 25ms
120K, 2mtorr, AGC 2e5 Isolation window 400 m/z
58 Proprietary & Confidential
400 500 600 700 800 900 1000 1100 1200 1300 1400m/z
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
1044.53
925.65 1132.39
1196.75
1112.18 1249.27970.871026.07
551.32 1358.47888.43452.25 1460.97846.92496.26 569.33 1260.65768.54712.35 1435.70
960.03
925.65 1044.53 1132.39
1168.98970.87 1055.94 1196.75551.32452.25 1071.09 1358.47888.63 1460.97827.91768.54496.26 712.35600.30 1260.65
Heavy Chain:CID, HCD Fragmentation
CID 35
HCD 10
120K, 2mtorr, AGC 2e5 Isolation window 400 m/z
59 Proprietary & Confidential
Combined Seq Cov: ETD, HCD, CID 25% Sequence Coverage
• ETD 10ms, 15ms, 25ms • CID 35%, HCD 10%
60 Proprietary & Confidential
p [ ]
600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rela
tive
Abun
danc
e
891.16907.65
942.56961.06
980.18859.93
1089.00844.83
1139.58830.51
1166.71
816.95
999.78
803.051195.10
1256.41
789.841360.94
1324.27
765.57 1441.09 1484.821580.76741.06 1753.591635.81680.82
Heavy Chain Isotopic Resolution: LC-MS
942.35 942.40 942.45 942.50 942.55 942.60 942.65 942.70 942.75m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
942.56942.54942.50
942.58 942.61942.48
942.42 942.52942.46
942.60942.39 942.67942.65942.44
942.69942.63942.73
942.40
942.36
942.71 942.75
Monoisotopic Mass 48929.58 Da
240K, 1mtorr, AGC 2e5 8ug sample on column, 5uscans, spectrum averaged over LC-MS peak
61 Proprietary & Confidential
• Sample load: 500ng
• Sigma mAb (p/n MSQC4) Reduced deglycosylated
• LC : Dionex Ultimate 3000
(Microflow)
• Column: ProSwift Monolithic RP-4H 200um x 25cm
Chromatography: Flow Rate 15ul/min
LC Gradient
62 Proprietary & Confidential
Light Chain: Top-down Fragmentation HCD 10: 22% Seq Cov CID 30: 22% Seq Cov
ETD 15ms: 46% Seq Cov ETD 25ms: 44% Seq Cov
63 Proprietary & Confidential
Light Chain Combined Coverage 70% Sequence Coverage
• ETD 15ms, 25ms • HCD 10 • CID 30
64 Proprietary & Confidential
400 600 800 1000 1200 1400 1600 1800 2000m/z
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
1194.29
1152.601326.99
1236.631033.53957.79
1492.61
896.47 1363.70772.43 1461.86708.36 1705.84
1544.80577.35864.44
486.23 1830.94 1960.531326.88
1194.29
1152.60 1363.70
1286.661492.61957.79
1403.72
1129.58
1002.81
772.43 1705.841524.78708.36 900.49645.35
1603.81877.46577.35
520.321738.88 1872.23 1985.8
Heavy Chain: ETD HD Fragmentation (CS 52+)
ETD Rxn 15ms
ETD Rxn 25ms
120K, 2mtorr
65 Proprietary & Confidential
Heavy Chain HCD Fragmentation (CS 52+)
500 600 700 800 900 1000 1100 1200 1300 1400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
925.66
551.32 1132.39970.77
569.331196.75
1025.93452.25
771.551070.95828.25
1249.271163.24809.23698.381083.56
997.51
903.46600.30470.26535.28 728.36
856.60
680.36423.23
1218.59
623.34 1270.781358.471317.23
1370.61
HCD 10 120K, 2mtorr
66 Proprietary & Confidential
Combined Sequence Coverage 31% Sequence Coverage
• ETD 15ms, 25ms • HCD 10%