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Intact and Top-Down Protein Analysis Guide: Orbitrap Fusion Lumos Seema Sharma October 2016

2

Orbitrap Fusion Lumos

Presenter

Presentation Notes

The new technology enhancements on Orbitrap Lumos as compared to Orbitrap Fusion are highlighted in blue. 1) The brighter ion source consists of the HCTT and EDIF, both are from the TSQ Quantiva product line. They increase the signal 2-5X. 2) The Advanced Active Ion Beam Guide (AABG) is from Q Exactive product line and keeps the Q1 clean(er). 3) A segmented quadrupole powered by the Advanced Quadrupole Technology is also from the Q Exactive product line. It allows for better transmission at higher resolutions, symmetric transmission across the isolation window, and improved transmission for m/z >1000. 4) The Advanced Vacuum Technology allows to manage gas flow in the IRM/Ion Trap and UHV regions, such that the higher vacuum is obtained between the C-trap and Orbitrap. The Lumos instrument inherits the winning tribrid architecture from the Orbitrap Fusion and all the proven hardware elements, such as ultra-high field Orbitrap, Ion Routing multipole, linear ion trap and Easy IC/ETD source. Note that the way we produce anions for ETD and IC has not changed. The ETD HD is an improved way to conduct ETD experiments in the linear ion trap, utilizing 3X more precursor ions. This results in much improved detection limits and dynamic range of the ETD reaction.

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



MS Protein <40kDa

MS/MS

MS Protein

40-50 kDa

Presenter

Presentation Notes

The Orbitrap Resolution and IRM pressure settings are dependent on the application of interest. Examples below: Orbitrap resolution 15000 and IRM pressure 8 mTorr (standard pressure mode) for average mass analysis of large proteins (>50 kDa) such as intact mAb. For isotopic resolution of intact proteins (mol. Wt < 40kDa), Orbitrap resolution 120K at IRM 2 mTorr (intact protein mode) is sufficient. However 240K resolution and 1mtorr IRM pressure is needed for proteins in the mass range 40-50 kDa. Often SIM and/or heavy averaging is required to build up signal. For Top-down fragmentation of intact proteins you can use Orbitrap resolution 120K at IRM 2 mTorr (intact protein mode). Note: If using 240K go down to 1 mTorr pressure for intact protein mode.

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

Presenter

Presentation Notes

Check the standard calibrations before proceeding to extended calibrations for intact protein analysis

5

Extended Calibrations MS

Protein >50 kDa

Normal Mass Range No Extended

Calibrations Required

High Mass Range Positive Extended: High Mass Range

CalMix & Enfuvirtide

Presenter

Presentation Notes

For proteins larger than 50kDa, no extended calibrations are needed if the spectra will be acquired in normal mass range. For proteins larger than 50kDa, instrument needs to be calibrated with calmix and Enfuvirtide prior to acquiring the spectra in high mass range.

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


Presenter

Presentation Notes

120K resolution at 2 mtorr is sufficient for proteins <40kDa, however 240K resolution and 1mtorr IRM pressure is needed for proteins in the mass range 40-50 kDa. For isotopic resolution of intact proteins, the mass spec needs to be calibrated in intact protein mode at the given IPM pressure value.

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).

Presenter

Presentation Notes

The IRM pressure for intact protein mode can be changed in diagnostics as shown. For example set ‘IRM_Pressure’ Parameter value 0.001 for resolution of 40-50 kDa intact proteins. Note that the instrument can only be calibrated for one IRM pressure value for standard protein mode plus one IRM pressure value for intact protein mode at a time.

8

Extended Calibrations Contd.

MS/MS Set IRM Pressure @

2mtorr


& ETD Extended:

Intact Protein Mode

Presenter

Presentation Notes

For top-down MS/MS characterization of intact proteins the extended calibrations needed are source to IRM, IRM to C-trap, IRM to Ion trap and the reagent ion source to IRM optics calibration in 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


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

Presenter

Presentation Notes

Data acquired for proteins at 120K or 240K resolution is isotopically resolved and can be processed using Xtract or THRASH algorithms to provide 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

Presenter

Presentation Notes

List of monoisotopic masses obtained from Xtract deconvolution was processed using ProSight Lite with fragment mass measurement tolerance 10ppm.

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

Presenter

Presentation Notes


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

Presenter

Presentation Notes



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

Presenter

Presentation Notes

Data acquired for large proteins at 15000 resolution is charge state resolved and can be processed using ReSpect deconvolution to provide Average Mass.

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

Presenter

Presentation Notes


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

Presenter

Presentation Notes


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

Presenter

Presentation Notes

ETD HD sequence coverage for Enolase CS 52+ obtained at different reaction times

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

Presenter

Presentation Notes

ETD HD sequence coverage for Enolase CS 63+ obtained at different reaction times

25

Enolase Combined Sequence Coverage 56% Seq Coverage

Presenter

Presentation Notes

Combined sequence coverage obtained from combining results from HCD, CID and ETD HD (CS 52+ and CS 63+ at different reaction times).

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


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


# Pr

otei

n G

roup

s

Fragmentation

E. coli lysate Precursor 120K

Precursor 15K

Higher number of proteins identified using High/High method

Presenter

Presentation Notes

For both bacterial lysates the High/High method with Full MS @ 120000 and MS2 @ 120000 gave higher number of protein identifications

36

Identification of Large Proteins Greater number of large proteins (>30kDa) identified using Med/High

0

20

40

60

80

100

120


# 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


# Pr

otei

n G

roup

s

Fragmentation

E. coli lysate <30kDa, Hi/Hi

<30kDa, Med/Hi

>30kDa, Hi/Hi

>30kDa, Med/Hi

Presenter

Presentation Notes

For both lysates, higher numbers of proteins (>30kDa) were identified using the Med/High method (Full MS @ 15000 resolution, MS2 @ 120000 resolution)

The world leader in serving science Proprietary & Confidential

High Flow LC-MS Analysis: Intact mAb

38 Proprietary & Confidential

Intact IgG: Seven Major Glycosylated Forms


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


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+


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


41+: Higher Resolution Reveals Multiple Isoforms

15K

30K

60K

Unique to Orbitrap Analyzer

G0F-G0F G0F-G1F

G1F-G1F

G1F-G2F G2F-G2F


• Sigma mAb (p/n MSQC4)

• Sample Load: 1ug

• LC : Dionex Ultimate 3000 • Column: MabPac RP 2.1 x 10 cm

Chromatography

LC Gradient


Instrument Method Full MS


Divert Valve Set-up


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


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


• 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


Divert Valve Set-up


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


Heavy Chain: ReSpect Sliding Window Deconvolution

Average Mass


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


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


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


Light Chain: ETD & EThcD 65% Sequence Coverage

• EThcD: 15ms, 25ms • ETD iw 400: 7ms, 15ms


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


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


Combined Seq Cov: ETD, HCD, CID 25% Sequence Coverage

• ETD 10ms, 15ms, 25ms • CID 35%, HCD 10%


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


• 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


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


Light Chain Combined Coverage 70% Sequence Coverage

• ETD 15ms, 25ms • HCD 10 • CID 30


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


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


Combined Sequence Coverage 31% Sequence Coverage

• ETD 15ms, 25ms • HCD 10%

Documents

Intact and Top-Down Protein Analysis Guide: …njms.rutgers.edu/proweb/documents/3IntactandTop-Down...The world leader in serving science Intact and Top-Down Protein Analysis Guide: