Improved Analysis of Biopharmaceutical Samples Using an MS-only Orbitrap Mass SpectrometerOlaf Scheibner; Eugen Damoc; Eduard Denisov; Jan-Peter Hauschild; Oliver Lange; Frank Czemper; Alexander Kholomeev; Alexander Makarov; Andreas Wieghaus; Maciej Bromirski

Thermo Fisher Scientific (Bremen) GmbH, Bremen, GERMANY

2 Improved Analysis of Biopharmaceutical Samples Using an MS-only Orbitrap Mass Sspectrometer

Humira_FullMS #1 RT: 4.84 AV: 1 NL: 3.92E5T: FTMS + p ESI Full ms [1000.00-4000.00]

2000 2500 3000 3500 4000m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

2794.982743.25

2904.542693.38

2962.592645.32

2598.93 3023.04

2554.14 3085.99

2510.87 3151.652469.04 3220.13

2428.593291.67

2389.433366.47

2314.79 3444.762244.68 3526.73

2147.123703.011975.45 3897.98

hela_lysc_wem_3e6_1e5_mz350_2000_fm150hcd_1 #19668 RT: 61.54 AV: 1 NL: 2.74E6T: FTMS + p NSI Full ms [350.00-2000.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

445.11856R=52407

z=1

536.16394R=47707

z=1 674.55774R=39206

z=5

1056.53552R=31307

z=2

950.84613R=32206

z=3825.46289R=36104

z=31191.24426

R=28006z=3 1826.72253

R=20702z=?

1577.42529R=19902

z=?

1478.22644R=20002

z=?

1322.64648R=22204

z=2

1972.63916R=16902

z=?

1694.47510R=18700

z=?

Improved Analysis of Biopharmaceutical Samples Using an MS-only Orbitrap Mass Sspectrometer Olaf Scheibner; Eugen Damoc; Eduard Denisov; Jan-Peter Hauschild; Oliver Lange; Frank Czemper; Alexander Kholomeev; Alexander Makarov; Andreas Wieghaus; Maciej BromirskiThermo Fisher Scientific (Bremen) GmbH, Bremen, GERMANY

Conclusion The described hardware changes improve the measurement performance for

large molecules significantly. This results in high abundant Full-MS spectra, e.g. of the Humira antibody with a mass accuracy better than 3 ppm for the deconvoluted spectrum. Using HCD fragmentation 138 matching fragments could be identified from a single experiment.

The C-Trap charge detector improves the automatic gain control for situations where the prescan AGC gets inaccurate. This is demonstrated for the case of partially digested proteins in a HeLa sample run.

AcknowledgementsWe would like to thank the research group of Professor Neil Kelleher from the Northwestern University (IL, USA) for confirming the sequence data using ProSight PC.

The research funding of the 7th European Framework Program is appreciated (Health-F4-2008-201648/PROSPECTS).

OverviewPurpose: Improve the performance of bench-top Orbitrap™ mass spectrometers for large molecules and complex samples.

Methods: The hardware of the Orbitrap assembly was improved to record the most abundant first section of the ion signal. The performance for complex samples was further improved by using an independent C-Trap charge detector.

Results: Measurements of the intact Humira™ antibody shows the performance increase after the optimization of the Orbitrap assembly. The improved behavior for complex samples is demonstrated using 60 minute gradient sample runs from HeLa cells.

IntroductionThis work is dedicated to improve capabilities of an MS-only bench-top Orbitrap mass spectrometer (Thermo Scientific Exactive Plus) for the analysis of very complex mixtures and biopharmaceutical samples.

Intact proteins create fast decaying beat patterns in Fourier-Transform (FT) image current detection systems. In order to have the ability to detect the most abundant signal from the very first beat, modifications to the instrument are required.

When dealing with very complex samples, a dedicated C-Trap charge detection (CTCD) system is shown to improve the accuracy of the prescan-based automated gain control (AGC). Together with the advanced signal processing, the hardware improvements show a significant improvement for several applications.

Large MoleculesImage Current Detection

Because image current detection is an interference detection method used in Fourier-Transform mass spectrometers (FTMS), isotopes within the instrument produce beat patterns. For larger molecules these beat patterns become visible in the time-domain transient signal as sketched in Figure 1. The larger (and cleaner) the molecule, the shorter the beats. For molecules comprising several tens of kilodaltons, the first beat is only visible for the first few milliseconds and the time between the beats can be more than a second. For bench-top systems offering only transient lengths up to 0.5 seconds, the complete detection of the very first beat is crucial.

C-Trap Charge Detection (CTCD)

To improve the analytical robustness of the AGC control scheme a C-Trap charge detection is used to monitor the AGC results every 5 to 10 seconds. During LC runs, the CTCD operation takes place in parallel to Orbitrap acquisition, see Figure 7: While the analytical scan is still being acquired, a few C-Trap injections are ejected to the collector to measure the C-Trap charge. From this, the total ion current (TIC) is calculated and compared to the TIC observed by the short transient AGC-scan. If necessary, the injection time is regulated downward to prevent the C-Trap from overfilling.

Humira is a registered trademark of Abbott Laboratories Inc. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

FIGURE 2. Typical transients: a) Former design (Exactive™), b) Improved design (Q Exactive™, Exactive ™ Plus)

FIGURE 1. Transient sketch of a decaying beat pattern resulting from a FTMS. FIGURE 3. Varied detect delay for 8 ms transient (development mode, 10x10µScans, 10 ms fixed inject time). The first five milliseconds show a significant signal contribution.

Technical Improvements

To inject ions into the Orbitrap analyzer, a voltage pulse of several kilovolts is applied to the central electrode. This voltage pulse propagates via both detection electrodes to the sensitive pre-amplifier, which creates a saturation condition of this pre-amplifier for several milliseconds in the former setup. Figure 2a) shows the resulting time-domain transient signal. Here the transient dwell time is about 5.5 milliseconds, so the detection is started with a detect delay of approximately 6.5 ms.

To avoid this effect, several countermeasures have been implemented: the Orbitrap assembly was made completely symmetrical and the dielectric materials were optimized. Additionally, the pre-amplifier circuit was changed to allow a faster recovery from the central electrode voltage pulse.

Having these changes in effect, the transient dwell time reduced to <0.25 milliseconds, as shown in Figure 2b).

Measurements

The overall performance was tested using Humira samples with the hardware changes implemented. A single spectrum was taken at a resolution setting of 17500 at m/z 200 to use the shortest available transient length; this will cover the entire first transient beat. This spectrum , shown in Figure 4a), was processed using Thermo Scientific ProMass Deconvolution, the result is shown in Figure 4b). The mass accuracy stays below 3 ppm and the different glycoforms are represented.

Figure 5a) shows the HCD fragment spectrum of this sample averaged over four minutes of the elution profile. A dense and well distributed spectrum is visible with the masses extending beyond 23 kDa. By processing this spectrum with Thermo Scientific ProSight PC, the molecule is sequenced. 138 matching fragments were found, giving a good sequence coverage for this single experiment, see Figure 5b).

Transient usedDetect delay

Transient used

Experimental Setup

• 5 µg Humira monoclonal antibody (mAb) (148 kDa, Abbott Lab.Inc.) loaded on a Thermo Scientific BioBasic-4 column of 1 mm i.d. and 100 mm length packed with 5 µm particle size

• 15 min run time with linear gradient from 20 to 80% of acetonitrile with 0.1% formic acid at a 150 μL/min flow rate Resulted in a sample elution time of ~5 min

2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000m/z

0

50

100

0

50

100

0

50

100

Rel

ativ

e A

bund

ance 0

50

100

0

50

1002795.144

N=14890.51

N=13217.72

N=11252.00

N=12931.95

NL: 1.58E7Humira_ResDependency_DetDelay#1224-1233 RT: 5.37-5.40 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1242-1251 RT: 5.45-5.49 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1261-1270 RT: 5.55-5.60 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1284-1293 RT: 5.69-5.76 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1303-1312 RT: 5.85-5.93 AV: 10 T: FTMS + p ESI Full ms

N=12683.94

0 ms

5 ms

10 ms

20 ms

30 ms

Detect delay

FIGURE 4. a) Humira (148kDa) spectrum, b) Deconvoluted spectrum using ProMass Deconvolution

a)

b)

Evaluation of signal intensity

To evaluate the signal intensity contained in the time-domain transient, a very short transient portion is used while varying the detect delay, see Figure 3. It is visible that the first five milliseconds contain a significant share of the total ion signal resulting in a better signal transmission for the improved design.

Hea

vy c

hain

Ligh

t cha

in

humira_IgG_std_HCD_pressure_AIF 11/17/2011 12:26:33 PM

humira_IgG_std_HCD_pressure_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.38E4T: FTMS + p ESI Full ms2 3100.00@hcd80.00 [290.00-4000.00]

600 800 1000 1200 1400 1600 1800 2000 2200 2400m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

1381.71984R=47623

z=91537.13718

R=46435z=8698.37761

R=76606z=1 948.45108

R=66140z=1

1756.58431R=45519

z=7785.41046R=74568

z=1

1243.64669R=48076

z=101049.53692R=63674

z=1

1821.90404R=45005

z=72125.90246

R=44833z=11

2338.29470R=46184

z=10

1990.99189R=44892

z=6

humira_IgG_std_HCD_pressure_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.25E3T: FTMS + p ESI Full ms2 3100.00@hcd80.00 [290.00-4000.00]

2334 2336 2338 2340 2342 2344 2346 2348 2350m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bund

anc

e

2341.79346R=46318

z=?

2344.98903R=45016

z=?

2338.99486R=46035

z=10

2336.69116R=41660

z=?2350.0114

R=43001z=?

2344.18644R=44816

z=?

2345.69079R=40774

z=?

FIGURE 5. a) Single experiment HCD fragmentation spectrum of Humira mAb. b) 138 matching fragments identified by ProSight PC.

Complex samplesAutomatic gain control (AGC)

To fully utilize the analytical performance and space charge capacity of the Orbitrap system, the number of ions injected to the C-Trap needs to be controlled. The measurement of the ion current is either done via a dedicated AGC-prescan, which records a very short transient, or by using the Scan-to-Scan AGC which uses the first short section of the previous analytical scan. The resulting ion current from this short transient acquisition is used to calculate the injection time for the next analytical scan. In some rare cases the number of ions can be underestimated because of the lower resolution and the lower signal response of this short transient acquisition. This is especially true for multiply charged ions and dense peaks below the noise threshold. To demonstrate this effect the AGC improvement described below was switched off and the maximum inject time was set untypically high. Figure 6 shows a 60 minute gradient LC chromatogram of a HeLa sample containing partially digested proteins and including the column wash stage (top). Nearing the end of the run at retention times between 62 and 72 minutes, the AGC becomes inaccurate. A single spectrum from this section shows multiply charged species that won’t be resolved in the short AGC-prescan and therefore will be underestimated (middle). The second spectrum shows the average of three minutes (bottom). Here, partially digested proteins become visible showing ions that also cannot be seen by the short acquisition of the AGC-prescan leading to further underestimation of the ion current. In this case, the inject time for the analytical scan will be too long causing overfilling of the C-Trap. A valid previous workaround was to reduce the AGC target and to set the maximum inject time carefully to a dedicated level for each sample class.

FIGURE 6. Chromatogram and mass spectra of a HeLa sample run with a 60 min. gradient. During the colum wash the AGC gets inaccurate. Both, multiply charged and peaks below the noise threshold appear in the corresponding retention time range.

FIGURE 8. With the CTCD active, the chromatogram does not show any C-Trap overfilling. The mass spectrum contains now analyte peaks of interest.

RT: 8.71 - 73.14

10 15 20 25 30 35 40 45 50 55 60 65 70Time (min)

0

1

2

3

4

Rel

ative

Abu

ndan

ce

53.96

62.1459.3670.3658.94

70.3354.3866.9762.28

64.10

NL: 1.53E9Base Peak F: ms MS hela_lysc_wem_3e6_1e5_mz350_2000_fm150hcd_1

FIGURE 7. Exactive Plus layout with C-Trap charge detector active while the analytical scan is acquired in the Orbitrap.

Measurement Results

Using the CTCD, the HeLa run is repeated and its chromatogram is shown in Figure 8. To emphasize the effect by getting closer to the upper C-Trap space charge limit, the AGC target was set to 3e6 for this experiment. Now the AGC stays accurate even during the column wash stage. The spectrum now shows several analyte peaks which can be used for further confirmation.

a)

b)

HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1 #19027-19394 RT: 65.00-68.00 AV: 293 NL: 4.71E3T: FTMS + p NSI Full ms [350.00-2000.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

946.55941R=33317

z=3

1542.77453R=26371

z=2

912.85357R=34397

z=31976.16243

R=14206z=1

1481.74376R=16917

z=1

1866.41480R=24006

z=6

1693.91682R=20607

z=?

1034.52732R=31884

z=3713.40758R=37751

z=?1318.66850

R=19466z=1

615.96752R=32939

z=1

1101.56294R=30690

z=5

445.12314R=51912

z=?

HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1 #18610 RT: 61.67 AV: 1 NL: 4.62E4T: FTMS + p NSI Full ms [350.00-2000.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

1350.11121R=25406

z=5921.32239R=31906

z=6 1125.25989R=26906

z=6

1381.47974R=24406

z=4

1549.78955R=21506

z=61977.32275

R=19600z=?

998.30304R=27506

z=4

1294.64734R=23002

z=?

722.46362R=33202

z=?

1697.28662R=14800

z=?

1824.30591R=16600

z=?

827.94812R=33700

z=?636.95868R=41204

z=3497.00040R=36500

z=?

RT: 8.71 - 73.14

10 15 20 25 30 35 40 45 50 55 60 65 70Time (min)

0

1

2

3

4

Rel

ative

Abu

ndan

ce

50.9354.33

57.65

57.6955.2759.56

60.3060.86 71.6864.90 67.76

NL: 1.82E9Base Peak F: ms MS HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1

a) b)

humira_IgG_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.38E4

humira_IgG_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.38E4

3Thermo Scientific Poster Note • PN63595_E 06/12S

Humira_FullMS #1 RT: 4.84 AV: 1 NL: 3.92E5T: FTMS + p ESI Full ms [1000.00-4000.00]

2000 2500 3000 3500 4000m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

2794.982743.25

2904.542693.38

2962.592645.32

2598.93 3023.04

2554.14 3085.99

2510.87 3151.652469.04 3220.13

2428.593291.67

2389.433366.47

2314.79 3444.762244.68 3526.73

2147.123703.011975.45 3897.98

hela_lysc_wem_3e6_1e5_mz350_2000_fm150hcd_1 #19668 RT: 61.54 AV: 1 NL: 2.74E6T: FTMS + p NSI Full ms [350.00-2000.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

445.11856R=52407

z=1

536.16394R=47707

z=1 674.55774R=39206

z=5

1056.53552R=31307

z=2

950.84613R=32206

z=3825.46289R=36104

z=31191.24426

R=28006z=3 1826.72253

R=20702z=?

1577.42529R=19902

z=?

1478.22644R=20002

z=?

1322.64648R=22204

z=2

1972.63916R=16902

z=?

1694.47510R=18700

z=?

Improved Analysis of Biopharmaceutical Samples Using an MS-only Orbitrap Mass Sspectrometer Olaf Scheibner; Eugen Damoc; Eduard Denisov; Jan-Peter Hauschild; Oliver Lange; Frank Czemper; Alexander Kholomeev; Alexander Makarov; Andreas Wieghaus; Maciej BromirskiThermo Fisher Scientific (Bremen) GmbH, Bremen, GERMANY

Conclusion The described hardware changes improve the measurement performance for

large molecules significantly. This results in high abundant Full-MS spectra, e.g. of the Humira antibody with a mass accuracy better than 3 ppm for the deconvoluted spectrum. Using HCD fragmentation 138 matching fragments could be identified from a single experiment.

The C-Trap charge detector improves the automatic gain control for situations where the prescan AGC gets inaccurate. This is demonstrated for the case of partially digested proteins in a HeLa sample run.

AcknowledgementsWe would like to thank the research group of Professor Neil Kelleher from the Northwestern University (IL, USA) for confirming the sequence data using ProSight PC.

The research funding of the 7th European Framework Program is appreciated (Health-F4-2008-201648/PROSPECTS).

OverviewPurpose: Improve the performance of bench-top Orbitrap™ mass spectrometers for large molecules and complex samples.

Methods: The hardware of the Orbitrap assembly was improved to record the most abundant first section of the ion signal. The performance for complex samples was further improved by using an independent C-Trap charge detector.

Results: Measurements of the intact Humira™ antibody shows the performance increase after the optimization of the Orbitrap assembly. The improved behavior for complex samples is demonstrated using 60 minute gradient sample runs from HeLa cells.

IntroductionThis work is dedicated to improve capabilities of an MS-only bench-top Orbitrap mass spectrometer (Thermo Scientific Exactive Plus) for the analysis of very complex mixtures and biopharmaceutical samples.

Intact proteins create fast decaying beat patterns in Fourier-Transform (FT) image current detection systems. In order to have the ability to detect the most abundant signal from the very first beat, modifications to the instrument are required.

When dealing with very complex samples, a dedicated C-Trap charge detection (CTCD) system is shown to improve the accuracy of the prescan-based automated gain control (AGC). Together with the advanced signal processing, the hardware improvements show a significant improvement for several applications.

Large MoleculesImage Current Detection

Because image current detection is an interference detection method used in Fourier-Transform mass spectrometers (FTMS), isotopes within the instrument produce beat patterns. For larger molecules these beat patterns become visible in the time-domain transient signal as sketched in Figure 1. The larger (and cleaner) the molecule, the shorter the beats. For molecules comprising several tens of kilodaltons, the first beat is only visible for the first few milliseconds and the time between the beats can be more than a second. For bench-top systems offering only transient lengths up to 0.5 seconds, the complete detection of the very first beat is crucial.

C-Trap Charge Detection (CTCD)

To improve the analytical robustness of the AGC control scheme a C-Trap charge detection is used to monitor the AGC results every 5 to 10 seconds. During LC runs, the CTCD operation takes place in parallel to Orbitrap acquisition, see Figure 7: While the analytical scan is still being acquired, a few C-Trap injections are ejected to the collector to measure the C-Trap charge. From this, the total ion current (TIC) is calculated and compared to the TIC observed by the short transient AGC-scan. If necessary, the injection time is regulated downward to prevent the C-Trap from overfilling.

Humira is a registered trademark of Abbott Laboratories Inc. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

FIGURE 2. Typical transients: a) Former design (Exactive™), b) Improved design (Q Exactive™, Exactive ™ Plus)

FIGURE 1. Transient sketch of a decaying beat pattern resulting from a FTMS. FIGURE 3. Varied detect delay for 8 ms transient (development mode, 10x10µScans, 10 ms fixed inject time). The first five milliseconds show a significant signal contribution.

Technical Improvements

To inject ions into the Orbitrap analyzer, a voltage pulse of several kilovolts is applied to the central electrode. This voltage pulse propagates via both detection electrodes to the sensitive pre-amplifier, which creates a saturation condition of this pre-amplifier for several milliseconds in the former setup. Figure 2a) shows the resulting time-domain transient signal. Here the transient dwell time is about 5.5 milliseconds, so the detection is started with a detect delay of approximately 6.5 ms.

To avoid this effect, several countermeasures have been implemented: the Orbitrap assembly was made completely symmetrical and the dielectric materials were optimized. Additionally, the pre-amplifier circuit was changed to allow a faster recovery from the central electrode voltage pulse.

Having these changes in effect, the transient dwell time reduced to <0.25 milliseconds, as shown in Figure 2b).

Measurements

The overall performance was tested using Humira samples with the hardware changes implemented. A single spectrum was taken at a resolution setting of 17500 at m/z 200 to use the shortest available transient length; this will cover the entire first transient beat. This spectrum , shown in Figure 4a), was processed using Thermo Scientific ProMass Deconvolution, the result is shown in Figure 4b). The mass accuracy stays below 3 ppm and the different glycoforms are represented.

Figure 5a) shows the HCD fragment spectrum of this sample averaged over four minutes of the elution profile. A dense and well distributed spectrum is visible with the masses extending beyond 23 kDa. By processing this spectrum with Thermo Scientific ProSight PC, the molecule is sequenced. 138 matching fragments were found, giving a good sequence coverage for this single experiment, see Figure 5b).

Transient usedDetect delay

Transient used

Experimental Setup

• 5 µg Humira monoclonal antibody (mAb) (148 kDa, Abbott Lab.Inc.) loaded on a Thermo Scientific BioBasic-4 column of 1 mm i.d. and 100 mm length packed with 5 µm particle size

• 15 min run time with linear gradient from 20 to 80% of acetonitrile with 0.1% formic acid at a 150 μL/min flow rate Resulted in a sample elution time of ~5 min

2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000m/z

0

50

100

0

50

100

0

50

100

Rel

ativ

e A

bund

ance 0

50

100

0

50

1002795.144

N=14890.51

N=13217.72

N=11252.00

N=12931.95

NL: 1.58E7Humira_ResDependency_DetDelay#1224-1233 RT: 5.37-5.40 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1242-1251 RT: 5.45-5.49 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1261-1270 RT: 5.55-5.60 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1284-1293 RT: 5.69-5.76 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1303-1312 RT: 5.85-5.93 AV: 10 T: FTMS + p ESI Full ms

N=12683.94

0 ms

5 ms

10 ms

20 ms

30 ms

Detect delay

FIGURE 4. a) Humira (148kDa) spectrum, b) Deconvoluted spectrum using ProMass Deconvolution

a)

b)

Evaluation of signal intensity

To evaluate the signal intensity contained in the time-domain transient, a very short transient portion is used while varying the detect delay, see Figure 3. It is visible that the first five milliseconds contain a significant share of the total ion signal resulting in a better signal transmission for the improved design.

Hea

vy c

hain

Ligh

t cha

in

humira_IgG_std_HCD_pressure_AIF 11/17/2011 12:26:33 PM

humira_IgG_std_HCD_pressure_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.38E4T: FTMS + p ESI Full ms2 3100.00@hcd80.00 [290.00-4000.00]

600 800 1000 1200 1400 1600 1800 2000 2200 2400m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

1381.71984R=47623

z=91537.13718

R=46435z=8698.37761

R=76606z=1 948.45108

R=66140z=1

1756.58431R=45519

z=7785.41046R=74568

z=1

1243.64669R=48076

z=101049.53692R=63674

z=1

1821.90404R=45005

z=72125.90246

R=44833z=11

2338.29470R=46184

z=10

1990.99189R=44892

z=6

humira_IgG_std_HCD_pressure_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.25E3T: FTMS + p ESI Full ms2 3100.00@hcd80.00 [290.00-4000.00]

2334 2336 2338 2340 2342 2344 2346 2348 2350m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bund

anc

e

2341.79346R=46318

z=?

2344.98903R=45016

z=?

2338.99486R=46035

z=10

2336.69116R=41660

z=?2350.0114

R=43001z=?

2344.18644R=44816

z=?

2345.69079R=40774

z=?

FIGURE 5. a) Single experiment HCD fragmentation spectrum of Humira mAb. b) 138 matching fragments identified by ProSight PC.

Complex samplesAutomatic gain control (AGC)

To fully utilize the analytical performance and space charge capacity of the Orbitrap system, the number of ions injected to the C-Trap needs to be controlled. The measurement of the ion current is either done via a dedicated AGC-prescan, which records a very short transient, or by using the Scan-to-Scan AGC which uses the first short section of the previous analytical scan. The resulting ion current from this short transient acquisition is used to calculate the injection time for the next analytical scan. In some rare cases the number of ions can be underestimated because of the lower resolution and the lower signal response of this short transient acquisition. This is especially true for multiply charged ions and dense peaks below the noise threshold. To demonstrate this effect the AGC improvement described below was switched off and the maximum inject time was set untypically high. Figure 6 shows a 60 minute gradient LC chromatogram of a HeLa sample containing partially digested proteins and including the column wash stage (top). Nearing the end of the run at retention times between 62 and 72 minutes, the AGC becomes inaccurate. A single spectrum from this section shows multiply charged species that won’t be resolved in the short AGC-prescan and therefore will be underestimated (middle). The second spectrum shows the average of three minutes (bottom). Here, partially digested proteins become visible showing ions that also cannot be seen by the short acquisition of the AGC-prescan leading to further underestimation of the ion current. In this case, the inject time for the analytical scan will be too long causing overfilling of the C-Trap. A valid previous workaround was to reduce the AGC target and to set the maximum inject time carefully to a dedicated level for each sample class.

FIGURE 6. Chromatogram and mass spectra of a HeLa sample run with a 60 min. gradient. During the colum wash the AGC gets inaccurate. Both, multiply charged and peaks below the noise threshold appear in the corresponding retention time range.

FIGURE 8. With the CTCD active, the chromatogram does not show any C-Trap overfilling. The mass spectrum contains now analyte peaks of interest.

RT: 8.71 - 73.14

10 15 20 25 30 35 40 45 50 55 60 65 70Time (min)

0

1

2

3

4

Rel

ative

Abu

ndan

ce

53.96

62.1459.3670.3658.94

70.3354.3866.9762.28

64.10

NL: 1.53E9Base Peak F: ms MS hela_lysc_wem_3e6_1e5_mz350_2000_fm150hcd_1

FIGURE 7. Exactive Plus layout with C-Trap charge detector active while the analytical scan is acquired in the Orbitrap.

Measurement Results

Using the CTCD, the HeLa run is repeated and its chromatogram is shown in Figure 8. To emphasize the effect by getting closer to the upper C-Trap space charge limit, the AGC target was set to 3e6 for this experiment. Now the AGC stays accurate even during the column wash stage. The spectrum now shows several analyte peaks which can be used for further confirmation.

a)

b)

HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1 #19027-19394 RT: 65.00-68.00 AV: 293 NL: 4.71E3T: FTMS + p NSI Full ms [350.00-2000.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

946.55941R=33317

z=3

1542.77453R=26371

z=2

912.85357R=34397

z=31976.16243

R=14206z=1

1481.74376R=16917

z=1

1866.41480R=24006

z=6

1693.91682R=20607

z=?

1034.52732R=31884

z=3713.40758R=37751

z=?1318.66850

R=19466z=1

615.96752R=32939

z=1

1101.56294R=30690

z=5

445.12314R=51912

z=?

HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1 #18610 RT: 61.67 AV: 1 NL: 4.62E4T: FTMS + p NSI Full ms [350.00-2000.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

1350.11121R=25406

z=5921.32239R=31906

z=6 1125.25989R=26906

z=6

1381.47974R=24406

z=4

1549.78955R=21506

z=61977.32275

R=19600z=?

998.30304R=27506

z=4

1294.64734R=23002

z=?

722.46362R=33202

z=?

1697.28662R=14800

z=?

1824.30591R=16600

z=?

827.94812R=33700

z=?636.95868R=41204

z=3497.00040R=36500

z=?

RT: 8.71 - 73.14

10 15 20 25 30 35 40 45 50 55 60 65 70Time (min)

0

1

2

3

4

Rel

ative

Abu

ndan

ce

50.9354.33

57.65

57.6955.2759.56

60.3060.86 71.6864.90 67.76

NL: 1.82E9Base Peak F: ms MS HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1

a) b)

humira_IgG_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.38E4

humira_IgG_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.38E4

4 Improved Analysis of Biopharmaceutical Samples Using an MS-only Orbitrap Mass Sspectrometer

Humira_FullMS #1 RT: 4.84 AV: 1 NL: 3.92E5T: FTMS + p ESI Full ms [1000.00-4000.00]

2000 2500 3000 3500 4000m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

2794.982743.25

2904.542693.38

2962.592645.32

2598.93 3023.04

2554.14 3085.99

2510.87 3151.652469.04 3220.13

2428.593291.67

2389.433366.47

2314.79 3444.762244.68 3526.73

2147.123703.011975.45 3897.98

hela_lysc_wem_3e6_1e5_mz350_2000_fm150hcd_1 #19668 RT: 61.54 AV: 1 NL: 2.74E6T: FTMS + p NSI Full ms [350.00-2000.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

445.11856R=52407

z=1

536.16394R=47707

z=1 674.55774R=39206

z=5

1056.53552R=31307

z=2

950.84613R=32206

z=3825.46289R=36104

z=31191.24426

R=28006z=3 1826.72253

R=20702z=?

1577.42529R=19902

z=?

1478.22644R=20002

z=?

1322.64648R=22204

z=2

1972.63916R=16902

z=?

1694.47510R=18700

z=?

Improved Analysis of Biopharmaceutical Samples Using an MS-only Orbitrap Mass Sspectrometer Olaf Scheibner; Eugen Damoc; Eduard Denisov; Jan-Peter Hauschild; Oliver Lange; Frank Czemper; Alexander Kholomeev; Alexander Makarov; Andreas Wieghaus; Maciej BromirskiThermo Fisher Scientific (Bremen) GmbH, Bremen, GERMANY

Conclusion The described hardware changes improve the measurement performance for

large molecules significantly. This results in high abundant Full-MS spectra, e.g. of the Humira antibody with a mass accuracy better than 3 ppm for the deconvoluted spectrum. Using HCD fragmentation 138 matching fragments could be identified from a single experiment.

The C-Trap charge detector improves the automatic gain control for situations where the prescan AGC gets inaccurate. This is demonstrated for the case of partially digested proteins in a HeLa sample run.

AcknowledgementsWe would like to thank the research group of Professor Neil Kelleher from the Northwestern University (IL, USA) for confirming the sequence data using ProSight PC.

The research funding of the 7th European Framework Program is appreciated (Health-F4-2008-201648/PROSPECTS).

OverviewPurpose: Improve the performance of bench-top Orbitrap™ mass spectrometers for large molecules and complex samples.

Methods: The hardware of the Orbitrap assembly was improved to record the most abundant first section of the ion signal. The performance for complex samples was further improved by using an independent C-Trap charge detector.

Results: Measurements of the intact Humira™ antibody shows the performance increase after the optimization of the Orbitrap assembly. The improved behavior for complex samples is demonstrated using 60 minute gradient sample runs from HeLa cells.

IntroductionThis work is dedicated to improve capabilities of an MS-only bench-top Orbitrap mass spectrometer (Thermo Scientific Exactive Plus) for the analysis of very complex mixtures and biopharmaceutical samples.

Intact proteins create fast decaying beat patterns in Fourier-Transform (FT) image current detection systems. In order to have the ability to detect the most abundant signal from the very first beat, modifications to the instrument are required.

When dealing with very complex samples, a dedicated C-Trap charge detection (CTCD) system is shown to improve the accuracy of the prescan-based automated gain control (AGC). Together with the advanced signal processing, the hardware improvements show a significant improvement for several applications.

Large MoleculesImage Current Detection

Because image current detection is an interference detection method used in Fourier-Transform mass spectrometers (FTMS), isotopes within the instrument produce beat patterns. For larger molecules these beat patterns become visible in the time-domain transient signal as sketched in Figure 1. The larger (and cleaner) the molecule, the shorter the beats. For molecules comprising several tens of kilodaltons, the first beat is only visible for the first few milliseconds and the time between the beats can be more than a second. For bench-top systems offering only transient lengths up to 0.5 seconds, the complete detection of the very first beat is crucial.

C-Trap Charge Detection (CTCD)

To improve the analytical robustness of the AGC control scheme a C-Trap charge detection is used to monitor the AGC results every 5 to 10 seconds. During LC runs, the CTCD operation takes place in parallel to Orbitrap acquisition, see Figure 7: While the analytical scan is still being acquired, a few C-Trap injections are ejected to the collector to measure the C-Trap charge. From this, the total ion current (TIC) is calculated and compared to the TIC observed by the short transient AGC-scan. If necessary, the injection time is regulated downward to prevent the C-Trap from overfilling.

Humira is a registered trademark of Abbott Laboratories Inc. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

FIGURE 2. Typical transients: a) Former design (Exactive™), b) Improved design (Q Exactive™, Exactive ™ Plus)

FIGURE 1. Transient sketch of a decaying beat pattern resulting from a FTMS. FIGURE 3. Varied detect delay for 8 ms transient (development mode, 10x10µScans, 10 ms fixed inject time). The first five milliseconds show a significant signal contribution.

Technical Improvements

To inject ions into the Orbitrap analyzer, a voltage pulse of several kilovolts is applied to the central electrode. This voltage pulse propagates via both detection electrodes to the sensitive pre-amplifier, which creates a saturation condition of this pre-amplifier for several milliseconds in the former setup. Figure 2a) shows the resulting time-domain transient signal. Here the transient dwell time is about 5.5 milliseconds, so the detection is started with a detect delay of approximately 6.5 ms.

To avoid this effect, several countermeasures have been implemented: the Orbitrap assembly was made completely symmetrical and the dielectric materials were optimized. Additionally, the pre-amplifier circuit was changed to allow a faster recovery from the central electrode voltage pulse.

Having these changes in effect, the transient dwell time reduced to <0.25 milliseconds, as shown in Figure 2b).

Measurements

The overall performance was tested using Humira samples with the hardware changes implemented. A single spectrum was taken at a resolution setting of 17500 at m/z 200 to use the shortest available transient length; this will cover the entire first transient beat. This spectrum , shown in Figure 4a), was processed using Thermo Scientific ProMass Deconvolution, the result is shown in Figure 4b). The mass accuracy stays below 3 ppm and the different glycoforms are represented.

Figure 5a) shows the HCD fragment spectrum of this sample averaged over four minutes of the elution profile. A dense and well distributed spectrum is visible with the masses extending beyond 23 kDa. By processing this spectrum with Thermo Scientific ProSight PC, the molecule is sequenced. 138 matching fragments were found, giving a good sequence coverage for this single experiment, see Figure 5b).

Transient usedDetect delay

Transient used

Experimental Setup

• 5 µg Humira monoclonal antibody (mAb) (148 kDa, Abbott Lab.Inc.) loaded on a Thermo Scientific BioBasic-4 column of 1 mm i.d. and 100 mm length packed with 5 µm particle size

• 15 min run time with linear gradient from 20 to 80% of acetonitrile with 0.1% formic acid at a 150 μL/min flow rate Resulted in a sample elution time of ~5 min

2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000m/z

0

50

100

0

50

100

0

50

100

Rel

ativ

e A

bund

ance 0

50

100

0

50

1002795.144

N=14890.51

N=13217.72

N=11252.00

N=12931.95

NL: 1.58E7Humira_ResDependency_DetDelay#1224-1233 RT: 5.37-5.40 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1242-1251 RT: 5.45-5.49 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1261-1270 RT: 5.55-5.60 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1284-1293 RT: 5.69-5.76 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1303-1312 RT: 5.85-5.93 AV: 10 T: FTMS + p ESI Full ms

N=12683.94

0 ms

5 ms

10 ms

20 ms

30 ms

Detect delay

FIGURE 4. a) Humira (148kDa) spectrum, b) Deconvoluted spectrum using ProMass Deconvolution

a)

b)

Evaluation of signal intensity

To evaluate the signal intensity contained in the time-domain transient, a very short transient portion is used while varying the detect delay, see Figure 3. It is visible that the first five milliseconds contain a significant share of the total ion signal resulting in a better signal transmission for the improved design.

Hea

vy c

hain

Ligh

t cha

in

humira_IgG_std_HCD_pressure_AIF 11/17/2011 12:26:33 PM

humira_IgG_std_HCD_pressure_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.38E4T: FTMS + p ESI Full ms2 3100.00@hcd80.00 [290.00-4000.00]

600 800 1000 1200 1400 1600 1800 2000 2200 2400m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

1381.71984R=47623

z=91537.13718

R=46435z=8698.37761

R=76606z=1 948.45108

R=66140z=1

1756.58431R=45519

z=7785.41046R=74568

z=1

1243.64669R=48076

z=101049.53692R=63674

z=1

1821.90404R=45005

z=72125.90246

R=44833z=11

2338.29470R=46184

z=10

1990.99189R=44892

z=6

humira_IgG_std_HCD_pressure_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.25E3T: FTMS + p ESI Full ms2 3100.00@hcd80.00 [290.00-4000.00]

2334 2336 2338 2340 2342 2344 2346 2348 2350m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bund

anc

e

2341.79346R=46318

z=?

2344.98903R=45016

z=?

2338.99486R=46035

z=10

2336.69116R=41660

z=?2350.0114

R=43001z=?

2344.18644R=44816

z=?

2345.69079R=40774

z=?

FIGURE 5. a) Single experiment HCD fragmentation spectrum of Humira mAb. b) 138 matching fragments identified by ProSight PC.

Complex samplesAutomatic gain control (AGC)

To fully utilize the analytical performance and space charge capacity of the Orbitrap system, the number of ions injected to the C-Trap needs to be controlled. The measurement of the ion current is either done via a dedicated AGC-prescan, which records a very short transient, or by using the Scan-to-Scan AGC which uses the first short section of the previous analytical scan. The resulting ion current from this short transient acquisition is used to calculate the injection time for the next analytical scan. In some rare cases the number of ions can be underestimated because of the lower resolution and the lower signal response of this short transient acquisition. This is especially true for multiply charged ions and dense peaks below the noise threshold. To demonstrate this effect the AGC improvement described below was switched off and the maximum inject time was set untypically high. Figure 6 shows a 60 minute gradient LC chromatogram of a HeLa sample containing partially digested proteins and including the column wash stage (top). Nearing the end of the run at retention times between 62 and 72 minutes, the AGC becomes inaccurate. A single spectrum from this section shows multiply charged species that won’t be resolved in the short AGC-prescan and therefore will be underestimated (middle). The second spectrum shows the average of three minutes (bottom). Here, partially digested proteins become visible showing ions that also cannot be seen by the short acquisition of the AGC-prescan leading to further underestimation of the ion current. In this case, the inject time for the analytical scan will be too long causing overfilling of the C-Trap. A valid previous workaround was to reduce the AGC target and to set the maximum inject time carefully to a dedicated level for each sample class.

FIGURE 6. Chromatogram and mass spectra of a HeLa sample run with a 60 min. gradient. During the colum wash the AGC gets inaccurate. Both, multiply charged and peaks below the noise threshold appear in the corresponding retention time range.

FIGURE 8. With the CTCD active, the chromatogram does not show any C-Trap overfilling. The mass spectrum contains now analyte peaks of interest.

RT: 8.71 - 73.14

10 15 20 25 30 35 40 45 50 55 60 65 70Time (min)

0

1

2

3

4

Rel

ative

Abu

ndan

ce

53.96

62.1459.3670.3658.94

70.3354.3866.9762.28

64.10

NL: 1.53E9Base Peak F: ms MS hela_lysc_wem_3e6_1e5_mz350_2000_fm150hcd_1

FIGURE 7. Exactive Plus layout with C-Trap charge detector active while the analytical scan is acquired in the Orbitrap.

Measurement Results

Using the CTCD, the HeLa run is repeated and its chromatogram is shown in Figure 8. To emphasize the effect by getting closer to the upper C-Trap space charge limit, the AGC target was set to 3e6 for this experiment. Now the AGC stays accurate even during the column wash stage. The spectrum now shows several analyte peaks which can be used for further confirmation.

a)

b)

HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1 #19027-19394 RT: 65.00-68.00 AV: 293 NL: 4.71E3T: FTMS + p NSI Full ms [350.00-2000.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

946.55941R=33317

z=3

1542.77453R=26371

z=2

912.85357R=34397

z=31976.16243

R=14206z=1

1481.74376R=16917

z=1

1866.41480R=24006

z=6

1693.91682R=20607

z=?

1034.52732R=31884

z=3713.40758R=37751

z=?1318.66850

R=19466z=1

615.96752R=32939

z=1

1101.56294R=30690

z=5

445.12314R=51912

z=?

HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1 #18610 RT: 61.67 AV: 1 NL: 4.62E4T: FTMS + p NSI Full ms [350.00-2000.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

1350.11121R=25406

z=5921.32239R=31906

z=6 1125.25989R=26906

z=6

1381.47974R=24406

z=4

1549.78955R=21506

z=61977.32275

R=19600z=?

998.30304R=27506

z=4

1294.64734R=23002

z=?

722.46362R=33202

z=?

1697.28662R=14800

z=?

1824.30591R=16600

z=?

827.94812R=33700

z=?636.95868R=41204

z=3497.00040R=36500

z=?

RT: 8.71 - 73.14

10 15 20 25 30 35 40 45 50 55 60 65 70Time (min)

0

1

2

3

4

Rel

ative

Abu

ndan

ce

50.9354.33

57.65

57.6955.2759.56

60.3060.86 71.6864.90 67.76

NL: 1.82E9Base Peak F: ms MS HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1

a) b)

humira_IgG_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.38E4

humira_IgG_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.38E4

5Thermo Scientific Poster Note • PN63595_E 06/12S

Humira_FullMS #1 RT: 4.84 AV: 1 NL: 3.92E5T: FTMS + p ESI Full ms [1000.00-4000.00]

2000 2500 3000 3500 4000m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

2794.982743.25

2904.542693.38

2962.592645.32

2598.93 3023.04

2554.14 3085.99

2510.87 3151.652469.04 3220.13

2428.593291.67

2389.433366.47

2314.79 3444.762244.68 3526.73

2147.123703.011975.45 3897.98

hela_lysc_wem_3e6_1e5_mz350_2000_fm150hcd_1 #19668 RT: 61.54 AV: 1 NL: 2.74E6T: FTMS + p NSI Full ms [350.00-2000.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

445.11856R=52407

z=1

536.16394R=47707

z=1 674.55774R=39206

z=5

1056.53552R=31307

z=2

950.84613R=32206

z=3825.46289R=36104

z=31191.24426

R=28006z=3 1826.72253

R=20702z=?

1577.42529R=19902

z=?

1478.22644R=20002

z=?

1322.64648R=22204

z=2

1972.63916R=16902

z=?

1694.47510R=18700

z=?

Improved Analysis of Biopharmaceutical Samples Using an MS-only Orbitrap Mass Sspectrometer Olaf Scheibner; Eugen Damoc; Eduard Denisov; Jan-Peter Hauschild; Oliver Lange; Frank Czemper; Alexander Kholomeev; Alexander Makarov; Andreas Wieghaus; Maciej BromirskiThermo Fisher Scientific (Bremen) GmbH, Bremen, GERMANY

Conclusion The described hardware changes improve the measurement performance for

large molecules significantly. This results in high abundant Full-MS spectra, e.g. of the Humira antibody with a mass accuracy better than 3 ppm for the deconvoluted spectrum. Using HCD fragmentation 138 matching fragments could be identified from a single experiment.

The C-Trap charge detector improves the automatic gain control for situations where the prescan AGC gets inaccurate. This is demonstrated for the case of partially digested proteins in a HeLa sample run.

AcknowledgementsWe would like to thank the research group of Professor Neil Kelleher from the Northwestern University (IL, USA) for confirming the sequence data using ProSight PC.

The research funding of the 7th European Framework Program is appreciated (Health-F4-2008-201648/PROSPECTS).

OverviewPurpose: Improve the performance of bench-top Orbitrap™ mass spectrometers for large molecules and complex samples.

Methods: The hardware of the Orbitrap assembly was improved to record the most abundant first section of the ion signal. The performance for complex samples was further improved by using an independent C-Trap charge detector.

Results: Measurements of the intact Humira™ antibody shows the performance increase after the optimization of the Orbitrap assembly. The improved behavior for complex samples is demonstrated using 60 minute gradient sample runs from HeLa cells.

IntroductionThis work is dedicated to improve capabilities of an MS-only bench-top Orbitrap mass spectrometer (Thermo Scientific Exactive Plus) for the analysis of very complex mixtures and biopharmaceutical samples.

Intact proteins create fast decaying beat patterns in Fourier-Transform (FT) image current detection systems. In order to have the ability to detect the most abundant signal from the very first beat, modifications to the instrument are required.

When dealing with very complex samples, a dedicated C-Trap charge detection (CTCD) system is shown to improve the accuracy of the prescan-based automated gain control (AGC). Together with the advanced signal processing, the hardware improvements show a significant improvement for several applications.

Large MoleculesImage Current Detection

Because image current detection is an interference detection method used in Fourier-Transform mass spectrometers (FTMS), isotopes within the instrument produce beat patterns. For larger molecules these beat patterns become visible in the time-domain transient signal as sketched in Figure 1. The larger (and cleaner) the molecule, the shorter the beats. For molecules comprising several tens of kilodaltons, the first beat is only visible for the first few milliseconds and the time between the beats can be more than a second. For bench-top systems offering only transient lengths up to 0.5 seconds, the complete detection of the very first beat is crucial.

C-Trap Charge Detection (CTCD)

To improve the analytical robustness of the AGC control scheme a C-Trap charge detection is used to monitor the AGC results every 5 to 10 seconds. During LC runs, the CTCD operation takes place in parallel to Orbitrap acquisition, see Figure 7: While the analytical scan is still being acquired, a few C-Trap injections are ejected to the collector to measure the C-Trap charge. From this, the total ion current (TIC) is calculated and compared to the TIC observed by the short transient AGC-scan. If necessary, the injection time is regulated downward to prevent the C-Trap from overfilling.

Humira is a registered trademark of Abbott Laboratories Inc. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

FIGURE 2. Typical transients: a) Former design (Exactive™), b) Improved design (Q Exactive™, Exactive ™ Plus)

FIGURE 1. Transient sketch of a decaying beat pattern resulting from a FTMS. FIGURE 3. Varied detect delay for 8 ms transient (development mode, 10x10µScans, 10 ms fixed inject time). The first five milliseconds show a significant signal contribution.

Technical Improvements

To inject ions into the Orbitrap analyzer, a voltage pulse of several kilovolts is applied to the central electrode. This voltage pulse propagates via both detection electrodes to the sensitive pre-amplifier, which creates a saturation condition of this pre-amplifier for several milliseconds in the former setup. Figure 2a) shows the resulting time-domain transient signal. Here the transient dwell time is about 5.5 milliseconds, so the detection is started with a detect delay of approximately 6.5 ms.

To avoid this effect, several countermeasures have been implemented: the Orbitrap assembly was made completely symmetrical and the dielectric materials were optimized. Additionally, the pre-amplifier circuit was changed to allow a faster recovery from the central electrode voltage pulse.

Having these changes in effect, the transient dwell time reduced to <0.25 milliseconds, as shown in Figure 2b).

Measurements

The overall performance was tested using Humira samples with the hardware changes implemented. A single spectrum was taken at a resolution setting of 17500 at m/z 200 to use the shortest available transient length; this will cover the entire first transient beat. This spectrum , shown in Figure 4a), was processed using Thermo Scientific ProMass Deconvolution, the result is shown in Figure 4b). The mass accuracy stays below 3 ppm and the different glycoforms are represented.

Figure 5a) shows the HCD fragment spectrum of this sample averaged over four minutes of the elution profile. A dense and well distributed spectrum is visible with the masses extending beyond 23 kDa. By processing this spectrum with Thermo Scientific ProSight PC, the molecule is sequenced. 138 matching fragments were found, giving a good sequence coverage for this single experiment, see Figure 5b).

Transient usedDetect delay

Transient used

Experimental Setup

• 5 µg Humira monoclonal antibody (mAb) (148 kDa, Abbott Lab.Inc.) loaded on a Thermo Scientific BioBasic-4 column of 1 mm i.d. and 100 mm length packed with 5 µm particle size

• 15 min run time with linear gradient from 20 to 80% of acetonitrile with 0.1% formic acid at a 150 μL/min flow rate Resulted in a sample elution time of ~5 min

2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000m/z

0

50

100

0

50

100

0

50

100

Rel

ativ

e A

bund

ance 0

50

100

0

50

1002795.144

N=14890.51

N=13217.72

N=11252.00

N=12931.95

NL: 1.58E7Humira_ResDependency_DetDelay#1224-1233 RT: 5.37-5.40 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1242-1251 RT: 5.45-5.49 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1261-1270 RT: 5.55-5.60 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1284-1293 RT: 5.69-5.76 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1303-1312 RT: 5.85-5.93 AV: 10 T: FTMS + p ESI Full ms

N=12683.94

0 ms

5 ms

10 ms

20 ms

30 ms

Detect delay

FIGURE 4. a) Humira (148kDa) spectrum, b) Deconvoluted spectrum using ProMass Deconvolution

a)

b)

Evaluation of signal intensity

To evaluate the signal intensity contained in the time-domain transient, a very short transient portion is used while varying the detect delay, see Figure 3. It is visible that the first five milliseconds contain a significant share of the total ion signal resulting in a better signal transmission for the improved design.

Hea

vy c

hain

Ligh

t cha

in

humira_IgG_std_HCD_pressure_AIF 11/17/2011 12:26:33 PM

humira_IgG_std_HCD_pressure_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.38E4T: FTMS + p ESI Full ms2 3100.00@hcd80.00 [290.00-4000.00]

600 800 1000 1200 1400 1600 1800 2000 2200 2400m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

1381.71984R=47623

z=91537.13718

R=46435z=8698.37761

R=76606z=1 948.45108

R=66140z=1

1756.58431R=45519

z=7785.41046R=74568

z=1

1243.64669R=48076

z=101049.53692R=63674

z=1

1821.90404R=45005

z=72125.90246

R=44833z=11

2338.29470R=46184

z=10

1990.99189R=44892

z=6

humira_IgG_std_HCD_pressure_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.25E3T: FTMS + p ESI Full ms2 3100.00@hcd80.00 [290.00-4000.00]

2334 2336 2338 2340 2342 2344 2346 2348 2350m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bund

anc

e

2341.79346R=46318

z=?

2344.98903R=45016

z=?

2338.99486R=46035

z=10

2336.69116R=41660

z=?2350.0114

R=43001z=?

2344.18644R=44816

z=?

2345.69079R=40774

z=?

FIGURE 5. a) Single experiment HCD fragmentation spectrum of Humira mAb. b) 138 matching fragments identified by ProSight PC.

Complex samplesAutomatic gain control (AGC)

To fully utilize the analytical performance and space charge capacity of the Orbitrap system, the number of ions injected to the C-Trap needs to be controlled. The measurement of the ion current is either done via a dedicated AGC-prescan, which records a very short transient, or by using the Scan-to-Scan AGC which uses the first short section of the previous analytical scan. The resulting ion current from this short transient acquisition is used to calculate the injection time for the next analytical scan. In some rare cases the number of ions can be underestimated because of the lower resolution and the lower signal response of this short transient acquisition. This is especially true for multiply charged ions and dense peaks below the noise threshold. To demonstrate this effect the AGC improvement described below was switched off and the maximum inject time was set untypically high. Figure 6 shows a 60 minute gradient LC chromatogram of a HeLa sample containing partially digested proteins and including the column wash stage (top). Nearing the end of the run at retention times between 62 and 72 minutes, the AGC becomes inaccurate. A single spectrum from this section shows multiply charged species that won’t be resolved in the short AGC-prescan and therefore will be underestimated (middle). The second spectrum shows the average of three minutes (bottom). Here, partially digested proteins become visible showing ions that also cannot be seen by the short acquisition of the AGC-prescan leading to further underestimation of the ion current. In this case, the inject time for the analytical scan will be too long causing overfilling of the C-Trap. A valid previous workaround was to reduce the AGC target and to set the maximum inject time carefully to a dedicated level for each sample class.

FIGURE 6. Chromatogram and mass spectra of a HeLa sample run with a 60 min. gradient. During the colum wash the AGC gets inaccurate. Both, multiply charged and peaks below the noise threshold appear in the corresponding retention time range.

FIGURE 8. With the CTCD active, the chromatogram does not show any C-Trap overfilling. The mass spectrum contains now analyte peaks of interest.

RT: 8.71 - 73.14

10 15 20 25 30 35 40 45 50 55 60 65 70Time (min)

0

1

2

3

4

Rel

ative

Abu

ndan

ce

53.96

62.1459.3670.3658.94

70.3354.3866.9762.28

64.10

NL: 1.53E9Base Peak F: ms MS hela_lysc_wem_3e6_1e5_mz350_2000_fm150hcd_1

FIGURE 7. Exactive Plus layout with C-Trap charge detector active while the analytical scan is acquired in the Orbitrap.

Measurement Results

Using the CTCD, the HeLa run is repeated and its chromatogram is shown in Figure 8. To emphasize the effect by getting closer to the upper C-Trap space charge limit, the AGC target was set to 3e6 for this experiment. Now the AGC stays accurate even during the column wash stage. The spectrum now shows several analyte peaks which can be used for further confirmation.

a)

b)

HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1 #19027-19394 RT: 65.00-68.00 AV: 293 NL: 4.71E3T: FTMS + p NSI Full ms [350.00-2000.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

946.55941R=33317

z=3

1542.77453R=26371

z=2

912.85357R=34397

z=31976.16243

R=14206z=1

1481.74376R=16917

z=1

1866.41480R=24006

z=6

1693.91682R=20607

z=?

1034.52732R=31884

z=3713.40758R=37751

z=?1318.66850

R=19466z=1

615.96752R=32939

z=1

1101.56294R=30690

z=5

445.12314R=51912

z=?

HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1 #18610 RT: 61.67 AV: 1 NL: 4.62E4T: FTMS + p NSI Full ms [350.00-2000.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

1350.11121R=25406

z=5921.32239R=31906

z=6 1125.25989R=26906

z=6

1381.47974R=24406

z=4

1549.78955R=21506

z=61977.32275

R=19600z=?

998.30304R=27506

z=4

1294.64734R=23002

z=?

722.46362R=33202

z=?

1697.28662R=14800

z=?

1824.30591R=16600

z=?

827.94812R=33700

z=?636.95868R=41204

z=3497.00040R=36500

z=?

RT: 8.71 - 73.14

10 15 20 25 30 35 40 45 50 55 60 65 70Time (min)

0

1

2

3

4

Rel

ative

Abu

ndan

ce

50.9354.33

57.65

57.6955.2759.56

60.3060.86 71.6864.90 67.76

NL: 1.82E9Base Peak F: ms MS HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1

a) b)

humira_IgG_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.38E4

humira_IgG_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.38E4

6 Improved Analysis of Biopharmaceutical Samples Using an MS-only Orbitrap Mass Sspectrometer

Humira_FullMS #1 RT: 4.84 AV: 1 NL: 3.92E5T: FTMS + p ESI Full ms [1000.00-4000.00]

2000 2500 3000 3500 4000m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

2794.982743.25

2904.542693.38

2962.592645.32

2598.93 3023.04

2554.14 3085.99

2510.87 3151.652469.04 3220.13

2428.593291.67

2389.433366.47

2314.79 3444.762244.68 3526.73

2147.123703.011975.45 3897.98

hela_lysc_wem_3e6_1e5_mz350_2000_fm150hcd_1 #19668 RT: 61.54 AV: 1 NL: 2.74E6T: FTMS + p NSI Full ms [350.00-2000.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

445.11856R=52407

z=1

536.16394R=47707

z=1 674.55774R=39206

z=5

1056.53552R=31307

z=2

950.84613R=32206

z=3825.46289R=36104

z=31191.24426

R=28006z=3 1826.72253

R=20702z=?

1577.42529R=19902

z=?

1478.22644R=20002

z=?

1322.64648R=22204

z=2

1972.63916R=16902

z=?

1694.47510R=18700

z=?

Improved Analysis of Biopharmaceutical Samples Using an MS-only Orbitrap Mass Sspectrometer Olaf Scheibner; Eugen Damoc; Eduard Denisov; Jan-Peter Hauschild; Oliver Lange; Frank Czemper; Alexander Kholomeev; Alexander Makarov; Andreas Wieghaus; Maciej BromirskiThermo Fisher Scientific (Bremen) GmbH, Bremen, GERMANY

Conclusion The described hardware changes improve the measurement performance for

large molecules significantly. This results in high abundant Full-MS spectra, e.g. of the Humira antibody with a mass accuracy better than 3 ppm for the deconvoluted spectrum. Using HCD fragmentation 138 matching fragments could be identified from a single experiment.

The C-Trap charge detector improves the automatic gain control for situations where the prescan AGC gets inaccurate. This is demonstrated for the case of partially digested proteins in a HeLa sample run.

AcknowledgementsWe would like to thank the research group of Professor Neil Kelleher from the Northwestern University (IL, USA) for confirming the sequence data using ProSight PC.

The research funding of the 7th European Framework Program is appreciated (Health-F4-2008-201648/PROSPECTS).

OverviewPurpose: Improve the performance of bench-top Orbitrap™ mass spectrometers for large molecules and complex samples.

Methods: The hardware of the Orbitrap assembly was improved to record the most abundant first section of the ion signal. The performance for complex samples was further improved by using an independent C-Trap charge detector.

Results: Measurements of the intact Humira™ antibody shows the performance increase after the optimization of the Orbitrap assembly. The improved behavior for complex samples is demonstrated using 60 minute gradient sample runs from HeLa cells.

IntroductionThis work is dedicated to improve capabilities of an MS-only bench-top Orbitrap mass spectrometer (Thermo Scientific Exactive Plus) for the analysis of very complex mixtures and biopharmaceutical samples.

Intact proteins create fast decaying beat patterns in Fourier-Transform (FT) image current detection systems. In order to have the ability to detect the most abundant signal from the very first beat, modifications to the instrument are required.

When dealing with very complex samples, a dedicated C-Trap charge detection (CTCD) system is shown to improve the accuracy of the prescan-based automated gain control (AGC). Together with the advanced signal processing, the hardware improvements show a significant improvement for several applications.

Large MoleculesImage Current Detection

Because image current detection is an interference detection method used in Fourier-Transform mass spectrometers (FTMS), isotopes within the instrument produce beat patterns. For larger molecules these beat patterns become visible in the time-domain transient signal as sketched in Figure 1. The larger (and cleaner) the molecule, the shorter the beats. For molecules comprising several tens of kilodaltons, the first beat is only visible for the first few milliseconds and the time between the beats can be more than a second. For bench-top systems offering only transient lengths up to 0.5 seconds, the complete detection of the very first beat is crucial.

C-Trap Charge Detection (CTCD)

To improve the analytical robustness of the AGC control scheme a C-Trap charge detection is used to monitor the AGC results every 5 to 10 seconds. During LC runs, the CTCD operation takes place in parallel to Orbitrap acquisition, see Figure 7: While the analytical scan is still being acquired, a few C-Trap injections are ejected to the collector to measure the C-Trap charge. From this, the total ion current (TIC) is calculated and compared to the TIC observed by the short transient AGC-scan. If necessary, the injection time is regulated downward to prevent the C-Trap from overfilling.

Humira is a registered trademark of Abbott Laboratories Inc. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.

FIGURE 2. Typical transients: a) Former design (Exactive™), b) Improved design (Q Exactive™, Exactive ™ Plus)

FIGURE 1. Transient sketch of a decaying beat pattern resulting from a FTMS. FIGURE 3. Varied detect delay for 8 ms transient (development mode, 10x10µScans, 10 ms fixed inject time). The first five milliseconds show a significant signal contribution.

Technical Improvements

To inject ions into the Orbitrap analyzer, a voltage pulse of several kilovolts is applied to the central electrode. This voltage pulse propagates via both detection electrodes to the sensitive pre-amplifier, which creates a saturation condition of this pre-amplifier for several milliseconds in the former setup. Figure 2a) shows the resulting time-domain transient signal. Here the transient dwell time is about 5.5 milliseconds, so the detection is started with a detect delay of approximately 6.5 ms.

To avoid this effect, several countermeasures have been implemented: the Orbitrap assembly was made completely symmetrical and the dielectric materials were optimized. Additionally, the pre-amplifier circuit was changed to allow a faster recovery from the central electrode voltage pulse.

Having these changes in effect, the transient dwell time reduced to <0.25 milliseconds, as shown in Figure 2b).

Measurements

The overall performance was tested using Humira samples with the hardware changes implemented. A single spectrum was taken at a resolution setting of 17500 at m/z 200 to use the shortest available transient length; this will cover the entire first transient beat. This spectrum , shown in Figure 4a), was processed using Thermo Scientific ProMass Deconvolution, the result is shown in Figure 4b). The mass accuracy stays below 3 ppm and the different glycoforms are represented.

Figure 5a) shows the HCD fragment spectrum of this sample averaged over four minutes of the elution profile. A dense and well distributed spectrum is visible with the masses extending beyond 23 kDa. By processing this spectrum with Thermo Scientific ProSight PC, the molecule is sequenced. 138 matching fragments were found, giving a good sequence coverage for this single experiment, see Figure 5b).

Transient usedDetect delay

Transient used

Experimental Setup

• 5 µg Humira monoclonal antibody (mAb) (148 kDa, Abbott Lab.Inc.) loaded on a Thermo Scientific BioBasic-4 column of 1 mm i.d. and 100 mm length packed with 5 µm particle size

• 15 min run time with linear gradient from 20 to 80% of acetonitrile with 0.1% formic acid at a 150 μL/min flow rate Resulted in a sample elution time of ~5 min

2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000m/z

0

50

100

0

50

100

0

50

100

Rel

ativ

e A

bund

ance 0

50

100

0

50

1002795.144

N=14890.51

N=13217.72

N=11252.00

N=12931.95

NL: 1.58E7Humira_ResDependency_DetDelay#1224-1233 RT: 5.37-5.40 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1242-1251 RT: 5.45-5.49 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1261-1270 RT: 5.55-5.60 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1284-1293 RT: 5.69-5.76 AV: 10 T: FTMS + p ESI Full ms

NL: 1.58E7Humira_ResDependency_DetDelay#1303-1312 RT: 5.85-5.93 AV: 10 T: FTMS + p ESI Full ms

N=12683.94

0 ms

5 ms

10 ms

20 ms

30 ms

Detect delay

FIGURE 4. a) Humira (148kDa) spectrum, b) Deconvoluted spectrum using ProMass Deconvolution

a)

b)

Evaluation of signal intensity

To evaluate the signal intensity contained in the time-domain transient, a very short transient portion is used while varying the detect delay, see Figure 3. It is visible that the first five milliseconds contain a significant share of the total ion signal resulting in a better signal transmission for the improved design.

Hea

vy c

hain

Ligh

t cha

in

humira_IgG_std_HCD_pressure_AIF 11/17/2011 12:26:33 PM

humira_IgG_std_HCD_pressure_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.38E4T: FTMS + p ESI Full ms2 3100.00@hcd80.00 [290.00-4000.00]

600 800 1000 1200 1400 1600 1800 2000 2200 2400m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

1381.71984R=47623

z=91537.13718

R=46435z=8698.37761

R=76606z=1 948.45108

R=66140z=1

1756.58431R=45519

z=7785.41046R=74568

z=1

1243.64669R=48076

z=101049.53692R=63674

z=1

1821.90404R=45005

z=72125.90246

R=44833z=11

2338.29470R=46184

z=10

1990.99189R=44892

z=6

humira_IgG_std_HCD_pressure_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.25E3T: FTMS + p ESI Full ms2 3100.00@hcd80.00 [290.00-4000.00]

2334 2336 2338 2340 2342 2344 2346 2348 2350m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bund

anc

e

2341.79346R=46318

z=?

2344.98903R=45016

z=?

2338.99486R=46035

z=10

2336.69116R=41660

z=?2350.0114

R=43001z=?

2344.18644R=44816

z=?

2345.69079R=40774

z=?

FIGURE 5. a) Single experiment HCD fragmentation spectrum of Humira mAb. b) 138 matching fragments identified by ProSight PC.

Complex samplesAutomatic gain control (AGC)

To fully utilize the analytical performance and space charge capacity of the Orbitrap system, the number of ions injected to the C-Trap needs to be controlled. The measurement of the ion current is either done via a dedicated AGC-prescan, which records a very short transient, or by using the Scan-to-Scan AGC which uses the first short section of the previous analytical scan. The resulting ion current from this short transient acquisition is used to calculate the injection time for the next analytical scan. In some rare cases the number of ions can be underestimated because of the lower resolution and the lower signal response of this short transient acquisition. This is especially true for multiply charged ions and dense peaks below the noise threshold. To demonstrate this effect the AGC improvement described below was switched off and the maximum inject time was set untypically high. Figure 6 shows a 60 minute gradient LC chromatogram of a HeLa sample containing partially digested proteins and including the column wash stage (top). Nearing the end of the run at retention times between 62 and 72 minutes, the AGC becomes inaccurate. A single spectrum from this section shows multiply charged species that won’t be resolved in the short AGC-prescan and therefore will be underestimated (middle). The second spectrum shows the average of three minutes (bottom). Here, partially digested proteins become visible showing ions that also cannot be seen by the short acquisition of the AGC-prescan leading to further underestimation of the ion current. In this case, the inject time for the analytical scan will be too long causing overfilling of the C-Trap. A valid previous workaround was to reduce the AGC target and to set the maximum inject time carefully to a dedicated level for each sample class.

FIGURE 6. Chromatogram and mass spectra of a HeLa sample run with a 60 min. gradient. During the colum wash the AGC gets inaccurate. Both, multiply charged and peaks below the noise threshold appear in the corresponding retention time range.

FIGURE 8. With the CTCD active, the chromatogram does not show any C-Trap overfilling. The mass spectrum contains now analyte peaks of interest.

RT: 8.71 - 73.14

10 15 20 25 30 35 40 45 50 55 60 65 70Time (min)

0

1

2

3

4

Rel

ative

Abu

ndan

ce

53.96

62.1459.3670.3658.94

70.3354.3866.9762.28

64.10

NL: 1.53E9Base Peak F: ms MS hela_lysc_wem_3e6_1e5_mz350_2000_fm150hcd_1

FIGURE 7. Exactive Plus layout with C-Trap charge detector active while the analytical scan is acquired in the Orbitrap.

Measurement Results

Using the CTCD, the HeLa run is repeated and its chromatogram is shown in Figure 8. To emphasize the effect by getting closer to the upper C-Trap space charge limit, the AGC target was set to 3e6 for this experiment. Now the AGC stays accurate even during the column wash stage. The spectrum now shows several analyte peaks which can be used for further confirmation.

a)

b)

HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1 #19027-19394 RT: 65.00-68.00 AV: 293 NL: 4.71E3T: FTMS + p NSI Full ms [350.00-2000.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

946.55941R=33317

z=3

1542.77453R=26371

z=2

912.85357R=34397

z=31976.16243

R=14206z=1

1481.74376R=16917

z=1

1866.41480R=24006

z=6

1693.91682R=20607

z=?

1034.52732R=31884

z=3713.40758R=37751

z=?1318.66850

R=19466z=1

615.96752R=32939

z=1

1101.56294R=30690

z=5

445.12314R=51912

z=?

HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1 #18610 RT: 61.67 AV: 1 NL: 4.62E4T: FTMS + p NSI Full ms [350.00-2000.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

1350.11121R=25406

z=5921.32239R=31906

z=6 1125.25989R=26906

z=6

1381.47974R=24406

z=4

1549.78955R=21506

z=61977.32275

R=19600z=?

998.30304R=27506

z=4

1294.64734R=23002

z=?

722.46362R=33202

z=?

1697.28662R=14800

z=?

1824.30591R=16600

z=?

827.94812R=33700

z=?636.95868R=41204

z=3497.00040R=36500

z=?

RT: 8.71 - 73.14

10 15 20 25 30 35 40 45 50 55 60 65 70Time (min)

0

1

2

3

4

Rel

ative

Abu

ndan

ce

50.9354.33

57.65

57.6955.2759.56

60.3060.86 71.6864.90 67.76

NL: 1.82E9Base Peak F: ms MS HeLa_LysC_noEM_1e6_1e5_mz350_2000_fm150HCD_1

a) b)

humira_IgG_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.38E4

humira_IgG_AIF #195-213 RT: 3.82-7.75 AV: 19 NL: 3.38E4

Thermo Fisher Scientific, San Jose, CA USA is ISO Certified.

PN63595_E 06/12S

Africa-Other +27 11 570 1840Australia +61 3 9757 4300Austria +43 1 333 50 34 0Belgium +32 53 73 42 41Canada +1 800 530 8447China +86 10 8419 3588Denmark +45 70 23 62 60

Europe-Other +43 1 333 50 34 0Finland/Norway/Sweden +46 8 556 468 00France +33 1 60 92 48 00Germany +49 6103 408 1014India +91 22 6742 9434Italy +39 02 950 591

Japan +81 45 453 9100Latin America +1 561 688 8700Middle East +43 1 333 50 34 0Netherlands +31 76 579 55 55New Zealand +64 9 980 6700Russia/CIS +43 1 333 50 34 0South Africa +27 11 570 1840

Spain +34 914 845 965Switzerland +41 61 716 77 00UK +44 1442 233555USA +1 800 532 4752

www.thermoscientific.com©2012 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.