simultaneous quantitative analysis and structure ... · pdf filesimultaneous quantitative...

Simultaneous Quantitative Analysis and Structure Elucidation of Lipidsfor Effective Biomarker Screening by Mass SpectrometryRoland Geyer 1, Uta Ceglarek 2; Ralf Bogumil 3, Axel Besa 4; 1Applied Biosystems Europe B.V. (part of Life Techno logies™), Rotkreuz, Switzerland, [email protected] ppliedbiosystems.com; 2ILM, University Hospital Leipzig, Leipzig, Germany; 3BIOCRATES Life Sciences AG, Innsbruck, Austria; 4Applied Biosystems (part of Life Technologies™), Da rmstadt, Germany

Figure 5. QTRAP ® 5500 MS/MS Sensitivity & Fragment Spectra Quality f or evaluation of Eicosanoid profiles fromLPS induced Monocyte cell cultures (MarkerView™ used for data assessment and analysis)

High sensitivity of the linear ion trap means more fragment ions available to compare and confirm with. On the QTRAP® 5500 fragment ions can be obtained also from underlying, very low abundant peaks � full fragment spectra at lower-limit-of-quantification (at S/N=10). PCA revealed relevant MRM transitions. Overall, MS/MS sensitivity and spectral quality leads to easier quantification & identification.

TRADEMARKS/LICENSINGFor research use only. Not for use in diagnostic procedures. AB (Design) and Applied Biosystems are registered trademarks of Applied Biosystems or its subsidiaries in the US and/or certain other countries. API 5000 and MarkerView are trademarks and QTRAP, Analyst, are registered trademarks of Applied Biosystems/MDS Analytical Technologies, a joint venture between Applied Biosystems and MDS Inc.

© 2008 Applied Biosystems and MDS Inc. Joint Owners. All rights reserved.

ABSTRACTDetection of eicosanoid profiles by ESI/MS/MS easily reveals stress response as shown for LPS treatment in monocyte cell cultures. Highest sensitivity offers significant advantages for their assessment, whereas fragment spectra generation enable easier identification or structure confirmation. Eicosanoids profiling is also suitable for revealing the inflammatory response processes in blood serum samples after liver transplantation. Fast tandem quadrupole measurements, together with the new collision cell of the QTRAP® 5000 allows precursor ion and constant neutral loss scans to be conducted at fast scan rates enhancing mass spectral quality. It also allows MRM measurements at short dwell times with no loss in sensitivity. This makes it possible to quantify hundreds of glycerophospholipids from minute sample amounts (GPCs from 10µL blood plasma sample extracted with AbsoluteIDQ™ kit). The QTRAP® 5000 also enables fast polarity switching with 50ms dwell time. Quantitative survey scans for glycerophosphocholines in the positive mode could be combined with structure elucidation in the negative mode. This will also work for other lipid classes. Overall, the new MS system offers substantial advantages for comprehensive profiling of a complex lipidome at the highest sensitivity.

INTRODUCTIONThe rapid recovery and analysis of regulatory lipids in biological samples offers great potential to provide a means of monitoring critical processes in responses to a multitude of factors such as inducing inflammatory stress. Such lipids may also serve as a prognostic marker for the outcome of a stress response (e.g. after surgery, tissue transplantation or perhaps even cancer treatment). Pivotal for quantitative profiling of hundreds of partly structural closely related lipids and revealing the potential biomarker are sample preparation, chromatographic separation and fast scanning capabilities of the mass spectrometry system. Molecules within a lipid class can have very different biological functions or activities that depend on their specific structure as well as concentration (e.g., glycerophospholipids, eicosanoids). Here we used the enhanced sensitivity, selectivity and speed of the new AB SCIEX QTRAP® 5500 System, a hybrid triple-quadrupole/linear-ion trap MS, to simultaneously obtain quantitative as well as qualitative information for a large number of lipid molecules. Suitability and advantages of the approach and technology is shown for e.g., monitoring inflammatory response processes in monocyte cell cultures after LPS treatment as well as for evaluating differences in sets of human serum samples.

MATERIALS AND METHODSEicosanoids were extracted by SPE from 100µL of monocyte cell culture supernatant treated with LPS or from serum samples after liver transplantation using protein precipitation and cleanup on StrataX SPE cartridges (Phenomenex). Phospholipids from plasma samples were extracted with the AbsoluteIDQ™ Kit (Biocrates, Innsbruck). Quantitative survey scans (MRM or Precursor scans) for certain lipid classes (e.g., GPC’s, eicosanoids) were directly coupled to a generation of fragment spectra from the two most abundant masses (information dependent data acquisition, IDA). For GPC’s this was combined with efficient polarity switching to obtain information on the acyl chains. Data processing software (MarkerView™) was used to extract, align, normalize and compare the complex MS data to reveal potential lipid biomarkers within sample sets. Structural characteristics of relevant lipid molecules were then evaluated based on the fragment spectra already acquired.

Acquire survey scan LC/MS Data

IDA-filtersno

Sample

Locate Peaks (m/z)

Simultaneously acquireLC/MS/MS Data

Identify / Confirm Metabolites

yes

(polarity switch)


IDA-filtersno

Sample

Locate Peaks (m/z)



yes

(polarity switch)


IDA-filtersIDA-filtersnono

Sample

Locate Peaks (m/z)



yes

Locate Peaks (m/z)



yes

(polarity switch)

XIC of -MRM (50 pairs): Exp 1, 355.3/275.2 Da from Sample 9 (Monozyten LPS 16h 6) of RG 09-24-08 LPS Stimulation Eicos API5K comparis... Max. 3.4e5 cps.

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

Inte

n...

3.77

4.041.91

3.273.00 4.72 5.34 10.41

-EPI (355.30) Charge (+0) FT (... Max. 3.6e8 cps.

100 150 200 250 300 350 400m/z, Da

0.0

1.0e8

2.0e8

3.0e8

3.6e8

Inte

nsity

, cps

354.8

93.1 195.4 273.6 289.4181.4


100 150 200 250 300 350 400m/z, Da

0.0

2.0e6

4.0e6

6.0e6

7.3e6

Inte

nsity

, cps

275.4 319.3 337.3

355.3193.4

179.3 237.6157.2113.2 311.4


100 150 200 250 300 350 400m/z, Da

0.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

Inte

nsity

, cps

275.5193.4 319.4

237.6

337.4 355.3

203.5157.3 255.6 311.2121.2107.0


100 150 200 250 300 350 400m/z, Da

0.0

1.0e7

2.0e7

3.0e7

4.0e7

5.0e7

5.9e7

Inte

nsity

, cps

307.2

229.4

273.5 351.2158.2137.2 233.3


100 150 200 250 300 350 400m/z, Da

0.0

5.0e5

1.0e6

1.5e6

1.9e6

Inte

nsity

, cps

271.4

315.5351.4

189.4229.4

307.3

217.6175.2 233.6 289.6158.395.0


100 150 200 250 300 350 400m/z, Da

0.0

2.0e5

4.0e5

6.0e56.9e5

Inte

nsity

, cps

351.4

271.4189.5

229.4 307.4

233.4

333.5203.3137.2 217.690.8 289.3

Eicosanoids from 100µL cell culture supernatant

S/N 15

Figure 1. AB SCIEX QTRAP ® 5500 System and IDA workflow

� Quantitate multiple targets

� Structure evaluation/verification

TIC of +Prec (184.10): from Sample 7 (Prec 184+ IDQ Plate H1_vial85 001) of RG 09-2008.wiff (Turbo Spray) Max. 6.6e7 cps.

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0Time, min

0.0

2.0e7

4.0e7

6.0e7

Inte

n...

3.363.55

3.27

3.802.40 4.112.09

0.44 4.441.34

+Prec (184.10): 3.822 min from Sample 7 (Prec 184+ IDQ Plate H1_vial85 001) of RG 09-2008.wiff (Turbo Spray) Max. 2.0e6 cps.

200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, Da

0.0

5.0e5

1.0e6

1.5e6

2.0e6

Inte

n...

790.9761.9

787.9

816.9

800.9773.9 825.0748.9 876.0

XIC of +Prec (1... Max. 4.8e6 cps.

2.0 3.0 4.0 5.0 6.0Time, min

0.0

1.0e6

2.0e6

3.0e6

4.0e6

4.8e6

Inte

nsity

, cps

3.393.55

3.81


2.0 3.0 4.0 5.0 6.0Time, min

0.0

1.0e6

2.0e6

3.0e6

4.0e6

4.7e6

Inte

nsity

, cps

3.43

3.84


2.0 3.0 4.0 5.0 6.0Time, min

0.0

2.0e6

4.0e6

6.0e6

8.0e6

9.4e6

Inte

nsity

, cps

3.79

4.09

3.43


2.0 3.0 4.0 5.0 6.0Time, min

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e62.8e6

Inte

nsity

, cps

4.12

4.44

3.66

Prec184+ mass at RT 3.8min

TIC Prec184+300ms dwell time

XICm/z 761

XICm/z 787

XICm/z 790

XICm/z 816


1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0Time, min

0.0

2.0e7

4.0e7

6.0e7

Inte

n...

3.363.55

3.27

3.802.40 4.112.09

0.44 4.441.34


200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, Da

0.0

5.0e5

1.0e6

1.5e6

2.0e6

Inte

n...

790.9761.9

787.9

816.9

800.9773.9 825.0748.9 876.0


2.0 3.0 4.0 5.0 6.0Time, min

0.0

1.0e6

2.0e6

3.0e6

4.0e6

4.8e6

Inte

nsity

, cps

3.393.55

3.81


2.0 3.0 4.0 5.0 6.0Time, min

0.0

1.0e6

2.0e6

3.0e6

4.0e6

4.7e6

Inte

nsity

, cps

3.43

3.84


2.0 3.0 4.0 5.0 6.0Time, min

0.0

2.0e6

4.0e6

6.0e6

8.0e6

9.4e6

Inte

nsity

, cps

3.79

4.09

3.43


2.0 3.0 4.0 5.0 6.0Time, min

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e62.8e6

Inte

nsity

, cps

4.12

4.44

3.66



XICm/z 761

XICm/z 787

XICm/z 790

XICm/z 816


1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0Time, min

0.0

2.0e7

4.0e7

6.0e7

Inte

n...

3.363.55

3.27

3.802.40 4.112.09

0.44 4.441.34


200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000m/z, Da

0.0

5.0e5

1.0e6

1.5e6

2.0e6

Inte

n...

790.9761.9

787.9

816.9

800.9773.9 825.0748.9 876.0


2.0 3.0 4.0 5.0 6.0Time, min

0.0

1.0e6

2.0e6

3.0e6

4.0e6

4.8e6

Inte

nsity

, cps

3.393.55

3.81


2.0 3.0 4.0 5.0 6.0Time, min

0.0

1.0e6

2.0e6

3.0e6

4.0e6

4.7e6

Inte

nsity

, cps

3.43

3.84


2.0 3.0 4.0 5.0 6.0Time, min

0.0

2.0e6

4.0e6

6.0e6

8.0e6

9.4e6

Inte

nsity

, cps

3.79

4.09

3.43


2.0 3.0 4.0 5.0 6.0Time, min

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e62.8e6

Inte

nsity

, cps

4.12

4.44

3.66



XICm/z 761

XICm/z 787

XICm/z 790

XICm/z 816

XICs of four mass transitions (Q1/184) already resolve 16 molecules; Short dwell time (300ms, mass range 200-1000amu) enables analysis of hundreds of molecules (lysoPCs, PCs and SMs) in a single run.

Liver transplantation: serum samples from 0, 24 & 240hours after surgery measured on 4000 QTRAP®

and API5000™

Monocyte stimulation with LPS: cell culture supernatant from 0h, LPS 4h, LPS 16h, control 4h & control 16h measured on QTRAP® 5500

5 µµµµL injectedL injectedL injectedL injected; 50 MRM transitions; 20ms dwell & 5ms pause time; Peaks (Q1/Q3 at RT) were extracted and evaluated with MarkerView™software including peak picking & PCA algorithm

Figure 2. Eicosanoid Extraction and Analysis

Capabilities of the QTRAP ® 5500 for lipidomics applications● Fast Tandem Quadrupole Scan Speeds

– up to 2‘000amu/sec for high quality Precursor Ion or Constant Neutral Loss Spectra

● Fast MRM Measurements

– 2 ms dwell times for multi-target profiling

● Fast Positive/Negative Ion Polarity Switching

– 50 ms switching times for covering both polarities (quantify/quantify or quantify/structure analysis)

● Fast Linear Ion Trap Scan Speeds

– up to 20‘000amu/sec for fast fragment spectra generation

XIC of +MRM (175 pairs): Exp 1, 162.1/85.1 Da from Sample 2 (081781_1_0400407385) of KIT1-30-3_081781_85_1_0400407385.wiff (Turbo ... Max. 1.6e5 cps.

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 7.5Time, min

0.0

2.0e6

4.0e6

6.0e6

8.0e6

Inte

n...

0.28

XIC of +MRM (175 pairs): Exp 1, 788.6/184.0 Da from Sample 2 (081781_1_0400407385) of KIT1-30-3_081781_85_1_0400407385.wiff (Turb... Max. 6.4e6 cps.

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 7.5Time, min

0.0

2.0e6

4.0e6

6.0e6

Inte

n...

3.91


200 400 600 800 1000m/z, Da

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.4e6

Inte

nsity

, cps

786.6

283.4

294.2699.6524.5484.6 768.5239.4


200 400 600 800 1000m/z, Da

0.0

5.0e5

1.0e6

1.5e6

Inte

nsity

, cps

786.6

283.3

768.6726.6153.1 523.6419.4


200 400 600 800 1000m/z, Da

0.0

2.0e5

4.0e5

6.0e5

8.0e5

9.7e5

Inte

nsity

, cps

786.6

281.4

768.7522.6390.5311.4 726.8

175 MRMs positive mode10µL plasma

fragmentsnegative modeEPI 786.6

XIC 788.6/184

positive mode

XIC of +MRM (175 pairs): Exp 1, 162.1/85.1 Da from Sample 2 (081781_1_0400407385) of KIT1-30-3_081781_85_1_0400407385.wiff (Turbo ... Max. 1.6e5 cps.

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 7.5Time, min

0.0

2.0e6

4.0e6

6.0e6

8.0e6

Inte

n...

0.28

XIC of +MRM (175 pairs): Exp 1, 788.6/184.0 Da from Sample 2 (081781_1_0400407385) of KIT1-30-3_081781_85_1_0400407385.wiff (Turb... Max. 6.4e6 cps.

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 7.5Time, min

0.0

2.0e6

4.0e6

6.0e6

Inte

n...

3.91


200 400 600 800 1000m/z, Da

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.4e6

Inte

nsity

, cps

786.6

283.4

294.2699.6524.5484.6 768.5239.4


200 400 600 800 1000m/z, Da

0.0

5.0e5

1.0e6

1.5e6

Inte

nsity

, cps

786.6

283.3

768.6726.6153.1 523.6419.4


200 400 600 800 1000m/z, Da

0.0

2.0e5

4.0e5

6.0e5

8.0e5

9.7e5

Inte

nsity

, cps

786.6

281.4

768.7522.6390.5311.4 726.8

175 MRMs positive mode10µL plasma

fragmentsnegative modeEPI 786.6

XIC 788.6/184

positive mode

Figure 6. Absolute IDQ™ Kit* used for phospholipid extraction

XIC of +MRM (175 pairs): 251.2/70.3 Da from Sample... Max. 3400.0 cps.

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 7.5Time, min

0.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

8.0e6

Inte

nsity

, cps

x 100.0

3.22 5.233.383.78 6.450.23 2.66 5.982.39 4.951.52 7.780.87 6.63

XIC of +MRM (175 pairs): 846.7/184.0 Da from Sample... Max. 6.8e6 cps.

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 7.5Time, min

0.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

8.0e6

Inte

nsity

, cps

4.35

3.71


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 7.5Time, min

0.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

8.0e68.5e6

Inte

nsity

, cps

4.36

3.923.72


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 7.5Time, min

0.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

8.0e6

Inte

nsity

, cps

3.712.87

blank(peak area 100x enlarged)

Standard Mix

plasma sampleReference

plasma sampleExperiment(1 out 40)Advantages of increased sensitivity for quantitative assessment of Eicosanoids include:

• broader coverage of profiles, • accessibility of low abundant Eicosanoids, • improved reproducibility, • smaller sample volume or • possibility for dilution to avoid matrix effects.Advantages of QTRAP®: fragmentation patterns for structure analysis and/or confirmation.

Figure 3. Increased Sensitivity of Eicosanoid detec tion in serumwith API 5000™ vs. 4000 QTRAP ® (XICs, day 10 after liver transplant)

100 µl serum

PGF2a8-iso-PGF2a

PGE2 PGD2

PGF2a8-iso-PGF2a

PGE2 PGD2

4. reconstitution in 100 µl MeOH

5. MS analysis6. data evaluation

LPS treatment, Surgery (e.g. liver transplantation)....

1. Protein precipitation2. SPE

3. Solvent evaporation

100 µl serum

PGF2a8-iso-PGF2a

PGE2 PGD2

PGF2a8-iso-PGF2a

PGE2 PGD2

4. reconstitution in 100 µl MeOH

5. MS analysis6. data evaluation

LPS treatment, Surgery (e.g. liver transplantation)....

1. Protein precipitation2. SPE

3. Solvent evaporation

TXB2

XIC’s311/267

XIC’s303/259

XIC’s327/116

± 50 MRMs

xn samples

automatedanalysis TXB2

XIC’s311/267XIC’s

311/267


303/259


327/116

± 50 MRMs

xn samples

automatedanalysis

t=0h

t=24h

t=240h

XIC of -MRM (50 pairs): 311.3/267.2 amu from Sample 17 (LTX_8-10) of 08-03-19.wiff (Turbo Spray) Max. 2.9e4 cps.

2 4 6 8 10 12 14 16 18Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

Inte

nsity

, cps

10.7311.33

XIC of -MRM (50 pairs): 311.3/267.2 amu from Sample 16 (LTX8-10 012) of RG_08-26-03 Eico Proben U. Ceglarek API5K.wiff (T... Max. 4.1e5 cps.

2 4 6 8 10 12 14 16 18Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

Inte

nsity

, cps

10.73

XIC of -MRM (50 pairs): 303.2/259.2 amu from Sample 17 (LTX_8-10) of 08-03-1... Max. 1.6e4 cps.

2 4 6 8 10 12 14 16 18Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

Inte

nsity

, cps

12.70

! !- Noise -

S/N = 196.8

Peak Int.(Subt.)=1.6e+4

3xStd.Dev.(Noise)=7.9e+1

XIC of -MRM (50 pairs): 303.2/259.2 amu from Sample 16 (LTX8-10 012) of RG_... Max. 6.5e5 cps.

2 4 6 8 10 12 14 16 18Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

6.5e5

Inte

nsity

, cps

12.71

! !- Noise -

S/N = 1565.9



XIC m/z 303/259arachidonic acid

API 5000™

> 25 peaks

4000 Q TRAP®

8 peaks

XIC of -MRM (50 pairs): 311.3/267.2 amu from Sample 17 (LTX_8-10) of 08-03-19.wiff (Turbo Spray) Max. 2.9e4 cps.

2 4 6 8 10 12 14 16 18Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

Inte

nsity

, cps

10.7311.33

XIC of -MRM (50 pairs): 311.3/267.2 amu from Sample 16 (LTX8-10 012) of RG_08-26-03 Eico Proben U. Ceglarek API5K.wiff (T... Max. 4.1e5 cps.

2 4 6 8 10 12 14 16 18Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

Inte

nsity

, cps

10.73

XIC of -MRM (50 pairs): 303.2/259.2 amu from Sample 17 (LTX_8-10) of 08-03-1... Max. 1.6e4 cps.

2 4 6 8 10 12 14 16 18Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

Inte

nsity

, cps

12.70

! !- Noise -

S/N = 196.8



XIC of -MRM (50 pairs): 303.2/259.2 amu from Sample 16 (LTX8-10 012) of RG_... Max. 6.5e5 cps.

2 4 6 8 10 12 14 16 18Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

6.5e5

Inte

nsity

, cps

12.71

! !- Noise -

S/N = 1565.9



XIC m/z 303/259arachidonic acid

API 5000™

> 25 peaks

4000 Q TRAP®

8 peaks

Figure 4. Evaluation of Eicosanoid concentrations i n serum before and after liver transplantation(API 5000™ & MarkerView™ used for data assessment and analysis)

Eicosanoid profiles directly reveal the timely response of the inflammation process.Left: overlay of chromatograms from replicate samples and different time points showing differences for target molecules (3 examples).Right: MarkerView™ evaluates sample grouping (score plot, upper left) and indicates the biomarker (loading plot, upper right). Abundance of eicosanoids that distinguish samples (e.g., TXB2 elevated at 24h, lower). Such eicosanoids may serve as prognostic marker.

LPS 16 h

LPS 4 h

1) Add 10 µL sample to deep-well plate (Blanks, Standards, QCs or blood plasma)

2) Dry samples (N2 flow) 3) Derivatize (PITC for amino acids)4) Dry samples (N2 flow) 5) Extract metabolites with solvent6) Centrifuge and dilute with Kit running solvent

* Kit is designed to identify and quantify >150 different metabolites in blood plasma. Here only extraction procedure of the kit was used and the provided MRM aquisition methods together with a fast gradient separation on a Synergi 2.5 µµµµm m m m LC column.

Figure 7. Multi-target profiling of metabolites in blood plasma, with > 100 mass transitions related to lysoGPCs, GP Cs or SMs

10µLblood

plasma

XIC of +MRM (175 pairs): 846.7/184.0 Da from Sample 1 (081781_1_0400407385) of KIT1-30-3_081781_85_1_0400407385.wiff (Turbo Spray) Max. 8.4e6 cps.

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 7.5Time, min

0.0

2.0e6

4.0e6

6.0e6

8.0e6

Inte

n...

4.36

3.923.72

398.2/184 496.3/184

522.3/184 563.4/184 788.6/184

703.6/184 760.6/184 816.7/184

846.6/184 815.7/184

XIC of +MRM (175 pairs): 846.7/184.0 Da from Sample 1 (081781_1_0400407385) of KIT1-30-3_081781_85_1_0400407385.wiff (Turbo Spray) Max. 8.4e6 cps.

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 7.5Time, min

0.0

2.0e6

4.0e6

6.0e6

8.0e6

Inte

n...

4.36

3.923.72

398.2/184 496.3/184

522.3/184 563.4/184 788.6/184

703.6/184 760.6/184 816.7/184

846.6/184 815.7/184

Figure 8. Multi-target profiling and automated data analysis of lysoGPCs, GPCs and SMs (>100MRMs)in blood plasma samples

Examples of XICs: single mass transitions can resolve in mutiple chromatographic peaks; left. Automated peak picking and data analysis by MarkerView™ software handles chromatographic peak individually. PCA analysis show the grouping of blood plasma samples (low and high) and standardized reference sample (Score plot) and the relative contribution of the molecules (Q1/Q3(RT)) to the grouping (Loading plot); right.

Score plot Loading plot

Figure 9. Fast Precursor Scanning ( m/z 184amu) for comprehensive lipid class profiling in blood plasma

Figure 10. Fragmentation reveals structural differences of isobaric compounds in blood serum*

Q1 isolation of m/z 482.4amu, fragmentation in Q2 (collision cell) and fragment ion scan with Q3 as linear ion trap.(*example generated with a 4000 QTRAP®)

TIC of +EPI (482.40) CE (40) CES (10): from Sample 26 (EPI... Max. 6.3e6 cps.

2 4 6Time, min

0.0

2.0e6

4.0e6

6.0e6

Inte

nsity

, cps

1.840.35 2.14

+EPI (482.40) CE (40) CES (10): 0.265 to 0.427 min from Sa... Max. 4.6e5 cps.

100 200 300 400 500m/z, amu

20%

40%

60%

80%

100%

Rel

. Int

. (%

)

482.3

143.2 424.3406.3


100 200 300 400 500m/z, amu

20%

40%

60%

80%

100%

Rel

. Int

. (%

)

184.1

482.3464.4

125.0 299.3


100 200 300 400 500m/z, amu

20%

40%

60%

80%

100%

Rel

. Int

. (%

)

482.4

184.1

125.0

TIC EPI 482.4 (MS2)

EPI 482.4RT 2.14min

EPI 482.4RT 0.35min

EPI 482.4RT 1.84min

TIC of +EPI (482.40) CE (40) CES (10): from Sample 26 (EPI... Max. 6.3e6 cps.

2 4 6Time, min

0.0

2.0e6

4.0e6

6.0e6

Inte

nsity

, cps

1.840.35 2.14


100 200 300 400 500m/z, amu

20%

40%

60%

80%

100%

Rel

. Int

. (%

)

482.3

143.2 424.3406.3


100 200 300 400 500m/z, amu

20%

40%

60%

80%

100%

Rel

. Int

. (%

)

184.1

482.3464.4

125.0 299.3


100 200 300 400 500m/z, amu

20%

40%

60%

80%

100%

Rel

. Int

. (%

)

482.4

184.1

125.0

TIC EPI 482.4 (MS2)

EPI 482.4RT 2.14min

EPI 482.4RT 0.35min

EPI 482.4RT 1.84min

Figure 11. Polarity switching for quantitation of c ompounds in both ionization modes in a single run without compromising sensitivity

Figure 12. Fast polarity switching enables quantita tion of compounds in positive ionization mode and structure evaluation in negative ionization mode to be done simultaneously in a single run

XIC of +Prec (184.10): Exp 1, 496.75 to 497.25... Max. 4.0e5 cps.

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0Time, min

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.3e6

Inte

nsity

, cps

3.18

4.30 8.366.49 6.835.222.49


1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0Time, min

0.00

2.00e5

4.00e5

6.00e5

8.00e5

1.00e6

Inte

nsity

, cps

4.36

8.295.233.102.76

+ER (535.4... Max. 9.0e6 cps.

530 540m/z, Da

0.0

2.0e6

4.0e6

6.0e6

8.0e69.0e6

Inte

nsity

, cps

534.6

535.5

536.5

+ER (761.3... Max. 8.5e6 cps.

760 765 770m/z, Da

0.0

2.0e6

4.0e6

6.0e6

8.0e6

Inte

nsity

, cps

760.8

761.7

762.7

+ER (720.2... Max. 2.3e6 cps.

715 720 725m/z, Da

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.3e6

Inte

nsity

, cps

721.7

722.7725.3720.0

+ER (761.3... Max. 8.5e6 cps.

780 785 790 795m/z, Da

0.0

1.0e6

2.0e6

3.0e6

3.5e6

Inte

nsity

, cps

786.8

787.7

788.7

-EPI (532.5... Max. 2.0e7 cps.

200 400 600m/z, Da

0.0

1.0e7

2.0e7

3.0e7

Inte

nsity

, cps 532.4

294.4

502.6

251.4 309.6 475.4

-EPI (758.7... Max. 2.4e7 cps.

200 400 600m/z, Da

0.0

1.0e7

2.0e7

3.0e7

Inte

nsity

, cps

758.4

281.2

255.2

362.2 714.8153.2 295.8 494.3

-EPI (719.7... Max. 3.0e7 cps.

200 400 600m/z, Da

0.0

1.0e7

2.0e7

3.0e7

Inte

nsity

, cps

701.4

719.4

279.2168.2 415.3 634.4

-EPI (784.7... Max. 1.5e7 cps.

200 400 600m/z, Da

0.0

1.0e7

2.0e7

3.0e7

Inte

nsity

, cps

784.6

281.3

522.5295.3255.2 622.7

Exp. 1: positive mode; PrecIon Scan of 184 amuOverlay of 30 XICs

Exp. 3: positive mode; Enhanced Resolution (2 most abundant Peaks at a given time)

Exp. 4&5: negative mode; Enhanced Product Ion Scan (2 most abundant Peaks at a given time)

Exp. 2: positive mode;PrecIon Scan of 264 amuOverlay of 16 XICs


1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0Time, min

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.3e6

Inte

nsity

, cps

3.18

4.30 8.366.49 6.835.222.49


1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0Time, min

0.00

2.00e5

4.00e5

6.00e5

8.00e5

1.00e6

Inte

nsity

, cps

4.36

8.295.233.102.76

+ER (535.4... Max. 9.0e6 cps.

530 540m/z, Da

0.0

2.0e6

4.0e6

6.0e6

8.0e69.0e6

Inte

nsity

, cps

534.6

535.5

536.5

+ER (761.3... Max. 8.5e6 cps.

760 765 770m/z, Da

0.0

2.0e6

4.0e6

6.0e6

8.0e6

Inte

nsity

, cps

760.8

761.7

762.7

+ER (720.2... Max. 2.3e6 cps.

715 720 725m/z, Da

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.3e6

Inte

nsity

, cps

721.7

722.7725.3720.0

+ER (761.3... Max. 8.5e6 cps.

780 785 790 795m/z, Da

0.0

1.0e6

2.0e6

3.0e6

3.5e6

Inte

nsity

, cps

786.8

787.7

788.7

-EPI (532.5... Max. 2.0e7 cps.

200 400 600m/z, Da

0.0

1.0e7

2.0e7

3.0e7

Inte

nsity

, cps 532.4

294.4

502.6

251.4 309.6 475.4

-EPI (758.7... Max. 2.4e7 cps.

200 400 600m/z, Da

0.0

1.0e7

2.0e7

3.0e7

Inte

nsity

, cps

758.4

281.2

255.2

362.2 714.8153.2 295.8 494.3

-EPI (719.7... Max. 3.0e7 cps.

200 400 600m/z, Da

0.0

1.0e7

2.0e7

3.0e7

Inte

nsity

, cps

701.4

719.4

279.2168.2 415.3 634.4

-EPI (784.7... Max. 1.5e7 cps.

200 400 600m/z, Da

0.0

1.0e7

2.0e7

3.0e7

Inte

nsity

, cps

784.6

281.3

522.5295.3255.2 622.7

Exp. 1: positive mode; PrecIon Scan of 184 amuOverlay of 30 XICs

Exp. 3: positive mode; Enhanced Resolution (2 most abundant Peaks at a given time)

Exp. 4&5: negative mode; Enhanced Product Ion Scan (2 most abundant Peaks at a given time)

Exp. 2: positive mode;PrecIon Scan of 264 amuOverlay of 16 XICs

Examples for separation and detection of compounds in positive mode based on 175 mass transitions (scan time = 0.82 sec/data point) and internal standards included in the kit.

Combination of multi-target profiling in the positive mode with fragmentation in the negative mode enables highest sensitivity and highest selectivity in phospholipid analysis (MRMs, left). Fast polarity switching (50ms) and IDA workflow also supports precursor ion scans to be combined with fragmentation in the opposite ionisation mode (Prec, right). The enhanced resolution scan (ER) determines here the mass for fragmentation as m/z+-2amu. Applications did run on a QTRAP® 5500 with different lipid extracts and LC methods used.

Standard Mix of AbsoluteIDQ™ Kit used to compare a positive mode run with a polarity switching run (left) and S/N values for 2 peaks (right)

XIC of +MRM (175 pairs): 403.3/85.1 Da from Sample 1 (0... Max. 6.1e5 cps.

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 7.5Time, min

0.0

1.0e5

2.0e5

3.0e5

4.0e5

5.0e5

6.0e5

Inte

nsity

, cps

2.10

! !- Noise -

S/N = 1874.2



XIC of +MRM (175 pairs): 622.4/184.0 Da from Sample 1 (... Max. 4.0e6 cps.

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 7.5Time, min

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

Inte

nsity

, cps

2.94

! !- Noise -

S/N = 6142.5Peak Int.(Subt.)=4.0e+6


XIC of +MRM (175 pairs): Exp 1, 403.3/85.1 Da from Samp... Max. 1.2e6 cps.

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 7.5Time, min

0.00

2.00e5

4.00e5

6.00e5

8.00e5

1.00e6

1.20e6

Inte

nsity

, cps

2.01

! !- Noise -

S/N = 2923



XIC of +MRM (175 pairs): Exp 1, 622.4/184.0 Da from Sam... Max. 3.7e6 cps.

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 7.5Time, min

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

Inte

nsity

, cps

2.84

! !- Noise -

S/N = 5005.6



Pos from pos/neg switch

Pos only

6.25pmol on column

XIC of +MRM (175 pairs): 622.4/184.0 Da from Sample 1 (081781_1_20000338) of KIT1-30-3_081781_25_1_200003... Max. 7.8e6 cps.

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 7.5Time, min

0.0

2.0e6

4.0e6

6.0e6

8.0e6

Inte

n...

2.94

XIC of +MRM (175 pairs): Exp 1, 622.4/184.0 Da from Sample 1 (081781_1_20000338) of KIT1-30-3_081781_25_1_2... Max. 8.5e6 cps.

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 7.5Time, min

0.0

2.0e6

4.0e6

6.0e6

8.0e6

Inte

n...

2.84

XIC of -MRM (15 pairs): Exp 2, 179.0/89.0 Da from Sample 1 (081781_1_20000338) of KIT1-30-3_081781_25_1_200... Max. 7.3e4 cps.

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 7.5Time, min

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

Inte

n...

0.28

175 MRMsonly positive mode

175 MRMs positive mode

& 15 MRMs negative mode

50ms constant polarity switch in 1 run

XIC of +MRM (175 pairs): 622.4/184.0 Da from Sample 1 (081781_1_20000338) of KIT1-30-3_081781_25_1_200003... Max. 7.8e6 cps.

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 7.5Time, min

0.0

2.0e6

4.0e6

6.0e6

8.0e6

Inte

n...

2.94

XIC of +MRM (175 pairs): Exp 1, 622.4/184.0 Da from Sample 1 (081781_1_20000338) of KIT1-30-3_081781_25_1_2... Max. 8.5e6 cps.

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 7.5Time, min

0.0

2.0e6

4.0e6

6.0e6

8.0e6

Inte

n...

2.84

XIC of -MRM (15 pairs): Exp 2, 179.0/89.0 Da from Sample 1 (081781_1_20000338) of KIT1-30-3_081781_25_1_200... Max. 7.3e4 cps.

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 7.5Time, min

0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

Inte

n...

0.28









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