FIGURE 9. Thermo Scientific TraceFinder Software for Targeted Analysis. A compound database is selected dependant on the compound class, tissue or pathway under

study . The customizable database contains all the information for batch acquisition and data

processing. Data review is efficient with flags indicating where there may be problems..

Conclusions

Ultra-high resolution accurate mass data from Orbitrap™

detectors provides the most reliable analysis of endogenous

metabolites and lipids

Component workflow decreases false positives due to chemical

background and redundant signals

Find the real differences in components across multiple sample

groups with SIEVE 2.0

MSn on hybrid Orbitraps and Mass Frontier 7.0 provide a complete

set of tools for structure elucidation

References1. D. A. Peake, J. Wang, P. Huang, A. Jochem, A. Higbee, D. J.

Pagliarini, Lipid Maps Annual Meeting, May 7-8, 2012, La Jolla,

California, USA

2. J. R. Bain, R. D. Stevens, B. R. Wenner, O. Ilkayeva, D. M. Muoio

and C. B. Newgard, Diabetes, 2009, 58, 2429-2443.

3. K Suhre and P Schmidt, MassTrix: Mass Translator Into Pathways,

Nucleic Acids Res, Vol. 36, Web Server Issue, W481-W484, 2008

Acknowledgements

We would like to thank Dr. Christopher Petucci from the Sanford-

Burnham Institute for supplying the ZDF rat data.

Introduction

Application of metabolomics to disease phenotype analysis and

identification of unique biomarkers to distinguish healthy individuals

compared to those with a disease has renewed the promise of

personalized medicine. We present here the application of a Thermo

Scientific benchtop quadrupole-Orbitrap mass spectrometer coupled to

a UHPLC and Thermo Scientific SIEVE software for several

metabolomics and lipidomics studies. The performance of the entire

analytical platform is illustrated with relevant examples including the

metabolomic analysis of lean and obese Zucker rat serum and

mitochondrial lipids in Yeast.

Methods

Sample Preparation

Metabolites were extracted from plasma with cold methanol and the

supernatant was removed, dried under nitrogen, and reconstituted with

80:20 water/methanol for LC/MS. Mitochondrial lipids were isolated

from wild-type (WT) yeast (S. cerevisiae) and a knockout (KO) strain

that does not produce coenzyme Q (CoQ), by extraction using

isopropanol (1).

Liquid Chromatography Mass Spectrometry

High-resolution accurate-mass full scan LC-MS and LC-MS/MS

analyses were performed with short UHPLC gradient at 70,000

resolving power on the Thermo Scientific Q Exactive mass

spectrometer in both positive and negative ion modes with

electrospray ionization source. MS-MS data was obtained at 35,000

resolution.

Data Analysis

All of the datasets were analyzed with the SIEVE™ 2.0 software with

the Component Extraction algorithm designed specifically for optimal

data analysis in untargeted metabolomics experiments. Components

that were significantly different between the sample groups were

detected and identified via local or online database search. MS/MS

data were then acquired and used to confirm the structure of these

components. Thermo Scientific Mass Frontier 7.0 RS1 software

provided spectral interpretation tools including MS/MS library search

and assigning structures to the fragment ions automatically in the

MS/MS spectrum. Thermo Scientific TraceFinder 2.1 software was

then used for streamlined targeted quantitative analysis.

Results

Analysis of the ZDF fatty rat serum compared to normal rat serum

served as a benchmarking study to determine the merits of the

technology platform. We observed an increase in acylcarnitines,

branched chain amino acids (2), phospholipids, fatty acids and

conjugated bile acids (P < 0.001) in fatty ZDF rat plasma relative to

normal ZDF rats (Table 1).

Preliminary analysis of yeast mitochondrial lipid extracts demonstrates

the ability to obtain statistically significant results with a single injection

of each biological replicate. The expected change in CoQ6 levels was

accompanied by changes in over 60 different components including

amino acids, acylglycerols, sterols, phospholipids and sphingolipids.

Mass Frontier is a trademark of High Chem Ltd. 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 1. Grouping of Adducts and Isotopes, m/z 169.0355, Uric Acid.Uric acid, identified by ChemSpider search, increased 3-fold in Obese vs. Normal rats.

Applying Q Exactive Benchtop Orbitrap LC-MS/MS and SIEVE Software for Cutting Edge

Metabolomics and Lipidomics Research

Michael Athanas, David A Peake, Mark Dreyer, Mark Sanders, Jessica Wang and Yingying Huang

Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, CA 95134, USA

FIGURE 8. Significant Metabolite Differences Found in WT vs. KO Yeast. Relative amounts of metabolites from KO Yeast (dark blue) vs. WT Yeast (light blue) increased

for sterols, ceramides and Co-Q9 whereas histidine, sphinganine and CoQ6 decreased in the

KO yeast.

FIGURE 5. ZDF Rat Metabolomics Data – Pathway Analysis (3)Annotated metabolites in Red were found from MW search using ChemSpider.

FIGURE 2. Workflow for Metabolomics and Lipidomics Experiments.

Component Component ExtractionExtraction

Pathway AnalysisPathway AnalysisLCLC--MSMS AnalysisAnalysis

AlignTotal Ion Current

Alignment

Detect Component Extraction

Identify Library search &

MS/MS confirmation

Features Components

FIGURE 4. LC-MS/MS of Kynurenine, m/z 209.1 from Rat Serum.

Mass Frontier software was used to annotate the accurately-measured fragment ions giving

excellent confirmation of the known structure from a single MS-MS scan.

AIM084_Plasma_pos_017_Target_35k_1 #133 RT: 2.39 AV: 1 NL: 4.19E5F: FTMS + p ESI Full ms2 209.09@hcd35.00 [50.00-230.00]

60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210

m/z

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

240000

260000

280000

300000

320000

340000

360000

380000

400000

Re

lative

Ab

un

da

nce

94.0651

C 6 H8 N

-0.0141 ppm

146.0601

C 9 H8 O N

0.3332 ppm

136.0757

C 8 H10 O N

0.2857 ppm

174.0549

C 10 H8 O2 N

-0.0468 ppm

192.0655

C 10 H10 O3 N

-0.1509 ppm120.0444

C 7 H6 O N

0.1693 ppm

150.0550

C 8 H8 O2 N

0.4542 ppm74.0239

C 2 H4 O2 N

3.2468 ppm 118.0651

C 8 H8 N

-0.3344 ppm99.0077

C 4 H3 O3

-0.0298 ppm163.0865

C 9 H11 O N2

-0.4427 ppm84.9597

132.0446

C 8 H6 O N

1.5984 ppm

104.0495

C 7 H6 N

-0.1385 ppm

ON 2H N 2H

O

OH

O

OH

O N 2H

O

O N 2H

N 2H O N 2H

N 2HO

OH

N 2HO

O 2H N 2H

O N 2H

N 2H

NH

N 2H

N 2H

O

OH

N 2H

O

FIGURE 3. Principal Components Analysis of Lean vs. Obese

ZDF rat serum. The Principle Components Analysis (PCA) results shows that

the LCMS analysis of the two ZDF rat serum groups are clearly different.

Lean Obese

CSID Compound Name MW MZ Time Ratio p-Value

63798 Imidazole propionic acid 140.0585 141.0658 0.83 0.418 2.45E-04

757 Homocysteine 135.0352 136.0425 0.68 0.485 8.32E-04

6050 Valine 117.0792 118.0865 0.61 0.655 5.44E-06

718 Glutamine 146.0690 147.0762 0.59 0.688 1.58E-04

96904 Propionyl carnitine 217.1314 218.1387 1.90 0.768 8.31E-04

2006614 Carnitine 161.1050 162.1123 0.62 1.203 5.44E-05

969 Phenylalanine 165.0789 166.0861 2.65 1.273 1.69E-04

834 Leucine 131.0946 132.1019 1.54 1.448 1.40E-04

1142 Uric acid 168.0282 169.0355 0.93 1.656 1.34E-06

24766528 C18:3 Lyso PC 517.3170 518.3243 9.78 1.980 2.18E-04

1141 Uracil 112.0275 113.0348 0.86 2.015 2.49E-06

5858 Adenonsine monophosphate 347.0625 348.0698 0.83 2.377 1.30E-05

388877 Butyl carnitine 231.1469 232.1541 3.54 2.967 2.73E-06

9931788 C20:5 Lyso PC 541.3168 542.3240 9.69 4.368 7.88E-04

Table 1. Compounds Identified via ChemSpider Database Search. Metabolites that significantly decrease (red) or increase (green) in ZDF Obese vs.

Normal Rats (p < 0.001).

FIGURE 6. Chromatographic Alignment of Yeast Lipid Extract LC-MS Data

Ergosta-5,7,22,24(28)-tetraen-3β-ol

Co-Q6 (oxid.) Co-Q9 (oxid.)

d18:0/16:0 Ceramide

Sphinganine

p = 0.003

p = 0.028

p = 0.020

p = 0.021

p = 0.006

p = 0.024

Histidine

p = 0.003

FIGURE 7. MS-MS Identification of PE 34:2, m/z 714.51

Excellent mass measurement and ID from a single MS-MS scan.

NL: 6.35 E+4

RT: 12.90 #4260

714.51 HCD@35.0

+ESI Full MS2

12WTPost_1 #4260 RT: 12.90 AV: 1 NL: 6.35E4F: FTMS + p ESI Full ms2 714.51@hcd35.00 [100.00-745.00]

250 300 350 400 450 500 550

m/z

0

10

20

30

40

50

60

70

80

90

100

Rela

tive A

bundance

573.4886C 37 H 65 O 4

1.5877 ppm

306.2793C 20 H 36 O N

0.6077 ppm237.2211C 16 H 29 O

-0.9389 ppm280.2638

C 18 H 34 O N

0.9713 ppm

337.2744C 21 H 37 O 3

1.8724 ppm

263.271

C 18 H 31 O

0.4498 ppm311.2577

C 19 H 35 O

-1.3222 ppm

PE (16:1/18:2)18:2

16:1

(M+H) –141.0191