8/14/2015

1

HPLC-MS/MS and Metabolomics

Yu Cao Ph.D.

CCIC MS&P Summer WorkshopAugust 17, 2015

Publication in Metabolomics

0

2000

4000

6000

8000

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014

Number of publications per yearSearch engine: Web of ScienceTopic: “metabolomics”

8/14/2015

2

Applications of metabolomics in cancer researchKathleen A. Vermeersch and Mark P. StyczynskiJ. Carcinogenesis, 2013, 12, doi: 10.4103/1477-3163.113622

Metabolomics short course at ASMS 2014

8/14/2015

3

INNOVATION

Metabolomics: the apogee of the omics trilogyGary J. Patti, Oscar Yanes and Gary SiuzdakMolecular Cell Biology, 2012, 13, 263-269

Sample preparation

8/14/2015

4

Sample Preparation Sample prep often most consuming (difficult) step

Sample prep/clean-up separates interfering species from analyte Example: analysis of drug and metabolites in plasma need to

remove protein interferences

Off-line or in-line from MS/MS detection

Sample prep/clean-up to concentrate analyte Example: Pesticides in drinking water

Basic principle of sample prep involves preferential binding of analyte over interfering species or vice versa, followed by elution to MS/MS

Separation technologies

essential in sample prep

“General Considerations” when dealing with samples for MS or MS/MS

Ionization method?

Instrument availability

Sensitivity/Quantification

Time of analysis

“In vivo” sample pre-MS treatment?

Chromatographic/extraction methods: the shorter, the better

GC, HPLC, zip-tip, solid phase extraction (SPE), dialysis, etc.

Derivatization: the simpler, the better

to increase volatility (GC); to study neutral loss; to increase ionization efficiency

8/14/2015

5

MethodSeparation based

onSeparation done

usingFurther steps

Liquid-liquid Extraction

Partitioning in one of two liquid phases

Glassware

Types of Separation Technologies for Molecules

Liquid-Liquid Extraction

An immiscible solvent

is added to the sample

which then separates into 2

distinct liquid phases.

Some sample analytes will

go into the bottom

phase (Aqueous), some will separate

into the top phase (Organic)

Large solvent consumption

Time/labor intensive

May need evaporation step

>1 extraction if mixture of analytes

Emulsions and contamination issues

8/14/2015

6

MethodSeparation based

onSeparation done

usingFurther steps

Liquid-liquid Extraction

Partitioning in one of two liquid phases

Glass ware

Solid-phase Extraction

Adsorption/ partitioning onto solid sorbent

Cartridges, disks, filters, plates

Types of Separation Technologies for Molecules

Solid-Phase Extraction

Uses chromatographic particles

Packed-bed column cartridges

or similar

Well established commercial

technology (1978)

1000s literature refs

Clean extracts

Good recovery for polar analytes

Sample must be in liquid state

Driving force: gravity, pressure, vacuum

Automation

cartridges

96 well plate

disk

http://solutions.3m.com/wps/portal/3M/en_US/Empore/extraction/

8/14/2015

7

Solid-Phase Extraction

Types of Chromatography

Normal Phase

Non-polar mobile phase

Polar stationary phase

Reversed Phase Most common

Polar mobile phase

Non-polar stationary phase

Ion Exchange

Buffer/Ionic mobile phase

Cationic/Anionic exchange stationary phase

Manufacturer Brand Name

Waters SEP-PAKOASIS

Varian BondElute

Baker BakerBond

3M Empore

Supelco Supelclean

+ Many Others

Solid-Phase Extraction - common protocol

Procedure

Sample

Prepare: Homogenize, suspend,

centrifuge, etc…

Load onto conditioned cartridge

Wash off weakly retained interferences

with weak solvent

Elute product with strong solvent

Analyze: HPLC, GC-MS, LC-MS/MS

8/14/2015

8

pure analyte(control)

engine oil contaminatedparking lot oil

same as b) after organicmatter removal by SPE

(KNO3)nK+

Gapeev, A. and Yinon, J. J. Forensic Sci. 2004, 49

MethodSeparation based

onSeparation done

usingFurther steps

Liquid-liquid Extraction

Partitioning in one of two liquid phases

Glass ware

Solid-phase Extraction

Adsorption/ partitioning onto solid sorbent

Cartriges, disks, filters, plates

Dialysis/UltrafiltrationMolecular weight/size

SlideAlyzer/tubing

Types of Separation Technologies for Molecules

8/14/2015

9

Tubing or Slide A-LyzerDiff MWCO ranges0.1– 0.5 mL capacityUseful for biologicals

Sample

loading

here

Dialysis

Spin filters

polyethersulfone membrane

(Vivaspin, ex)

volumes from 100 μl to 20 ml,

with a range of molecular

weight cutoff values from Mr = 3 000 - 100 000

MethodSeparation based

onSeparation done

usingFurther steps

Liquid-liquid Extraction

Partitioning in one of two liquid phases

Glass ware

Solid-phase Extraction

Adsorption/ partitioning onto solid sorbent

Cartriges, disks, filters, plates

Dialysis/UltrafiltrationMolecular weight/size

SlideAlyzer, tubing, spin filter

Precipitation Solubility

Types of Separation Technologies for Molecules

8/14/2015

10

HPLC

Mechanism, Method Setup, Examples

Retention Mechanisms

Phenomenex

8/14/2015

11

Retention Mechanisms

Phenomenex

Mobile Phase Composition

Phenomenex

8/14/2015

12

Phenomenex

Phenomenex

8/14/2015

13

Generic LC Method Development

Column selection

Bedding chemistry, dimension, bead size

Buffers (mobile phase)

Ionic strength, pH, pairing reagent

Fine tune the method

Temperature

Gradient slope

pH Selectivity

Waters Corporation

8/14/2015

14

pH Selectivity

Waters Corporation

Organic Modifier Selection

Waters Corporation

8/14/2015

15

Chart of Mobile Phase Selection

Waters Corporation

Column Configurations and Applications

Column Type

ID (mm) Length (mm)

Particle Size (m)

Flow Rate Ranges

Applications Sensitivity Increase

Nano 0.1-0.075 150 3.5 100-600 nL/min

Proteomics, Sample Limited PTM Characterization

2000-3700

Capillary 0.3, 0.5 35-250 3.5, 5 1-10 L/min

Peptide Mapping LC/MS

100

Micro Bore 1.0 30-150 3.5, 5 30-60 L/min

High Sensitivity LC/MS

20

Narrow Bore

2.1 15-150 3.5, 5 0.1-0.3 mL/min

Sample Limited. LC/MS

5

Analytical 4.6 15-250 3.5, 5 1-4 mL/min

Analytica; 1

Semi-prep 9.4 50-250 5 4-10 mL/min

Small Scale protein purification

--

Preparative 21.2 50-250 5, 7 20-60 mL/min

CombiChem purification

--

8/14/2015

16

Column Comparison by Vendors

Imtakt

Phenomenex

Waters

Others

Details refer to the pdf files

http://www.imtaktusa.com/products/

http://phx.phenomenex.com/lib/po26681014_w.pdf

http://www.waters.com/webassets/cms/library/docs/720002241en.pdf

LC Tips on Peak Shape Issues

8/14/2015

17

LC Tips on Peak Shape Issues

LC Tips on Peak Shape Issues

8/14/2015

18

Metabolomics in MS&P

Projects going on

Eerie lake project

Amino acid analysis in animal models

Oligonucleotide project

Unknown identification in smoker plasma

Honey bee project

Drug stability

TMAO analysis for breast cancer study

Sample type:

Plant extractCoating materialEnvironmentalBio-fluidTissues

etc.

Mass spec analysis

Untargeted metabolomics: Q-TOF

Targeted metabolomics: Q-TOF and QQQ

8/14/2015

19

Q-TOF

Benefits:

Higher resolution & mass accuracy

All ions recorded in parallel

Ref: Chemushevich, 2001

Q1 q2 TOF

Chromatogram of Oligonucleotides stdon Q-TOF

Sample: Bruker standard 217028 and 206200 (MW: 1K~10K Da)

0 5 10 15 20 25 30 Time [min]

0.5

1.0

1.5

2.0

4x10Intens.

Olistd_040815_03_BE4_01_171.d: BPC -All MS

1

2

3

4

5

6

7

8

8/14/2015

20

MS of Peak 1 at 2.4 min

402.9755

585.6075 1172.2196

1300.1652

‐MS, 2.4min #145

1172.2196

1172.7220

1173.2211

1174.22371175.2245

‐MS, 2.4min #145

585.6075

586.1094

586.6119587.1097

‐MS, 2.4min #145

0.00

0.25

0.50

0.75

4x10Intens.

0.00

0.25

0.50

0.75

1.00

4x10

0.0

0.2

0.4

0.6

0.8

4x10

400 600 800 1000 1200 1400 m/z

1171 1172 1173 1174 1175 1176 1177 m/z

585.0 585.5 586.0 586.5 587.0 587.5 588.0 m/z

MS of Peak 6 at 19.0 min

402.9783555.7557

667.1091834.1345

981.9848

1112.5167

1395.7077

1669.2783

‐MS, 19.0min #1138

1668.7765

1669.2783

1669.7776

1670.2807

1670.78581671.2796 1671.7823

‐MS, 19.0min #1138

1112.1838

1112.5167

1112.6720

1112.8509

1113.1849

1113.5211

1113.8554 1114.1830

‐MS, 19.0min #1138

0.00

0.25

0.50

0.75

4x10Intens.

0.00

0.25

0.50

0.75

1.00

4x10

0

2000

4000

500 750 1000 1250 1500 1750 2000 2250 2500 2750 m/z

1668.0 1668.5 1669.0 1669.5 1670.0 1670.5 1671.0 1671.5 1672.0 1672.5 m/z

1111.5 1112.0 1112.5 1113.0 1113.5 1114.0 1114.5 1115.0 1115.5 m/z

8/14/2015

21

Untargeted metabolomicsRPLC-MS

L_6996_062014_01_BB5_01_1132.d: BPC -All MS

L_6996_061914_01_BB5_01_1108.d: BPC +All MS0

2

4

6

5x10Intens.

0.0

0.2

0.4

0.6

0.8

6x10Intens.

0 10 20 30 40 50 Time [min]

MS -

MS +

311.1698

452.2813

540.3352

L_6996_062014_01_BB5_01_1132.d: ‐MS, 32.7min #1963

0.0

0.2

0.4

0.6

0.8

1.0

5x10Intens.

200 400 600 800 1000 1200 1400 m/z

RPLC-MS at 32.7 min

454.2978

496.3465

620.4440

664.4708

708.4972

991.6849

L_6996_061914_01_BB5_01_1108.d: +MS, 32.7min #1965

0

2

4

6

5x10Intens.

200 400 600 800 1000 1200 1400 m/z

MS -

MS +

8/14/2015

22

Untargeted HILIC-LC-MS

L_6996_062214_01_BB5_01_1181.d: BPC -All MS

L_6996_062314_01_BB5_01_1206.d: BPC +All MS0

2

4

6

5x10Intens.

0.0

0.2

0.4

0.6

0.8

6x10Intens.

0 5 10 15 20 25 30 35 Time [min]

MS -

MS +

133.0133

242.0782

302.0988

611.1400

L_6996_062214_01_BB5_01_1181.d: ‐MS, 18.2min #1092

0.0

0.5

1.0

1.5

2.0

4x10Intens.

200 400 600 800 1000 1200 1400 m/z

198.1228

258.1098

515.2127

L_6996_062314_01_BB5_01_1206.d: +MS, 18.2min #1093

0

1

2

3

5x10Intens.

200 400 600 800 1000 1200 1400 m/z

HILIC-MS at 18.2 min

MS -

MS +

8/14/2015

23

Chromatogram of m/z at 258.1098– RPLC v.s. HILIC

L_6996_061914_01_BB5_01_1108.d: EIC 258.1100±0.1 +All MS

L_6996_062314_01_BB5_01_1206.d: EIC 258.1100±0.1 +All MS0.0

0.5

1.0

1.5

4x10Intens.

0

1

2

3

5x10Intens.

0 10 20 30 40 50 Time [min]

Targeted metabolomics

min5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80

%

0

100

F3:MRM of 1 channel,ES+TIC

12182014_30349_STD_GS_IS L_50 sample after GS, centrifuge, filter, fresh prep in 50%B

2.174e+007IS;6.25;2232479.75;21550402

min

%

0

100

F1:MRM of 1 channel,ES+TIC

12182014_30349_STD_GS_IS L_50 sample after GS, centrifuge, filter, fresh prep in 50%B

3.462e+006Clo;6.14;382144.81;3449917

min

%

0

1004.446e+006Imi;6.24;439232.47;4237493

5.84N+

O

HNN

NN

Cl

Imidacloprid

Imidacloprid-d4 (Int. Std.)

N+

O O-

HNN

NN

Cl

D DDD

NH

NH

NN+O

-OS

NCl

Clothianidin

O-

256.0/209.0

250.0/169.0

260.0/213.0

What are we detecting and which instrument is used???

8/14/2015

24

QQQ

Benefits:

Simple, ion filter

Good for quantification

Q1 q2

Q3

MS/MS Scan Modes

Select

Select

Select Select

Scan

Scan

Scan Scan

Dissociate

Dissociate

Dissociate

Dissociate

Product Ion Scan

Neutral Loss Scan

Precursor Ion Scan

Selected ReactionMonitoring (SRM)

8/14/2015

25

Selected Reaction Monitoring

Q1 Q3(gas)Source Detector

Select one m/z

(fixed Vac/Vdc)

MS/MS at different collision energies

Thermo provided data base

8/14/2015

26

Quantitation upon AUC

Compound name: ImiCorrelation coefficient: r = 0.998517, r^2 = 0.997036Calibration curve: 0.730897 * x + 0.110503Response type: Internal Std ( Ref 4 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Include, Weighting: 1/x, Axis trans: None

ng/mL-0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500

Re

spo

nse

-0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

360

Compound name: CloCorrelation coefficient: r = 0.999227, r^2 = 0.998455Calibration curve: 0.67092 * x + 0.0383766Response type: Internal Std ( Ref 4 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Include, Weighting: 1/x, Axis trans: None

ng/mL-0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500

Re

spo

nse

-0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

Std curveQCsSamplesBlanks

Bioinformatics in Metabolomics

8/14/2015

27

ESI-MSn Libraries# Spectra # Compounds Notes

METLIN > 68,000 13,048 Public, no download

mzCloud > 416,000 2,975 Public, no download

MoNA > 236,000 69,946 Public, no download

MassBank > 28,100 > 43,000 Public, downloadable

HMDB 9,500 Public, downloadable

GNPS > 9,000 ~2200 NP Public, downloadable

ReSpect (MSn) > 9,000 3,595 Public, downloadable

NIST14 MS/MS > 234,000 9,344 Commercial

Wiley MSforID > 10,000 > 1,200 Commercial

MetabolomicsWorkBench Public, no download

LipidBlast 200,000 Public, downloadable

Lipid Maps 37,500 Public, downloadable

LipidSearch >1500,000 lipid ions Commercial

Database search – Progenesis QI

http://www.nonlinear.com/progenesis/qi/how-it-wor

8/14/2015

28

Import data

Data alignment

MS -MS +

8/14/2015

29

Peak picking

MS -MS +

Review deconvolution

8/14/2015

30

Identify compounds

Review compounds

8/14/2015

31

Pathway analysis

Other Computational Tools for Metabolomics

MS-DIAL

Data dependent and/or independent MS/MS experiments

MS-FINDER

GC/MS data alignment, database search (commercial)

8/14/2015

32

Anal. Chem. 2014, 86, 9583-9589

Anal. Chem. 2014, 86, 9358-9361

8/14/2015

33

Thank you!