HPLC-MS/MS and Metabolomics - Ohio State University › sites › default › files › CCIC_metabolomics.… · Waters SEP-PAK OASIS Varian BondElute Baker BakerBond 3M Empore Supelco

HPLC-MS/MS and

Metabolomics

Yu Cao, Ph.D.

CCIC MS&P Summer Workshop

July 2016

Publication in Metabolomics

Number of publications per year

Search engine: Web of Science

Topic: “metabolomics”

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2000

4000

6000

8000

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015

Applications of metabolomics in cancer researchKathleen A. Vermeersch and Mark P. Styczynski

J. Carcinogenesis, 2013, 12, doi: 10.4103/1477-3163.113622

Metabolomics short course at ASMS 2014

INNOVATION

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

Sample Preparation

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

MethodSeparation based

on

Separation 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


on

Separation 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


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/

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-PAK

OASIS

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

pure analyte

(control)

engine oil contaminated

parking lot oil

same as b) after organic

matter removal by SPE

(KNO3)nK+

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


on

Separation 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/sizeSlideAlyzer/tubing


Tubing or Slide A-Lyzer

Diff MWCO ranges

0.1– 0.5 mL capacity

Useful 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


on

Separation 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


Blood – Plasma and Serum (~80% in metabolomics study)

Whole

blo

od

anti-coagulant

(EDTA or heparin

or citrate)

unclotted blood

clotted blood

natural

process of

clotting at room

temperature, 30-

60 min

• 60% in metabolomics

• Plasma collection can be more reproducible

• More proteins

• Anticoagulants may affect separation and detection

• 20% in metabolomics

• Possible enzymatic

degradation

• Possible loss of some

metabolites during clot

Dajana Vuckovic (2012) Anal Bioanal Chem, 403, 1523

adapted from Junhua Wang, Ph.D. at Thermo

The Choice of Anti-coagulants Affect the Analysis

http://www.bioreclamationivt.com/search/all/rat%20plasma

Citrate peaksevere suppression of the underneath peak

using both reversed-phase HPLC and HILIC

Vuckovic, (2011) Anal. Chem.

EDTA peak

large peak with a retention time of ~10 min

cause severe ionization suppression

Pereira, (2010) Metabolomics

Lithium Heparin

significant loss of features in the 500–650-s

region in negative ESI mode

Wedge, (2011) Anal. Chem.

Example: commercially available rat plasma


Serum and Plasma – Deproteinization

http://www.ionsource.com/tutorial/ms

quan/prep.htm

Want, Siuzdak (2006) Anal. Chem. 78:743

Protein removal efficiency Metabolite

coverage

methanol (98 %) High-2056

ethanol (96 %) Higher-1919

acetonitrile (94 %) Medium-1606

acetone/methanol (95 %) High-2136

heat and acid treatment (98 %) Low-942

1:3 plasma: solvent (v/v)

1:2 to1:5 used in

literatures


95% water

typically protein free

urea, creatinine and uric acid typically present as products of nitrogen metabolism and needed to be removed from the body

urea present at up to 2%

salts Na, Cl, K, PO4, SO3 I, NH3

pH 4-8,

meat diets tend to produce a lower pH than vegetarians

other metabolites – typically lower MW species such as amino and organic acids and lower concentrations of high MW lipids (cf serum)

A relatively “simple” biofluid

Urine (7-8% in metabolomics study)

No, still a lot of proteins! If you dry down, you will see them!

Actually detect many species

that are different from blood.

adapted from Warwick Dunn, University of Birmingham, UK

Gika, (2008) J. Sep. Sci. Hydrophilic interaction and reversed-phase ultra-performance liquid chromatography TOF-MS for metabonomic analysis of Zucker rat urine.

Callahan, (2009) J. Sep. Sci. Profiling of polar metabolites in biological extracts using diamond hydride-based aqueous normal phase chromatography.

Collection

Dilution and

centrifugation

(high water)

RP-LC-MS

analysis

Dilution and

centrifugation

(high organic)

HILIC-LC-MS

analysis

Urine - LC-MS analysis

Recommend: using 5k MWCO

Lyophilization


Brain contains about 160 ml on average

0.8% proteins

Water

Inorganic salts

Metabolites including neurotransmitters and GABA, glutamate,

glutamine etc

Wishart , (2008) J Chromatogr B. The human cerebrospinal fluid metabolome

http://www.csfmetabolome.ca/scripts/CSF_browse.cgi

Collect, typically a lumbar puncture, freeze and store at -80ºC

Cerebrospinal Fluid (CSF) (~2% in metabolomics study)

(1) Quench the metabolism; (2) prepare and analyze as for serum/plasma!


Mammalian Tissues (~5% in

metabolomics study) Tissues are an ensemble of interacting cells

Blood presence in tissue, influences metabolome of tissue!

Different types of tissues

nervous tissue, central nervous system (brain, spinal cord, periphereal nerves)

epithelial tissue, layers of cells that cover organs such as skin

muscle tissue, smooth, skeletal and cardiac, produces force and causes motion

connective tissues are fibrous tissues


Mammalian Tissues Extraction 10-200 mg of tissue extracted

Extraction typically involves homogenisation of tissue

using a homogeniser or a shaker with stainless steel balls

manual with a mortar and pestle in liquid nitrogen

Typically perform a modified Folch extraction

methanol/water/chloroform

two phases collected (polar and non-polar)

Wu, (2008) Anal Biochem. 15;372(2):204

Lyophilisation, grinding and extract powder is a second option

Bobeldijk, (2008) J Chromatogr B, 871(2):306

Wheaton Glass Tissue Grinder

0.2 mL, minimal sample loss


Mammalian Tissues: Folch extraction

Collect and freeze immediately (10-200mg)

Thaw on ice and wash in cold saline

Homogenise in appropriate solvent at 4ºC

(2:2:1.8 methanol:chloroform:water)

Shake at 4ºC

Centrifuge, two phase supernatant (chloroform at bottom)

Transfer two phases to separate tubes for analysis

Add 1:1 chloroform:water


A Fully Miscible “Cocktail” Solvent Mixture

for Tissue Extraction

“Metabolomics Approach

Studies the Metabolic

Perturbation Induced

Molecular Changes in Retinal

Degeneration Disease”

Wang, Siuzdak et al

ACN/MeOH

163 x 6 x

57 x 17 xinfinity

276 x

Rats’ eyes for retinal

degeneration disease.


Cell Line Sample Preparation

(cold methanol; methanol/ammonium

bicarbonate; liquid nitrogen into

cells/methanol)

Centrifugation

(with filtration)

LC/IC-MS

experiment

Wash (fast <10s,

physiological buffer+H2O )

adherent mammalian cells

suspension mammalian cells

metabolism

quenching/extraction

scrap/suspend/centrifuge

suspend/centrifuge

LC/IC-MS

experiment


HPLC

Mechanism, Method Setup, Examples

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

Retention Mechanisms

Phenomenex

Retention Mechanisms

Phenomenex

Mobile Phase Composition

Phenomenex

Chart of Mobile Phase Selection

Waters Corporation

pH Selectivity

Waters Corporation

Organic Modifier Selection

Waters Corporation

Column Configurations and

ApplicationsColumn

Type

ID (mm) Length

(mm)

Particle

Size (mm)

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

mL/min

Peptide Mapping

LC/MS100

Micro Bore 1.0 30-150 3.5, 5 30-60

mL/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

--

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

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



Watch the Sample Carry-over

(Contamination) All the Time

http://www.sielc.com/Products_Autosamplers.html

Quantitation

Qualitative studies

Other separation techniques

2D LC-MS

Waters M-class

Thermo RSLC

Different MS

instrument

2D GC-MS

http://www.chromacademy.com/essential-guide/sept_2011/2d_gc_figure15.jpg

CE-ESI-MS

Ile

Leu

Ion Mobility

ASMS Ion Mobiilty Short Course 2016

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 extract

Coating material

Environmental

Bio-fluid

Tissues

etc.

Mass spec analysis

Untargeted metabolomics: Q-TOF

Targeted metabolomics: Q-TOF and QQQ

Q Exactive plus

15T FT ICR

Q-TOF

Benefits:

Higher resolution & mass accuracy

All ions recorded in parallel

Ref: Chemushevich, 2001

Q1 q2 TOF

Chromatogram of Oligonucleotides std

on 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

4x10

Intens.

Olistd_040815_03_BE4_01_171.d: BPC -All MS

1

2

3

4

5

6

7

8

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

4x10

Intens.

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

4x10

Intens.

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

Untargeted Metabolomics

RPLC-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

5x10

Intens.

0.0

0.2

0.4

0.6

0.8

6x10

Intens.

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

5x10

Intens.

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

5x10

Intens.

200 400 600 800 1000 1200 1400 m/z

MS -

MS +

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

5x10

Intens.

0.0

0.2

0.4

0.6

0.8

6x10

Intens.

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

4x10

Intens.

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

5x10

Intens.

200 400 600 800 1000 1200 1400 m/z

HILIC-MS

at 18.2

minMS -

MS +

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

4x10

Intens.

0

1

2

3

5x10

Intens.

0 10 20 30 40 50 Time [min]

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 Reaction

Monitoring (SRM)

D

Selected Reaction Monitoring

Q1 Q3(gas)Source Detector

Select one m/z

(fixed Vac/Vdc)

MS/MS at Different Collision Energies

Thermo provided database

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


TIC

12182014_30349_STD_GS_IS L_50


3.462e+006Clo;6.14;382144.81;3449917

min

%

0

100


TIC

12182014_30349_STD_GS_IS L_50


4.446e+006Imi;6.24;439232.47;4237493

5.84

min

%

0

100


TIC

12182014_30349_STD_GS_IS L_50 Smooth(Mn,2x3)


5.093e+006Thi;5.83;1057297.75;5049061

256.0/209.0

250.0/169.0

260.0/213.0

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

Quantitation upon AUC

Compound name: Imi

Correlation coefficient: r = 0.998517, r^2 = 0.997036

Calibration curve: 0.730897 * x + 0.110503

Response 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

sp

on

se

-0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

360

Compound name: Clo

Correlation coefficient: r = 0.999227, r^2 = 0.998455

Calibration curve: 0.67092 * x + 0.0383766

Response 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

sp

on

se

-0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

Std curve

QCs

Samples

Blanks

Bioinformatics in

Metabolomics

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-works/

Import Data

Data Alignment

MS -MS +

Peak Picking

MS -MS +

Review Deconvolution

Identify Compounds

Review Compounds

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)

Anal. Chem. 2014, 86, 9583-9589

Anal. Chem. 2014, 86, 9358-9361

Thank you!

Documents

HPLC-MS/MS and Metabolomics - Ohio State University › sites › default › files › CCIC_metabolomics.… · Waters SEP-PAK OASIS Varian BondElute Baker BakerBond 3M Empore Supelco