HPLC-MS/MS and Metabolomics Kathleen A. Vermeersch and ... · Analyze: HPLC, GC -MS, LC MS/MS Solid Phase Extraction- common protocol a) pure analyte (control) b) engine oil contaminated

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HPLC-MS/MS and Metabolomics

MATTHEW BERNIER, PH.D.

CCIC MS&P SUMMER WORKSHOP

JULY 2018

Thanks to Yu Cao

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

Importance of the metabolome

◦ Encompasses biomolecules intracellular and extracellular that aren’t proteins/nucleic acids◦ Lipids (Lipidomics)

◦ Carbohydrates (Glycomics)

◦ Small peptides (Peptidomics)

◦ Other small molecules

◦ Single Amino Acids◦ Cytokines◦ Phosphates

◦ Many many more…

Publications on “Metabolomics”

0

500

1000

1500

2000

2500

3000

2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016

Number of publications per yearSearch engine: Web of Science Topic “metabolomics”

# of hits

Year

Metabolomics short course at ASMS 2014

Why LC ESI-MS for Metabolomics?

A “feature” is simply a signal that relates to one unique m/z and retention time (for MS)

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

Sample Preparation

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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 or MS/MS analysis Separation technologies

essential in sample prep

Sample Preparation

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

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

Method Separation based on Separation done using Further steps

Liquid-liquid ExtractionPartitioning in one of two liquid phases

Glassware

Types of Separation Technologies

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

Liquid-liquid Extraction

faculty.ksu.edu.sa



Glass ware

Solid-phase ExtractionAdsorption/ partitioning onto solid sorbent

Cartridges, disks, filters, plates


cartridges

96 well plate

disk

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

◦ Uses chromatographic particles

◦ Packed-bed column cartridges or similar

◦ Well established commercial technology (1978)

◦ 1000s of literature references

◦ Clean extracts

◦ Good recovery for polar analytes

◦ Sample must be in liquid state

◦ Driving force: gravity, pressure, vacuum

◦ Automation possible

Solid Phase Extraction

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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 ExtractionProcedure:

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

Solid Phase Extraction- common protocol

a) pure analyte (control)

b) engine oil contaminatedparking lot oil

c) same as b) after organicmatter removal by SPE

(KNO3)nK+

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

Solid Phase Extraction – Example



Glass ware


Cartriges, disks, filters, plates

Dialysis/UltrafiltrationMolecular weight/size

SlideAlyzer/tubing


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

Tubing or Slide A-Lyzer

(Different MWCO Ranges

0.1 – 0.5 mL capacity

Useful for biologicals Method Separation based on Separation done using Further steps


Glass ware


Cartriges, disks, filters, plates

Dialysis/UltrafiltrationMolecular weight/size

SlideAlyzer, tubing, spin filter

Precipitation Solubility


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adapted from Junhua Wang, Ph.D. at Thermo

Wh

ole

blo

od

anti-coagulant (EDTA or heparin or citrate)

unclotted blood

clotted blood

naturalprocess 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

Blood – Plasma and Serum (~80% of metabolomics studies)

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


Anti-coagulant Choice - Effects

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

Plasma and Serum - Deproteinization


95% water

“Relatively” 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

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

Urine (7-8 % of metabolomics studies)

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.

CollectionDilution and centrifugation(high water)

RP-LC-MS analysis

Dilution and centrifugation(high organic)

HILIC-LC-MS analysis

Recommend: using 5k MWCO

Lyophilization


Urine – LC/MS Analysis ProtocolsBrain 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

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


Cerebrospinal Fluid (CSF) (~2% of metabolomics studies)

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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 (~5% of metabolomics studies)

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 Grinder0.2 mL, minimal sample loss


Mammalian Tissue Extraction

Collect and freeze immediately (10-200mg)

Thaw on ice and wash in cold saline

Homogenise in appropriate solvent at 4ºC(2:2:1 methanol:chloroform:water)

Shake at 4°C

Centrifuge, two phase supernatant (chloroform at bottom)

Transfer two phases to separate tubes for analysis

Add 1:1 chloroform:water


Mammalian Tissues: Folch 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.


Solvent Mixtures for Tissue Extraction

(cold methanol; methanol/ammonium bicarbonate; liquid nitrogen into cells/methanol; sonication)

Centrifugation (with filtration)

LC/IC-MS experiment

Wash (fast <10s, physiological buffer+H2O )

adherent mammalian cells

suspension mammalian cells

metabolism quenching/extraction/lysis

scrap/suspend/centrifuge

suspend/centrifuge

LC/IC-MS experiment


Cell Line Samples Preparations

High-Performance Liquid Chromatography (HPLC)

MECHANISM, METHOD SETUP, EXAMPLES

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Retention Mechanisms

Phenomenex

Retention Mechanisms

Phenomenex

Mobile Phase Composition

Phenomenex Phenomenex

Mobile Phase Composition

(John Dolan) http://www.hplc.eu/Downloads/ACE_Guide_BufferSelection.pdf

Column selection◦ Bedding chemistry, dimension, bead size

Buffers (mobile phase)◦ Ionic strength, pH, pairing reagent

Fine tune the method◦ Temperature

◦ Gradient slope

LC Method Development/Optimization pH Selectivity

Waters Corporation

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pH Selectivity

Waters Corporation Waters Corporation

Organic Modifier Selection

Column

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/MS

100

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 Configurations/Applications

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

Column Comparisons by Vendors

LC Tips on Peak Shape Issues

Agilent Technologies


Agilent Technologies

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

Quantitation

Qualitative studies

Watch for Sample Carry-Over (Contamination)

On-going Projects:

Quantification of target metabolite from infected tissue

Amino acid analysis in fish embryos

Detection/quant. of modified carbohydrate phytochemicals in broccoli extracts

Lipidomics of fatty tissue

TMAO/TMA analysis from plasma and urine

Sample type:

Plant extractsCoating materialEnvironmentalBio-fluidTissues

etc.

Metabolomics in MS&P

Mass spec analysis

U NTA R G E T E D M E TA B O LO M ICS : Q - TO F

TA R G E T E D M E TA B O LO M ICS : Q Q Q

Q E X A C T IV E P LU S

1 5 T F T IC R

Bruker maXis User Manual

TOF

Quadrupole Time-of-Flight (Q-TOF)Benefits:

Higher resolution & mass accuracy

All ions recorded in parallel

Q1 q2

Oligonucleotide Standard BPC

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

ID m/z

Oligo 4 1173.8

Oligo 5 1487.0

Oligo 7 2105.4

Oligo 9 2722.8

Oligo 11 3341.2

Oligo 12 3645.4

Oligo 20 6117.0

Oligo 30 9191.0

RPLC – with Hexafluoro isopropanol (HFIP) 400 mM & TEA 15 mMBruker Standards 217028 and 206200

1.

2.

3.

4.

5.

6.

7.

8.

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Mass Spectrum at 2.4 min

ID m/z

Oligo 4 1173.8

Oligo 5 1487.0

Oligo 7 2105.4

Oligo 9 2722.8

Oligo 11 3341.2

Oligo 12 3645.4

Oligo 20 6117.0

Oligo 30 9191.0

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

1.0015 m/z

0.5019 m/z

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 +

Untargeted Metabolomics (RPLC-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

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 +

RPLC-MS at 32.7 min

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]

Untargeted HILIC-LC-MS

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 min

MS -

MS +

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]

RPLC vs HILIC (Comparison of 258.1098 Elution)

RPLC

HILIC

Pyro-L-glutamine-L-glutamine

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Benefits:

Simple, ion filter

Ideal for quantification

Q1 q2

Q3

Triple Quadrupole MS (QQQ)

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

QQQ Scan Modes for MSMS Analysis

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

Targeted Metabolomics

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 curveQCsSamplesBlanks

Quantitation Using Area Under the Curve (AUC)

Thank you!• Yu Cao• Arpad Somogyi• Vicki Wysocki• Entire CCIC MSP Facility

Documents

HPLC-MS/MS and Metabolomics Kathleen A. Vermeersch and ... · Analyze: HPLC, GC -MS, LC MS/MS Solid Phase Extraction- common protocol a) pure analyte (control) b) engine oil contaminated