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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
Method Separation based on Separation done using Further steps
Liquid-liquid ExtractionPartitioning in one of two liquid phases
Glass ware
Solid-phase ExtractionAdsorption/ partitioning onto solid sorbent
Cartridges, disks, filters, plates
Types of Separation Technologies
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
Method Separation based on Separation done using Further steps
Liquid-liquid ExtractionPartitioning in one of two liquid phases
Glass ware
Solid-phase ExtractionAdsorption/ partitioning onto solid sorbent
Cartriges, disks, filters, plates
Dialysis/UltrafiltrationMolecular weight/size
SlideAlyzer/tubing
Types of Separation Technologies
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
Liquid-liquid ExtractionPartitioning in one of two liquid phases
Glass ware
Solid-phase ExtractionAdsorption/ partitioning onto solid sorbent
Cartriges, disks, filters, plates
Dialysis/UltrafiltrationMolecular weight/size
SlideAlyzer, tubing, spin filter
Precipitation Solubility
Types of Separation Technologies
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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
adapted from Junhua Wang, Ph.D. at Thermo
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
adapted from Junhua Wang, Ph.D. at Thermo
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
adapted from Warwick Dunn, University of Birmingham, UK
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!
adapted from Warwick Dunn, University of Birmingham, UK
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
adapted from Warwick Dunn, University of Birmingham, UK
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
adapted from Warwick Dunn, University of Birmingham, UK
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
adapted from Warwick Dunn, University of Birmingham, UK
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.
adapted from Junhua Wang, Ph.D. at Thermo
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
adapted from Junhua Wang, Ph.D. at Thermo
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
LC Tips on Peak Shape Issues
Agilent Technologies
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LC Tips on Peak Shape Issues
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
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
100
F2:MRM of 1 channel,ES+
TIC
12182014_30349_STD_GS_IS L_50
sample after GS, centrifuge, filter, fresh prep in 50%B
4.446e+006Imi;6.24;439232.47;4237493
5.84
min
%
0
100
F4:MRM of 1 channel,ES+
TIC
12182014_30349_STD_GS_IS L_50 Smooth(Mn,2x3)
sample after GS, centrifuge, filter, fresh prep in 50%B
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
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-0
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40
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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
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se
-0
20
40
60
80
100
120
140
160
180
200
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Std curveQCsSamplesBlanks
Quantitation Using Area Under the Curve (AUC)
Thank you!• Yu Cao• Arpad Somogyi• Vicki Wysocki• Entire CCIC MSP Facility
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