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High Throughput Workflow for Global Metabolomic Profiling of Biological Samples
with RapidFire online SPE/MS system
Michelle Romm, Ph.D.
RapidFire Senior Scientist,
Agilent Technologies, Inc.
Sumit Shah, B.Pharm., Ph.D.
Metabolomics Applications Scientist,
Agilent Technologies, Inc.
10/8/2014
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Metabolomics/Lipidomics Platforms
Separate
Detect
Analyze
LC SFC
TOF QTOFIMS
QTOF
ICP
MS
NMR
GC CE
MassHunterQual
Quant
Profinder
Mass Profiler
Professional
QQQ
Online
SPE
SQ
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RapidFire 365 Online SPE/MS System
For Research Use Only. Not for Use in Diagnostic Procedures.
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3
~12sec cycle time BenchBot plate handling robot
63 plate capacity (384 well plates = 24,192 samples)
Off-deck reach capability
Quaternary pumps
Solvent switching capability
Capable of running multiple disparate methods in one batch
Automated Method Development
Perform multiple injections of a single sample with different SPE cartridges and mobile phases
C18, C8, C4, CN, Phenyl, HILIC, Graphitic Carbon
Simply run multiple samples overnight and select your ideal conditions the next morning
12 position automated cartridge changer
Aspirate Load/Wash Elute Re-equilibrate
600ms 3000ms 8000ms 500ms
High Throughput Metabolomics Profiling Workflow
Batch Data
ProcessingData Analysis
Verification/
Confirmation/
Validation
High Throughput
Quantitation
High Throughput
Acquisition
MassHunter
Profinder(NEW)
.d data input
Recursive
MFE
Features
.cef output
Mass Profiler
Professional (MPP)
Statistics
Database Search
Pathway Analysis
Potential
Targets
Pooled Sample/
Random Samples
LC/MS
AcquisitionFindbyFormula
Verify Targets
LC/MS/MS
AcquisitionMetlin Library/
SimLipid/
MSC
Confirm Targets
Validate with
Reference
Standard
RapidFire 365
iFunnel
6550 Q-TOF
RapidFire 365
iFunnel
6490 QQQ
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Rapid Method
Development
Large Batch
Profiling
Targeted
Profiling
Life Sciences
Pharma
BioPharma
Food/Nutrition
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Human Urine Metabolomics Profiling
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Human Urine Sample Prep
Human urine, collected and pooled from healthy subjects (males and females),
was purchased from Innovative Research Inc. (Novi, MI)
Filter 400µL urine through a 0.22µm Ultrafree-MC® centrifugal filter units
(Millipore) by centrifugation @ 12,000xg for 20min @ 4°C
Pipette 50µL of filtered urine (10 replicates) into the Amicon Ultra-0.5mL
Centrifugal Filter (3kDa Cut-off Ultracel-3 membrane) from Millipore
Dilute with 350µL of Milli-Q quality water containing 0.2% Acetic acid, 1mM
Ammonium acetate, and centrifuge @ 12000xg, 4°C for 30min to concentrate
The flow-through represents the metabolomics sample
Inject 10µL of flow-through for SPE/MS as well as LC/MS analysis
Reverse-phase and HILIC chromatography were used for metabolite separation
Human urine equivalent to 1.25µL was used for the metabolomics profiling studies
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Ionization Positive ESI (Dual AJS)
Drying Gas Temp. 225°C
Drying Gas Flow 17 L/min
Sheath Gas Temp. 275°C
Sheath Gas Flow 12 L/min
Nebulizer 35 psi
Capillary Voltage 3500 V
Nozzle Voltage 1000 V
Fragmentor 100V
Scan Mode Extended Dynamic Range (2GHz)
MS Scan Rate 5 spectra/sec (RF-MS); 2 spectra/sec (LC-MS)
Mass Range 50-1700 m/z
MS Source Conditions
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Reverse Phase RapidFire Method
Column Agilent C, C18 [Part # G9205A]
Injection Volume 10 µL
Flow-rate 0.6 mL/min
Mobile Phase A 0.1% Formic Acid in Water
Mobile Phase B 0.1% Formic Acid in Methanol
Aspirate Load/Wash Elute Re-equilibrate
600ms 3000ms 8000ms 500ms
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HILIC RapidFire Method
Column Agilent H, HILIC [Part # G9209A ]
Injection Volume 10 µL
Flow-rate 0.6 mL/min
Mobile Phase A Hexane
Mobile Phase B 0.1% Formic Acid in Methanol
Aspirate Load/Wash Elute Re-equilibrate
600ms 3000ms 8000ms 1000ms
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EIC Overlay of a Single Feature in Urine on C18 RapidFire SPE/MS
EIC (C18, Pos ESI)
Male Urine
Female Urine
Female Urine
Male Urine
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EIC of a Single Injection of Urine Sample
on C18 RapidFire SPE/MS
Female Urine
+ESI
Male Urine
+ESI
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Profinder for High Throughput Batch Analysis of Samples
Female Urine
Male UrineFemale Urine
Male Urine
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C18-PosESI
Mass Profiler Professional (MPP)
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734 features (all 10 reps, CV < 15%) 593 features (un-paired t-test, p < 0.05)
138 features (Fold-change > 5)Cluster Analysis (138 features)
Annotation of Creatinine in Female Urine Samples
by C18 RapidFire SPE/MS
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Compound (RF-C18)
Hippuric acid
AFMK
Ser Arg Ser
4-Oxo-4-(3-pyridyl)-butanoic acid
Phe Asn Leu
Glycerol
DL-Methionine sulfoxide
Creatinine
N(alpha)-t-Butoxycarbonyl-L-leucine
4-Chloroacetophenone
D-Xylulose
N,N-Hexamethyleneamiloride
1-Nitronaphthalene-7,8-oxide
L-Tryptophan
Onitin
Nap-Gly-OH
Gly Cys Cys
2-Methylguanosine
6-Hydroxykynurenate
Pro Phe
Indoleacrylic acid
Phe-Abu-OH
Tegaserod
1-Hydro-1,1a-dihydroxy-9-fluorenone
Dopamine
Compound (RF-HILIC)
Glycerol
Mycophenolic Acid
Glu Glu
3-Methyl-L-histidine
Creatinine
Aniline
Pro Ile
1-Aminocyclohexanecarboxylic acid
Ser Arg Ser
Asp Pro Arg
L-Histidine
7,8-Diaminononanoate
Quinone
Hippuric acid
N-Nitrosodi-n-butylamine
Asp Arg Tyr
L-Glutamic acid n-butyl ester
N1-Acetylspermidine
Pro Gln Gly
Phe Asn Leu
Thr His
3-hexanoyl-NBD Cholesterol
2-Hydroxy-6-oxonona-2,4-diene-1,9-dioate
3,4-Dihydroxyphenylpropanoate
AFMK
Select High Abundant Metabolites in Human Urine Annotated by SPE/MS
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Glucose Homeostasis Pathway (WP661_45308)
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Dynamic Range of Annotated Metabolites: C18 SPE/MS
Ser-Arg-Ser
Male Urine
Female Urine
Female Urine
Male Urine
Threo-Isocitric acid
Female Urine
Male Urine
N-Acetylputrescine
Compound RF-C18-Male-Avg RF-C18-Female-Avg
Ser Arg Ser 1156619 872143
threo-Isocitric acid 4027 13795
N-Acetylputrescine 207 811
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Dynamic Range of Annotated Metabolites: HILIC SPE/MS
3-Methyl-L-histidine
Male Urine
Female Urine
1-Methylhistamine
Male Urine
Female Urine
S-Adenosylhomocysteine
Female Urine
Male Urine
Compound RF-HILIC-Male-Avg RF-HILIC-Female-Avg
3-Methyl-L-histidine 1496426 1144011
1-Methylhistamine 41234 27579
S-Adenosylhomocysteine 72 2701
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LC/MS/MS Confirmation of L-HistidineCID@10
CID@20
CID@40
L-Histidine
MS/MS @ 40
Metlin Spectral Library
MFG for MS/MS Spectral Peaks
L-Histidine
RapidFire HILIC SPE/MS +ESI
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Male Urine
Female Urine
Metabolome Profiling: C18 vs. HILIC SPE/MS (+ESI)
Filter features present in all replicates in a group with CV<15%
Features searched against Metlin® Database
526 metabolites annotated by C18 SPE/MS
270 metabolites annotated by HILIC SPE/MS
49 annotated metabolites common between HILIC and C18 SPE/MS
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HILIC
221
C18
47749
Utilize multiple unique chemistries to increase the metabolite coverage
747 unique metabolites
Human Serum Lipidomics Profiling
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Human serum, collected and pooled from healthy subjects (males and females),
was purchased from Innovative Research Inc. (Novi, MI)
Filter 400µL serum through a 0.22µm Ultrafree-MC® centrifugal filter units
(Millipore) by centrifugation @ 12,000xg for 20min @ 4°C
Pipette 100µL filtered serum into a Protein LoBind® Eppendorf tube
Add 700µL ice-cold Methanol:Isopropanol, 1% Acetic Acid, vortex 30sec
Equilibrate for 10min at room temp and Centrifuge at ~12000xg, 20min, 4°C
The organic supernatant represents the lipidomics sample
Inject 10µL for SPE/MS as well as LC/MS analysis
Reverse-phase chromatography were used for resolving the lipids
Human Serum Sample Prep
Human serum equivalent to 1.25µL was used for the lipidomics studies
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Ionization Positive or Negative ESI (Dual AJS)
Drying Gas Temp. 275°C
Drying Gas Flow 15 L/min
Sheath Gas Temp. 325°C
Sheath Gas Flow 12 L/min
Nebulizer 45 psi
Capillary Voltage 4000 V
Nozzle Voltage 1000 V
Fragmentor 100V
Scan Mode Extended Dynamic Range (2GHz)
MS Scan Rate 5 spectra/sec (RF/MS); 2 spectra/sec (LC/MS)
Mass Range 100-3000 m/z
MS Source Conditions
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Reverse Phase RF Method
Column Agilent C, C18 [Part # G9205A]
Injection Volume 10 µL
Flow-rate 0.6 mL/min
Mobile Phase A 0.1% Acetic Acid in Water, 1mM Ammonium Acetate
Mobile Phase B 0.1% Acetic Acid in Methanol:IPA (30:70), 1mM Ammonium
Acetate
Aspirate Load/Wash Elute Re-equilibrate
600ms 3000ms 8000ms 500ms
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EIC of a Single Injection of Serum Sample
on C18 RapidFire SPE/MSFemale Serum
+ESI
Male Serum
+ESI
Female Serum
-ESI
Male Serum
-ESI
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Profinder for High Throughput Batch Analysis of Samples
Female Serum
Male Serum Female Serum
Male Serum
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Annotation of a Carnitine in Female Serum Samples
by C18 RapidFire SPE/MS in +ESI
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Compound (-ESI)
Palmitic acid
16-methyl-6Z,9Z-heptadecadienoic acid
18-methyl-5Z,8Z,11Z,14Z-nonadecatetraenoic acid
16-methyl-6Z,9Z,12Z-heptadecatrienoic acid
7-Methoxy-9-methyl-4E,8E-hexadecadienoic acid
PE(18:0/0:0)
7,4'-Dihydroxyflavan
PS(16:0/22:1(11Z))
2,4-dimethyl-2E-tetradecenoic acid
Ximaosteroid D
PS(17:0/21:0)
PS(18:0/22:1(11Z))
1a,1b-dihomo-15-deoxy-delta-12,14-PGJ2
PA(16:0/0:0)
PA(18:2(9Z,12Z)/0:0)
8Z-decen-4,6-diynoic acid
PE(20:0/0:0)
PS(18:1(9Z)/22:2(13Z,16Z))
4,8,12,15,18-eicosapentaenoic acid
PC(O-12:0/O-12:0)
PS(18:0/22:2(13Z,16Z))
18-methyl-8Z,11Z,14Z-nonadecatrienoic acid
7E,9E,11-Dodecatrienyl acetate
PS(20:1(11Z)/22:2(13Z,16Z))
PI(18:0/20:4(5Z,8Z,11Z,14Z))
Select High Abundant Lipids in Human Serum (C18 SPE/MS)
Compound (+ESI)
PC(16:0/18:1(9Z))
PC(18:0/18:2(10Z,12Z))
PC(O-16:0/2:0)
PC(16:0/18:2(10E,12Z))
PC(10:0/24:0)
PC(O-16:1(11Z)/2:0)
SM(d18:1/16:0)
PE(19:1(9Z)/22:4(7Z,10Z,13Z,16Z))
TG(17:0/17:2(9Z,12Z)/18:0)[iso6]
TG(17:1(9Z)/17:2(9Z,12Z)/18:0)[iso6]
PE(19:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z))
PC(18:2(2E,4E)/0:0)
3a-Hydroxy-6-oxo-5beta-cholan-24-oic Acid
PE(17:1(9Z)/22:4(7Z,10Z,13Z,16Z))
PC(20:3(8Z,11Z,14Z)/0:0)
SM(d18:1/24:1(15Z))
DG(18:2(9Z,12Z)/20:0/0:0)[iso2]
TG(17:2(9Z,12Z)/17:2(9Z,12Z)/18:0)[iso3]
TG(16:0/17:1(9Z)/17:1(9Z))[iso3]
SM(d18:2/24:1)
TG(16:0/16:0/18:1(11E))
PC(18:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z))
PC(O-16:0/20:4(5E,8E,11E,14E))
NeuAcalpha2-3Galbeta-Cer(d18:1/18:0)
PE(O-20:0/17:2(9Z,12Z))
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Lipidomics Profiling of Human Serum by C18 SPE/MS
Filter features present in all replicates in a group with CV<15%
Features searched against SimLipid® Database
169 lipids annotated in +ESI mode
275 lipids annotated in –ESI mode
29 annotated lipids common between +ESI and –ESI modes
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+ESI
140
-ESI
24629
415 unique lipids
LC/MS/MS Identification of 1,2-dipalmitoyl-sn-glycero-3-PC
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31
LC/MS/MS Confirmation of 1,2-dipalmitoyl-sn-glycero-3-PC
1,2-dipalmitoyl-sn-glycero-3-PC
MS/MS @ 40
Metlin Spectral Library
1,2-dipalmitoyl-sn-glycero-3-PC
RapidFire C18 SPE/MS +ESI
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1,2-dipalmitoyl-sn-glycero-3-PC
C18 LC/MS +ESI
Female Serum
Male Serum
Female
Serum
Male
Serum
CID@10
CID@20
CID@40
CID@0
SUMMARY
Performed “high throughput” profiling of biological samples with
RapidFire SPE/MS
747 metabolites were annotated in human urine
415 lipids were annotated in human serum
RapidFire data were fully integrated and compatible with Agilent
software solutions (MassHunter, ProFinder, MPP, Metlin,
SimLipid*)
Demonstrated reproducibility and fidelity of the current high
throughput workflow for data processing and analysis
Verified the annotated metabolites by LC/MS analysis
Confirmed select metabolites/lipids by MS/MS analysis
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The Bottom Line…..
RapidFire Urine Study
Time
40 Inj = 40 x 14sec
~10min
Solvent
40 Inj = 0.6mL/min x 10min
~6mL
Equivalent LC/MS Study
Time
40 Inj = 40 x 30min
~20hr
Solvent
40 Inj = 0.3mL/min x 1200min
~360mL
Actual LC/MS Study
Time
2 Inj = 2 x 30min
~1hr
Solvent
2 Inj = 0.3mL/min x 60min
~18mL
120X Faster
60X Less
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The Big Picture
The RapidFire work-flow is not meant to replace LC/MS based
work-flows.
It is meant to streamline your profiling/discovery platform by
finding the differences between large experimental groups in
the highest throughput, highest capacity, and most
ECONOMICAL manner.
The RapidFire data should then be confirmed and validated by
conventional LC/MS.
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MassHunter Qual: Data Mining Process
Monoisotopic
mass
(varies in ppm)
Isotope spacing (varies in ppm)
Isotope distribution (varies in %)
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![Electronic Supplementary Information … · Electronic Supplementary Information ... 80, Sheath Gas Flow Rate (arb): 40, Aux Gas ... [220.00-390.00] 220 230 240 250 260 270 280 290](https://img.dokumen.tips/doc/110x75/5bbefc1109d3f208478d03c0/electronic-supplementary-information-electronic-supplementary-information-.jpg)
