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Uplc Ms Mayo2017 DCG
Uplc Ms Mayo2017 DCG
Bienvenidos! 1
UPLC-MS
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1. General background
Metabolomics
Mass spectrometry
Samples and chromatography
Metabolic profiling (UPLC-MS)
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W. Johannsen, 1911
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Genotype + Environment Phenotype
Organism status
Healthy At Risk Disease
Dietary inputs
Genome
Microbiome Metabolic phenotype
Epigenetic
Pollutants Normal Intermediate Pathological
SNP variations
Parasites
Biomarkers: disease risk,
disease, nutrition...
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P e R S Metabolomics
PeCtiVeS
a Targeted metabolomics
Question:
at are t e le el o pecic
etabolite in a a ple?
Standard LC/MS of Selected reaction Optimization Samples: LC/MS of Data analysis by Quantication
metabolites standard monitoring and standard Tissue lysates metabolite comparison of o pecic
metabolites curve for Cells extracts sample groups metabolites
+ uantication Blood and and/or standards in biological
+
ot er bio uid samples
Intensity
+ Standard
+ metabolite
++
Time Group 1 Group 2
b Untargeted metabolomics
Question:
What is the global metabolic
prole o a a ple?
Samples: LC/MS of Overlayed extracted Data alignment and analysis Validation with Global
Tissue lysates metabolite ion chromatograms MS/MS from metabolic
Cells extracts + + standards prole o
Blood and + biological
ot er bio uid samples
Intensity
+
++
Time m/z
Figure 1 | The targeted and untargeted workflow for LC/MS-based first isolated from biological samples and subsequently analysed by liquid
metabolomics. a | In the triple quadrupole (QqQ)-based targeted metab- chromatography followed by mass Nature Reviews(LC/MS).
spectrometry | Molecular Cell
After Biology
data acqui-
olomic workflow, standard compounds for the metabolites of interest are sition, the results are processed by using bioinformatic software such as
first used to set up selected reaction monitoring methods. Here, optimal XCMS to perform nonlinear retention time alignment and identify peaks
instrument voltages are determined and response curves are generated that are changing between the groups of samples measured. The m/z
for absolute quantification. After the targeted methods have been estab- values for the peaks of interest are searched in metabolite databases to
lished on the basis of standard metabolites, metabolites are extracted from obtain putative identifications. Putative identifications are then confirmed
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tissues, biofluids or cell cultures and analysed. The data output provides by comparing tandem mass spectrometry (MS/MS) data and retention time
quantification only of those metabolites for which standard methods have data to that of standard compounds. The untargeted workflow is global in
Working principle of the UPLC-MS system
Vacuum system
Protonation
Deprotonation
Cationionization
Electron ejection
Electron capture
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Scan mode
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2. Sample analysis
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Start up guide
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on/standby
Figure 30. Status view of front lens adjustment process in the Tune
Figure 31. ESI Source dialog box with typical parameters post-tuning par
profile/centroid
positive/negative
ion mode
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Figure 30. Status view of front lens adjustment process in the Tune
ion padre
C.E. en general empezar en 20 e ir
aumentando hasta que el ion parental
este al 15% de intensisdad del ion hijo 14
5 Acquiring ESI Sample Data By Using Tune Plus
Figure 30. Status view of front lens adjustment process in the Tune
Setting Up to Acquire Full-Scan MS/MS Data
b. If you are infusing reserpine, verify that the values in the dialog box are the same as
those shown in Figure 47 on page 80. If you are infusing a different analyte, ensure
that the Parent Mass (m/z) box contains the correct value and that the scan range is
appropriate.
c. Under MSn Settings, start with a relatively wide isolation width of 2 Da.
d. After entering the values, click Apply, and leave the Define Scan dialog box open.
5. Turn on the LC pump and specify a flow rate of 0.4 mL/min.
6. Verify that the inlet plumbing connections do not leak.
7. Click the Syringe Pump button.
The Syringe Pump dialog box appears (Figure 48).
Figure 48. Syringe Pump dialog box
Checar:
Tipo y volumen de la
jeringa en uso
* seleccionar un flujo
8. Turn on the syringe pump and set an infusion flow rate of 5 L/min as follows:
a. Under Flow Control, select the On option. 15
b. In the Flow Rate (L/min) box, type 5.
Figure 30. Status view of front lens adjustment process in the Tune
4. Pumb ready 16
Figure 30. Status view of front lens adjustment process in the Tune
Figure 56. Acquire Data dialog box showing typical settings for acquiring data
Start to acquire
DIESI-MS
In general, many
samples =
#scans/sample
7. Specify the acquisition parameters as follows:
a. In the File Name box, type a name for the file.
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b. In the Sample Name box, type text to identify the sample.
Cmo crear un programa?
Xcalibur
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Accela AS Method Suficiente muestra > full loop
Poca muestra No waste
Tray temperature
(5C para
muestras
sensibles)
Column oven
control
>25
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Sample preparation Wash needle (outside)
Flus to waste (inside)
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Pump general
Pressure stability
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Gradient program
Do not forget
to change the
flow you want
Final
condition
equal to initial
condition
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Check list
Injection volume
Injection mode
Tray temperature
Column oven
temperature
Pressure stability
Flow rate
(L/min)
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Ones you select experiment type you can
not change it
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1. Select samples you want to run
2. click
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3. Data analysis
Storage
Basic statistics
Databases
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Storage
Transfer big files
Folder all samples and
replicates Meta information:
Date
Order
conversion: .mzXML, .mzML,
.abf
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Raw data methods
Filtering
Crop filter:
Retention time
Polarity
Scans
m/z range
Baseline correction
Peak detection 30
Crop filter
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Baseline correction
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Peak detection (GridMass)
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Peak list methods
Alignment
RANSAC
Filtering
Duplicate peak filter:
m/z tolerance
retention time tolerance
Gap filling
Sam RT and m/z gap filler
Export/Import
Export to CSV file 34
RANSAC alignment
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Duplicate peak filter
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Gap filling
Same RT and m/z range gap filler
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Export to CSV file
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Project
Set sample parameters
Add new parameter
Add value
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Raw data methods
Crop filter According to your runs
Baseline correction Parameters recommended
Peak detection You can change the height to
detect peaks (200.00 is in the
example
Peak list methods
Ransac alignment Parameters recommended
Filter
Gap filling
Export to csv You can select the options you
want to save
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Gracias por su atencin!
If you educate one person, youve planted a seed that will grow
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