UPLC-MS

Daniel Czarez Garca

Departamento de biotecnologa y bioqumica
CINVESTAV-IPN Irapuato
18 de mayo de 2017

Bienvenidos! 1
 UPLC-MS

1. General 2. Sample 3. Data

background analysis analysis

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1. General background

Metabolomics
Mass spectrometry
Samples and chromatography
Metabolic profiling (UPLC-MS)

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 W. Johannsen, 1911

The phenotypes of all species can be

characterized by direct inspection or by
finer methods of measuring or
description.

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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...

Profiling with e.g. Mass

spectrometry techniques

Modified from (Holmes et al., 2008)

Mass spectrometry

John B. Fenn, 2002

"Mass spectrometry
is the art of
measuring atoms
and molecules to
determine their
molecular weight."

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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

Flies Polarity of Molecular

extract metabolites weight (Da)
Polar 701-1000
Stationary
501-700
phase 301-500
Non Polar 100-300

Vacuum system

Sample Ionization Mass Mass

inlet source analyzer detector
(Suizdak, 2008. Richard F. Venn, 2000)
Ionization methods

Protonation

Deprotonation

Cationionization

Electron ejection

Electron capture
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Scan mode

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2. Sample analysis

LCQ fleet ion trap

Xcalibur
Run samples

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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

3 Automatic Tuning and Calibrati

Tuning the MS Detector Automati

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

4 Tuning with an Analyte in the LC/ESI/MS Mo

Setting Up to Tune the MS Detector with Your Anal

Defining the Scan Parameters

To define the scan parameters, scan type, and scan polarity for the analyte
1. On the Instrument Control toolbar, click the Define Scan ( ) button.
The Define Scan dialog box appears (Figure 42).
Figure 42. Define Scan dialog box showing typical settings for acquiring reserpine data of the SIM type

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

Take pump control

1. Flow: empezar con flujo bajo (100 L/min) y 100%A
por 1-2 min.
2. Cambiar temperatura de columna (eg. 30).
3. Cambiar a flujo utilizado (eg. 400 L/min). Puedes
hacer un gradiente manual o dejar acondicionar la
columna con las condiciones iniciales de tu programa

4. Pumb ready 16
 Figure 30. Status view of front lens adjustment process in the Tune

5 Acquiring ESI Sample Data By Using Tune Plus

Acquiring ESI/MS Data in the SIM Scan Type

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

Random sample order

(check position in tray)
Sample type

Path (folder Instrument

to store data) method that
you create

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3. Data analysis

Storage

Data pre treatment

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

Raw data import

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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