(full width half maximum at m/z ¼ 400) or low resolution
(LTQ).
5. Use normalized collision energies of 40% and 30–35% in HCD
(higher energy collisional dissociation) and MS
n experiments,
respectively (see Note 5).
4 Notes
1. The described method is aimed at rapid screening of lipid
molecular species across a broad range of lipid classes. While
use of positive ion mode is described, the same platform can
also be applied using electrospray ionization in negative ion
mode, which will lead to better sensitivity for specific phospholipid classes such as phosphatidylinositols and phosphatidylserines
as
well
as
glycosylated
sphingolipids
and
sulfoquinovosyldiacylglycerols (SQDGs). In negative ion
mode, a better ionization efficiency is achieved when the
UPLC eluents are prepared without formic acid. In the case
of polar, glycosylated sphingolipids, another extraction method
is needed in order to achieve sufficient extraction recoveries.
2. The method can be transferred to a different LC-MS system.
Ideally, the system used should provide a high mass accuracy
and have capability to obtain MS/MS data. For example, a
UPLC-Synapt TQ-S (Waters) instrument with capability of
parallel MS/MS experiments by utilizing its MS
E capability
has been utilized.
3. The updated version of the open source MZmine 2 software,
including tutorials, is available at http://mzmine.github.io/.
The data processing parameters need to be optimized for a
specific analytical system used in order to account for different
peak shapes and lengths as well as different mass spectrometer
resolution. Differential profiling of multiple samples requires
steps such as peak detection, alignment (matching of peaks
across multiple samples), and normalization using internal
standards.
4. Some algae species/cells contain significant amounts of TAG
(17:0/17:0). If new types of samples are analyzed, it is recommended first to run a sample extract without any internal standard
addition and check the profile. If significant amounts of TAG
(17:0/17:0) are detected, another internal standard should be
chosen instead (preferable a stable isotope-labeled compound).
5. In general, the MS/MS or MS
n experiments can be performed
on the same instrument where the LC-MS data was acquired
(e.g., Q-Tof or ion trap instruments) or, as in this case, on
separate instruments. If a separate instrument is used, it is
Liquid Chromatography-Mass Spectrometry (LC-MS)-Based Analysis of Molecular. . .
221
(LTQ).
5. Use normalized collision energies of 40% and 30–35% in HCD
(higher energy collisional dissociation) and MS
n experiments,
respectively (see Note 5).
4 Notes
1. The described method is aimed at rapid screening of lipid
molecular species across a broad range of lipid classes. While
use of positive ion mode is described, the same platform can
also be applied using electrospray ionization in negative ion
mode, which will lead to better sensitivity for specific phospholipid classes such as phosphatidylinositols and phosphatidylserines
as
well
as
glycosylated
sphingolipids
and
sulfoquinovosyldiacylglycerols (SQDGs). In negative ion
mode, a better ionization efficiency is achieved when the
UPLC eluents are prepared without formic acid. In the case
of polar, glycosylated sphingolipids, another extraction method
is needed in order to achieve sufficient extraction recoveries.
2. The method can be transferred to a different LC-MS system.
Ideally, the system used should provide a high mass accuracy
and have capability to obtain MS/MS data. For example, a
UPLC-Synapt TQ-S (Waters) instrument with capability of
parallel MS/MS experiments by utilizing its MS
E capability
has been utilized.
3. The updated version of the open source MZmine 2 software,
including tutorials, is available at http://mzmine.github.io/.
The data processing parameters need to be optimized for a
specific analytical system used in order to account for different
peak shapes and lengths as well as different mass spectrometer
resolution. Differential profiling of multiple samples requires
steps such as peak detection, alignment (matching of peaks
across multiple samples), and normalization using internal
standards.
4. Some algae species/cells contain significant amounts of TAG
(17:0/17:0). If new types of samples are analyzed, it is recommended first to run a sample extract without any internal standard
addition and check the profile. If significant amounts of TAG
(17:0/17:0) are detected, another internal standard should be
chosen instead (preferable a stable isotope-labeled compound).
5. In general, the MS/MS or MS
n experiments can be performed
on the same instrument where the LC-MS data was acquired
(e.g., Q-Tof or ion trap instruments) or, as in this case, on
separate instruments. If a separate instrument is used, it is
Liquid Chromatography-Mass Spectrometry (LC-MS)-Based Analysis of Molecular. . .
221