We have earlier utilized a lipidomics platform for clinical samples [6], and the UHPLC-MS-based lipidomics platform has
shown to be a valuable tool for investigating lipids in many different
sample matrices [7] including algae [8]. Here we describe a simple
UPLC-MS-based global analysis platform for algae lipids. Lipid
composition of algal biomass consists mainly of neutral lipids such
as triacylglycerols (TAGs), glycolipids MGDGs, DGDGs, and sulfoquinovosyl diacylglycerols (SQDGs) as well as phospholipids
(mainly phosphatidylcholines, phosphatidylglycerols, phosphatidylinositols, and phosphatidylethanolamines [5]). The simple platform in which the specific properties of each lipid class are tackled
by using class-specific internal standards provides a relatively comprehensive lipid profile of algae. Additionally, an UPLC-MS/MS
platform for lipid characterization is described. In the characterization stage, a LTQ-Orbitrap or another instrument offering a good
mass accuracy and mass resolution is utilized. Lipid annotations are
based on accurate mass of the molecular ion and on MS/MS
fragmentation.
2 Materials
2.1 Samples
Samples: 5 mg freeze-dried algae material, e.g., Euglena gracilis or
Chlorella pyrenoidosa biomass cultivated in bioreactors.
2.2 Standards and
Chemicals
The internal standard mixture contains compounds from several
different lipid classes: phosphatidylcholines (PC), phosphatidylethanolamines (PE), a ceramide (Cer), a phosphatidylglycerol
(PG), a glucosylceramide (GlcCer), as well as di- and triacylglycerols (DAG and TAG, respectively). Lipids are denoted by their
molecular composition as follows::
indicates a phosphatidylcholine comprising two C17 fatty acids
with no double bonds.
1. Standard mixture 1 added to the samples before extraction: PC
(17:0/0:0), PC(17:0/17:0), PE (17:0/17:0), PG(17:0/17:0)
[rac], Cer(d18:1/17:0), GlcCer(d8:1/12:0), and DAG(17:0/
17:0/0:0)[rac] and TAG(17:0/17:0/17:0). The concentration level of each standard is approximately 0.2 μg/sample.
2. Standard mixture 2 added after extraction: PC (16:1/0:0-D 3 ),
PC(16:1/16:1-D 6 ), and TAG(16:0/16:0/16:013
C3). The
concentration level of each standard is 0.2 μg/sample.
3. Extraction solvents: HPLC-grade chloroform/methanol (2:1,
v/v).
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shown to be a valuable tool for investigating lipids in many different
sample matrices [7] including algae [8]. Here we describe a simple
UPLC-MS-based global analysis platform for algae lipids. Lipid
composition of algal biomass consists mainly of neutral lipids such
as triacylglycerols (TAGs), glycolipids MGDGs, DGDGs, and sulfoquinovosyl diacylglycerols (SQDGs) as well as phospholipids
(mainly phosphatidylcholines, phosphatidylglycerols, phosphatidylinositols, and phosphatidylethanolamines [5]). The simple platform in which the specific properties of each lipid class are tackled
by using class-specific internal standards provides a relatively comprehensive lipid profile of algae. Additionally, an UPLC-MS/MS
platform for lipid characterization is described. In the characterization stage, a LTQ-Orbitrap or another instrument offering a good
mass accuracy and mass resolution is utilized. Lipid annotations are
based on accurate mass of the molecular ion and on MS/MS
fragmentation.
2 Materials
2.1 Samples
Samples: 5 mg freeze-dried algae material, e.g., Euglena gracilis or
Chlorella pyrenoidosa biomass cultivated in bioreactors.
2.2 Standards and
Chemicals
The internal standard mixture contains compounds from several
different lipid classes: phosphatidylcholines (PC), phosphatidylethanolamines (PE), a ceramide (Cer), a phosphatidylglycerol
(PG), a glucosylceramide (GlcCer), as well as di- and triacylglycerols (DAG and TAG, respectively). Lipids are denoted by their
molecular composition as follows:
indicates a phosphatidylcholine comprising two C17 fatty acids
with no double bonds.
1. Standard mixture 1 added to the samples before extraction: PC
(17:0/0:0), PC(17:0/17:0), PE (17:0/17:0), PG(17:0/17:0)
[rac], Cer(d18:1/17:0), GlcCer(d8:1/12:0), and DAG(17:0/
17:0/0:0)[rac] and TAG(17:0/17:0/17:0). The concentration level of each standard is approximately 0.2 μg/sample.
2. Standard mixture 2 added after extraction: PC (16:1/0:0-D 3 ),
PC(16:1/16:1-D 6 ), and TAG(16:0/16:0/16:013
C3). The
concentration level of each standard is 0.2 μg/sample.
3. Extraction solvents: HPLC-grade chloroform/methanol (2:1,
v/v).
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