Identification of a novel lipid family containing a modified head group
Seetaramanjaneyulu Gundimeda, Arunkumar Padmanaban.
Agilent Technologies India Pvt. Ltd, Bangalore, Karnataka, India
Introduction
Experimental
Processing
the data
Results
and Discussion
Work Flow
Step 1. Identification of a characteristic feature in the
compound class
Extraction of
lipids from cells
Agilent Bond
Elut aminopropyl cartridge
PS
negative
Bond-Elut
Aminopropyl
cartridge
D E F….J
Polarity
+
Figure 1. Schematic diagram showing fractionation of lipid
extract using solid phase extraction
CH3
negative 97 or 153
O
PA
OH
negative 241
O
-
PI
60 (w/formate adduct)
-
O
OH
P
O
O
CH3
87
Table 1. Characteristic features of different lipid classes in
their MS/MS spectra
Step 2. Filtering the MS/MS data with a specific
characteristic feature
MS/MS Spectra can be filtered either using a fragment
ion or a neutral loss. Compound list containing the
characteristic feature of interest in their MS/MS spectra
can be obtained by filtering the data in MassHunter as
explained in figure2. the generated list is converted to
CEF file and exported to SimLipid.
Convert the compounds list into CEF file
and exporting to SimLipid
Total lipid
extracts Organic Aqueous
ABC
Choose a suitable characteristic feature to
identify a lipid class from ‘Filter results by
fragments’ option
negative 140
-
Lipids were extracted from cells/plasma using Folch’s
method involving methanol/water/chloroform (1:1:2 v/v).
The total lipid extract was loaded on Agilent Bond-Elut
aminopropyl cartridge to separate the mixture into
individual classes2.
PE
O
Experimental
negative
P
Find compounds through MassHunter
Qualitative analysis using ‘Find compounds
by Auto MS/ MS’
PC
Neutral loss
O
1290 Infinity UHPLC, 6550 LC/Q-TOF
Characteristic
Fragment Ion
(m/z)
CH3
Agilent
Eclipse RRHD
C18 column
Identification of Novel Lipids
Using the work flow given in steps 1, 2 and 3, different
classes of phospholipids were identified. During the search,
we identified a lipid family with novel fragmentation
pattern.
Step 3. Selecting relevant lipid class in SimLipid
Before filtering the data, knowledge of characteristic
fragment ion or neutral loss for every lipid class is
necessary. Some of the widely accepted characteristic
features are given in table 1.
Lipid Polarity
class
Agilent
Eclipse RRHD
C18 column
Results and Discussion
O
Lipids are functionally important molecules. Lipid analysis
can help reveal information about biologic systems. Lipids
occupy a major portion of the human metabolome.
Dysfunctional lipid metabolism has been a signature in
many metabolic disorders. Altered lipid metabolism is
reported in cancer, Alzheimer’s, Parkinson’s, etc1. Novel
lipid identification has been a topical area globally and
helped research scientists in understanding lipid
biosynthesis in a better way. Identification of a new class of
oxidized phosphatidyl cholines (PC) helped research
scientists in
identifying the mechanism behind the
scavenging of oxidized low density lipoprotein (LDL).
Accurate mass information and MS/MS information are
necessary for an unambiguous identification of lipids. Due
to the variations in ionization efficiencies among lipid
classes, it may be essential to acquire the data both in
positive and negative modes. For lipid profiling studies, total
lipid extract can be used without any further fractionation.
However, this can severely hamper the detection of low
ionizing species like ether lipids. Fractionation of lipid
extract will reduce the sample complexity enabling
identification of low ionizing lipids. In the present study, we
carried out lipid profiling of THP-1 cells with and without
any sample pretreatment prior to LC-MS analysis.

ASMS 2016
Poster TP518
Figure 3, Selection of lipid class to search for matching
spectra after uploading the CEF file to SimLipid
Figure 5. MS/MS spectra of ion m/z 409 from THP-1 cells
The CEF file from MassHunter was exported to SimLipid
and processed for lipid identification. A Partial list of lipids
identified by SimLipid is given in table 2.
m/z
744.556
746.5153
748.5303
748.5303
750.546
750.546
750.546
750.546
No. of hits
19
1
2
2
6
6
6
6
Name of the compound
PE(18:0/18:1(9Z))
PE(P-16:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z))
PE(O-16:0/22:6(4Z,7Z,10Z,13Z,16Z,19Z))
PE(P-18:0/20:5(5Z,8Z,11Z,14Z,17Z))
PE(O-16:0/22:5(4Z,7Z,10Z,13Z,16Z))
PE(P-16:0/22:4(7Z,10Z,13Z,16Z))
PE(P-18:0/20:4(5Z,8Z,11Z,14Z))
PE(P-18:0/20:4(5Z,8Z,11Z,14Z))
Score
0.2772
0.2625
0.2417
0.2452
0.1548
0.2131
0.2726
0.2131
Obtain the search results from SimLipid by
selecting the lipid class relevant to the
characteristic feature chosen
 Low ionizing lipids such as ether lipids were identified
from the total lipid extract using 6550 iFunnel Q-TOF
mass spectrometer without any fractionation
Figure 6. MS/MS spectra of ion m/z 437 from THP-1 cells
327.2337
1
0
Figure 2. Filtering the data using a
specific fragment ion
283.2439
436.2836
746.5143
100 150 200 250 300 350 400 450 500 550 600 650 700 750
Counts vs. Mass-to-Charge (m/z)
Figure 4. MS/MS spectra of ion m/z 746 containing
signature ion m/z 140 for PE
 Lipids with this modified head group found to contain
alkyl and acyl hydrophobic chains similar to known
classes of lipids
1.
2.
3.
4.
59.0140 140.0124 196.0385
50
 A novel lipid with a modification on the phosphate head
group is identified
References
3
2
Conclusions
 SimLipid could be used to identify the lipids from their
MS/MS spectra using characteristic fragment ions
Table 2. Partial list of lipids with fragment ion m/z 140 in
the MS/MS spectra as identified by SimLipid
4
x10 -ESI Product Ion (746.515)
4
Phospholipids typically give fragment ions at m/z 153, 97
and 79. Among these, fragment ion 97 corresponds to
phosphate moiety. MS/MS spectra of two precursor ions at
m/z 409 and 437 showed an ion at m/z 111, at a shift of 14
Da from the expected ion at m/z 97. This can be correlated
to the presence of an additional CH2 in the head group. The
spectra also showed another uncommon neutral loss of 32
Da from the parent ion (Figures 6 and 7) These two
observations strongly indicated the presence of a P-OMe
group in the phosphate head3. The spectra did not show
any other intense ion related to hydrophobic chain. In the
absence of any such dominant peak corresponding to the
hydrophobic chain, precursor ion 409 may contain an ether
linked alkyl chain4. Accurate mass measurement confirmed
the proposed structure with a methyl hydrogen phosphate
head group and C16 ether linked hydrophobic chain for ion
409 and C18 chain for another ion. Another lipid at m/z 449
(Figure 8) also showed similar fragmentation pattern but
with an acyl chain indicated by a dominant ion at m/z 281.
Figure 7. MS/MS spectra of ion m/z 449 from THP-1 cells
Zhao YY et al. Chem Biol Interact. 240, 2015, 220-238
Facciotti F et al. Nat. Immunology, 13, 2012, 474-480
Aloki J et al. The J. Biochem. 146, 2009, 283-293.
Lepore M et al. JEM 211, 2014, 1363-1377
For Research Use Only. Not for use in diagnostic procedures.