Structural characterization of oxidized glycerophosphatidylserine: Evidence of polar head Oxidation

Article abstract of DOI:10.1007/s13361-011-0194-9

Non-oxidized phosphatidylserine (PS) is known to play a key role in apoptosis but there is considerable research evidence suggesting that oxidized PS also plays a role in this event, leading to the increasing interest in studying PS oxidative modifications. In this work, different PS (1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (PLPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) were oxidized in vitro by hydroxyl radical, generated under Fenton reaction conditions, and the reactions were monitored by ESI-MS in negative mode. Oxidation products were then fractionated by thin layer chromatography (TLC) and characterized by tandem mass spectrometry (MS/MS). This approach allowed the identification of hydroxyl, peroxy, and keto derivatives due to oxidation of unsaturated fatty acyl chains. Oxidation products due to oxidation of serine polar head were also identified. These products, with lower molecular weight than the non-modified PS, were identified as [M - 29 - H]^- (terminal acetic acid), [M - 30 - H] ^- (terminal acetamide), [M - 13 - H]^- (terminal hydroperoxyacetaldehyde), and [M - 13 - H]^- (terminal hydroxyacetaldehyde plus hydroxy fatty acyl chain). Phosphatidic acid was also formed in these conditions. These findings confirm the oxidation of the serine polar head induced by the hydroxyl radical. The identification of these modifications may be a valuable tool to evaluate phosphatidylserine alteration under physiopathologic conditions and also to help understand the biological role of phosphatidylserine oxidation in the apoptotic process and other biological functions. American Society for Mass Spectrometry, 2011.

Full text of DOI:10.1007/s13361-011-0194-9

B American Society for Mass Spectrometry, 2011
J. Am. Soc. Mass Spectrom. (2011) 22:1804Y1814
DOI: 10.1007/s13361-011-0194-9
RESEARCH ARTICLE
Structural Characterization of Oxidized
Glycerophosphatidylserine: Evidence of Polar
Head Oxidation
Elisabete Maciel, Raquel Nunes da Silva, Cláudia Simões, Pedro Domingues,
M. Rosário M. Domingues
Mass Spectrometry Centre, QOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
Abstract
Non-oxidized phosphatidylserine (PS) is known to play a key role in apoptosis but there is
considerable research evidence suggesting that oxidized PS also plays a role in this event,
leading to the increasing interest in studying PS oxidative modifications. In this work, different
PS (1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (PLPS), 1-palmitoyl-2-oleoyl-sn-glyc-
ero-3-phospho-L-serine (POPS), and 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS)
were oxidized in vitro by hydroxyl radical, generated under Fenton reaction conditions, and
the reactions were monitored by ESI-MS in negative mode. Oxidation products were then
fractionated by thin layer chromatography (TLC) and characterized by tandem mass
spectrometry (MS/MS). This approach allowed the identification of hydroxyl, peroxy, and keto
derivatives due to oxidation of unsaturated fatty acyl chains. Oxidation products due to oxidation
of serine polar head were also identified. These products, with lower molecular weight than the
non-modified PS, were identified as [M – 29 – H]– (terminal acetic acid), [M – 30 – H]– (terminal
acetamide), [M – 13 – H]– (terminal hydroperoxyacetaldehyde), and [M – 13 – H]– (terminal
hydroxyacetaldehyde plus hydroxy fatty acyl chain). Phosphatidic acid was also formed in these
conditions. These findings confirm the oxidation of the serine polar head induced by the hydroxyl
radical. The identification of these modifications may be a valuable tool to evaluate
phosphatidylserine alteration under physiopathologic conditions and also to help understand
the biological role of phosphatidylserine oxidation in the apoptotic process and other biological
functions.
Key words: Phosphatidylserine, Oxidation, Hydroxyl radical, Electrospray, Mass spectrometry
the plasma membrane is lost and PS is translocated to the
outer leaf of the plasma membrane [1–3]. The external-
Introduction
hosphatidylserine (PS) is a phospholipid (PL) that is
widely distributed among all mammalian cells. It
ization of PS is an early indicator of apoptosis and is
essential for the recognition and removal of apoptotic cell.
Phagocytes recognize PS as a signal initiator of apoptosis [1,
4–8]. Tyurina et al. reported that inhibition of amino-
phospholipid translocase, which selectively pumps phospha-
tidylserine and phosphatidylethanolamine from the outer to
the inner plasma membrane monolayer [9], results in
externalization of phosphatidylserine, and activation of
macrophages [10]. PS asymmetry has also been associated
with clinical situations in which apoptosis plays a relevant
role, as in cancer, chronic autoimmunity, and infections [3].
P
comprises approximately 10% of the total phospholipid of
the cell and is found preferentially in the inner leaflet of the
plasma membrane and in endocytic membranes. PS plays
important roles in many biological processes, with emphasis
on blood clotting and apoptosis [1]. In some physiologic
processes, the asymmetric distribution of phospholipids of
Correspondence to: M. Rosario M. Domingues; e-mail: mrd@ua.pt
Received: 18 April 2011
Revised: 7 June 2011
Accepted: 8 June 2011
Published online: 19 July 2011

E. Maciel, et al.: Oxidation of PS polar head by TLC and MS
1805
Although non-oxidized PS are major ligands recognized by
macrophages on the apoptotic cell, there is growing evidence
that oxidized PS (oxPS) also can have an active role [11–
14].
(DPPS), and palmitoyloleoylphosphatidylethanolamine were
obtained from Avanti Polar Lipids, Inc. (Alabaster, AI, USA)
and used without further purification. FeCl2, EDTA and H2O2
(30%, w/w) were acquired from Merck (Darmstadt, Germany).
Triethylamine (Acros Organics, Geel, Belgium), chloroform
(HPLC grade), methanol (HPLC grade). and ethanol absolute
(Panreac) were used without further purification. TLC silica gel
60 plates with concentrating zone (2.5×20 cm) were purchased
from Merck.
When the phospholipids undergo oxidation, a variety of
oxidized products can be formed, mainly due to the
modification of unsaturated fatty acyl chains [15]. These
modifications can be induced in vivo by enzymatic
(cytochrome c, myeloperoxidase) or non-enzymatic reac-
tions (•OOH,•OH, Fe2+, Cu+, radiation). Depending on the
predominating oxidative process, different PLs oxidation
products can be observed [16]. It is thought that oxidized
PLs may have new biological properties, participating in
various biological processes such as the immune response,
inflammation, apoptosis, and age-related diseases [16].
However, little is known about the relationship between
specific oxidation products and their biological effects.
Mass spectrometry (MS) has been used to identify non-
oxidized and oxidized phospholipids, including native and
oxidized PS [17–21]. PS hydroperoxy and hydroxy species
were identified by Kagan and co-authors using electrospray
mass spectrometry (ESI-MS) and tandem mass spectrometry
(ESI-MS/MS) in negative mode. Oxidized PS has been
identified in several pathological conditions: Tyurina et al.
identified mono-hydroperoxy derivatives of PS after intesti-
nal injury induced by γ-irradiation, [22, 23]; Bayir et al. also
proposed the presence of PS hydroperoxides derivatives in
traumatic brain injury after controlled cortical impact [24];
hydroperoxyl and hydroxyl PS derivates were also identified
by Tyurin et al. during apoptosis induced in neurons by
staurosporine [25] and in cells and tissues after pro-apoptotic
and pro-inflammatory stimuli [26]; the same group showed
that the pattern of phospholipid oxidation during apoptosis is
non-random and that PS is one of the preferred peroxidation
substrates [8].
Although several studies have produced evidence on the
role of oxidation of PS in inflammation and apoptosis, there is
still insufficient data on the mechanisms of oxidation and on
the nature of the oxidized species formed. The objective of
this research is to evaluate the molecular changes induced in
phosphatidylserines when subjected to oxidative stress,
focusing on the oxidation products resulting from oxidation
of the polar head. Different PS species were subjected to
oxidation induced by the hydroxyl radical generated under
Fenton reaction (H2O2/Fe2+) conditions and the oxidation
products were subsequently separated by thin layer chroma-
tography (TLC) and analyzed by electrospray (ESI), mass
spectrometry (MS), and tandem mass spectrometry (MS/MS).
Oxidation of Phosphatidylserine by Fenton
Reaction
Ammonium hydrogen carbonate buffer (5 mM, pH 7.4) was
added to 1 mg of phospholipid (2 mg/mL) and the solution
was vortex-mixed and sonicated for the formation of
vesicles. Oxidative treatments using Fe (II) and H2O2 were
carried out by adding 40 μM FeCl2/EDTA (1:1) and 10 mM
of H2O2 to total a volume of 500 μL of solution. The
mixture was left to react at 37 °C in the dark for several days
with agitation. Controls were performed by replacing H2O2
with water and without H2O2 and Fe2+.
ESI-MS Conditions (Linear Ion Trap)
The extent of oxidation was monitored by electrospray mass
spectrometry in a linear ion trap mass spectrometer LXQ
(ThermoFinnigan, San Jose, CA, USA). The LXQ linear ion
trap mass spectrometer was operated in negative mode. ESI
conditions were as follows: electrospray voltage was 4.7 kV;
capillary temperature was 275 °C, and the sheath gas flow was
25 U. An isolation width of 0.5 Da was used with a 30 ms
activation time for MS/MS experiments. Full scan MS spectra
and MS/MS spectra were acquired with a 50 ms and 200 ms
maximum ionization time, respectively. For MS/MS experi-
ments, normalized collision energy (CE) was applied in the
range of 17 to 20 (arbitrary units) for MS/MS. Data acquisition
was carried out on an Xcalibur data system (ver. 2.0).
Exact Mass Measurement and Elemental
Composition
Identification of the oxidation product ions corresponding to
the oxidation in the polar head group was confirmed by
exact mass measurement and elemental composition deter-
mination in a MALDI-TOF/TOF mass spectrometer. Ele-
mental composition of the ion at m/z 721.5 (observed for
DPPS) was confirmed by exact mass measurement and
elemental composition determination using a ESI-Q-TOF2
mass spectrometer due to the presence of a contaminate with
the same m/z value in the MALDI-MS spectrum.
Experimental
Materials
MALDI-MS Conditions
1-Palmitoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine
(PLPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-ser-
ine (POPS), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine
MALDI mass spectra were acquired using a MALDI-TOF/
TOF Applied Biosystems 4800 Proteomics Analyzer

1806
E. Maciel, et al.: Oxidation of PS polar head by TLC and MS
(Applied Biosystems, Framingham, MA, USA) instrument
equipped with a nitrogen laser emitting at 337 nm and
operating in a reflectron mode. Full scan mass spectra
ranging from m/z 650 to 4000 were acquired in the negative
mode. All spectra were acquired with 2,5-dihydroxybenzoic
acid (DHB) matrix. The matrix solution was prepared by
dissolving 10 mg of DHB in a 1 mL mixture of methanol:
aqueous (1:1, vol/vol). For the accurate mass measurements,
the lock mass in each mass spectrum was the calculated
monoisotopic mass/charge of the non-modified phosphati-
dylserine and palmitoyloleoylphosphatidylethanolamine as
internal standard.
ions of oxidation products. The ESI-MS spectra of PLPS
(Figure 2a), acquired after exposure to oxidative conditions,
show a larger number of new molecular ions. This is due to
the presence in this molecule of two double bonds in the sn-
2 fatty acyl chain, increasing the number of possible
oxidation sites. Lower number of ions were observed for
POPS (Figure 2c) and even lower for DPPS (Figure 2e). In
the spectra of oxPLPS and oxPOPS, some oxidation
products can be observed at higher m/z values than the
non-modified PS, corresponding to the oxidation products
with insertion of oxygen atoms as hydroperoxides ([M – H +
2O]–), hydroxide ([M – H + O]–), and keto ([M – H + O –
2 Da]–) derivatives. These oxidation products will be briefly
discussed in this work since they have already been
described and fully characterized [13, 23, 25].
ESI-MS Conditions (ESI-Q-TOF2)
For the analysis in the ESI-QTOF2 instrument (Micromass,
Manchester, UK), the flow rate was 10 μL min–1, the needle
voltage was set at 3 kV, the cone voltage at 30 V, the ion
source set at 80 °C, and the desolvation temperature at 150 °C.
Mass spectra were averaged for 1 min. For the accurate mass
measurements, the lock mass in each mass spectrum was the
calculated monoisotopic mass/charge of the native phospho-
lipid (non-modified phospholipid).
In the spectrum obtained after oxidation of the PLPS
(Figure 2a) we can observe new ions at m/z 666 and 682
(666+16), with lower m/z than the non-modified PLPS.
These ions correspond to oxidation products with shortened
acyl chain at C9 and a carboxylic acid terminal. Short chain
oxidation products originated from cleavage of fatty acyl
chains have been described for PS [13] and have been
studied in phosphatidylcholine, phosphatidylserine, and
cardiolipin phospholipids [27–29]. The shortened oxidation
products are generated after abstraction of the bis-allylic
hydrogen atoms by the hydroxyl radical, which through a β-
scission mechanism, break down to short-chain phospholipid
products with terminal aldehydic or carboxylic acid func-
tion. As reviewed elsewhere, these are well known products
of lipid peroxidation [30, 31]. No short chain oxidation
products were formed during POPS oxidation, similar to
previously observed POPC oxidation, using the same
oxidative conditions [31].
There are other ions observed at lower m/z than the non-
modified PS in MS spectra of oxidized PLPS, POPS, and
DPPS (Figure 2a, c, and e), which were not assigned as short-
chain compounds. These oxidation products, with less 29, 30,
and 13 Da compared with the native PS, occur due to oxidative
modification in PS polar head, as confirmed by separation
using thin layer chromatography (TLC) (Figure 3), and
analysis by tandem mass spectrometry will be described latter.
Elemental composition determination for the ions with less
29 Da (for DPPS: C37H70O10 P, error 18.7 ppm; for POPS
C39H72O10P, error 27.8 ppm, for PLPS C39H70O10P, error
−15.4 ppm), with less 30 Da (for DPPS, C37H71NO9P error
10.3 ppm; for POPS C39H73NO9P, error −1.3 ppm; for PLPS
C39H71NO9P error −32.1 ppm), and with less 13 Da (for DPPS
C37H70O11P error −9.9; for POPS C39H72O11P error
20.9 ppm) confirmed the modification of serine polar head.
Mass spectrometry analysis, in positive mode, of these
oxidized PS, showed molecular ions that correspond to the
same oxidation products, but observed as low abundant [M +
H]+, [M + Na]+, and [M – H + 2Na]+ ions (data not shown).
However, the oxidation product resulting from loss of 29 Da
was not observed. We propose that this oxidation product
contains a carboxylic acid group polar head, as will be
Separation of Oxidation Products of PS by Thin
Layer Chromatography
The oxidation products were separated by thin layer
chromatography (TLC) using silica gel 60 plates with
concentrating zone 2.5×20 cm (Merck KGaA). Prior to
separation, plates were treated with boric acid 2.3% in
ethanol. The plates were developed with solvent mixture
chloroform/ethanol/water/triethylamine (30:35:7:35, vol/vol/
vol/vol). Lipid spots on TLC plates were observed by
exposure to primuline. The TLC spots identified as oxidized
PS were scraped from the plates and extracted using
chloroform/methanol (2:1, vol/vol). The oxidation products
separated by thin layer chromatography (TLC) were ana-
lyzed by electrospray (ESI), mass spectrometry (MS), and
tandem mass spectrometry (MS/MS).
Results and Discussion
This paper focuses on the ESI-MS analysis of PS that was
submitted to oxidation induced by the hydroxyl radical
generated under Fenton reaction (H2O2/Fe2+). Selected PS
were dipalmitoyl-phosphatidylserine (DPPS; C16:0/C16:0;
m/z [M – H]–=734), 1-palmitoyl-2-oleoyl-phosphatidylser-
ine (POPS; C16:0/C18:1; m/z [M – H]– =758), and the 1-
palmitoyl-2-linoleoyl phosphatidylserine (PLPS; C16:0/
C18:2; m/z [M – H]-=756) (Figure 1). Since DPPS contains
saturated acyl chains, it is considered as a model for
studying oxidation of the phospholipid polar head [15].
Comparing the ESI-MS spectra of the three PS obtained
before and after oxidation (Figure 2), new ions can be
observed. These ions correspond to the [M – H]– molecular

E. Maciel, et al.: Oxidation of PS polar head by TLC and MS
1807
Figure 1. Molecular structures of PLPS, POPS, and DPPS
described latter, forming, preferentially, negative ions. To
better characterize these new oxidation products, they were
further analyzed by TLC, MS, and MS/MS in negative mode.
oxidation products because it has two saturated fatty acyl
chains that do not oxidize [15]. ESI-MS/MS spectra of PLPS
and POPS oxidation products show formation of hydroxy [M
–H + O]–, keto [M – H + O – 2 Da]–, peroxy [M – H + 2O]–,
and hydroxy peroxy [M – H + 3O]– derivatives, as
summarized in Table 1. In all these MS/MS spectra, we can
observe a major product ion formed by loss of 87 Da, due to
loss of phospholipid polar head. This is an abundant loss,
typical of PS [18]. MS/MS analysis (Table 1) shows that
insertion of oxygen atoms occur in the linoleic (for PLPS) or
Phosphatidylserine Oxidation in Fatty Acyl
Chains—Analysis by ESI-MS/MS
Hydroperoxide and hydroxide derivatives are primary oxida-
tion products of lipids and phospholipids, and occur during
PLPS and POPS oxidation. DPPS does not yield these
Figure 2. ESI-MS spectra of PLPS (a), (b), POPS (c), (d) and DPPS (e), (f). Spectra a, c, and e were acquired after exposing
samples to oxidative stress. Spectra b, d, and f were acquired from control samples

1808
E. Maciel, et al.: Oxidation of PS polar head by TLC and MS
Figure 3. TLC of oxidation products of phosphatidylserines with modifications in the polar head (oxPLPS (lines 1, 2, and 3),
oxPOPS (lines 4, 5, and 6), and oxDPPS (lines 8, 9, and 10). PS and PE standards were applied in line 7
oleic acids (for PLPS), as confirmed by the observation of the
carboxylate anions of modified fatty acyl chains R'COO–, in
agreement with previous published work [26, 28, 31–33].
In the TLC of the oxidized PS, five new spots were
resolved using the same conditions used to separate
phospholipid classes, demonstrating that significant changes
of polarity have occurred, presumably with formation of new
compounds with different polar head structures. The new
spots were scraped from the plate, the OxPL extracted, and
further analyzed by ESI-MS and MS/MS. Table 2 summa-
rizes are all molecular ions observed in MS spectra for each
spot of TLC and the proposed structure.
In spots #1 (Figure 3, Table 2) we have identified the
non-modified PS together with the hydroperoxides, keto,
and hydroxides oxidation products (Table 1), in agreement
with previous results [26]. In the spots #2 (Figure 3, Table 2)
we identified, for all PS, oxidation products with polar head
modifications [M – H – 29]–. Spots #3 (Figure 3, Table 2)
shows a Rf similar to the phosphatidic acid (PA) standard.
The MS spectra acquired from samples of these spots show
[M – H – 87]– ions, thus indicating that PA is generated
during PS oxidation, with complete loss of polar head. PA
was also observed during cardiolipin oxidation by γ-
irradiation [34, 35]. The oxidation products from spots #4
(Figure 3, Table 2) contained a head modification, with a
mass difference of less 30 Da. It is also possible to observe
spots #5 (Figure 3, Table 2) in the DPPS and POPS.
Analysis of the MS spectrum of these samples shows the
presence of ions at m/z 747 and 763 for POPS, and an ion at
m/z 721 for DPPS. These ions have a difference of 13 Da
compared with the non-modified PS. In the case of PLPS,
these oxidation products were not observed, probably
because they might decompose to fatty acyl short chain
Phosphatidylserine Oxidation with Modification
of in Polar Head—Analysis by TLC and ESI-MS/
MS
Oxidative modifications in the PS polar head should change
significantly the polarity of these species. Consequently,
their separation may be possible through simple techniques
such as TLC, which is widely used for separation of
different classes of phospholipids. Thus, oxidative mixtures
of the three PS under study were fractioned by TLC, as is
illustrated in Figure 3.
Table 1. Product Ions Observed in ESI-MS/MS Spectra of Oxidized PLPS
and POPS, which were Determined by Tandem Mass Spectrometry to
Contain Modified Fatty Acyl Chains
Modifications
[M–H]–
R2'COO–
[M–R1COOH–87–H]–
PLPS
+1 O
–OH
– =O
774
772
790
806
666
682
776
774
792
-
295
293
311
327
187
203
297
295
313
-
431
429
447
463
323
339
433
431
449
-
+2 O
+3 O
Short (C9)*
Short (C9) + O
POPS +1 O
+2 O
-
DPPS
*Short chain oxidation product in C9 with carboxylic acid terminal.

E. Maciel, et al.: Oxidation of PS polar head by TLC and MS
1809
Table 2. Molecular Ions Observed in the ESI-MS Spectra from the Different Spots Identified in the TLC Plate for Each PS. The Table Shows the m/z Value
of the [M – H]– Ions, their Most Probable Identification, Including the Modified Polar Headgroup, and the Typical Neutral Loss, Observed in the MS/MS
Spectra
[M H]
Spots
#
Polar
head
Neutral
loss
Modification
PLPS POPS DPPS
–
+14
+16
+30
+32
+46
+48
29
29+14
29+16
29+30
29+32
87
87+14
87+16
87+30
87+32
30
30+14
30+16
30+30
30+32
13
758
772
774
788
790
804
806
729
743
745
759
761
671
685
687
701
703
728
742
744
758
760
–
760
774
776
790
792
–
734
–
–
–
–
–
–
705
–
–
–
–
647
–
–
–
–
704
–
–
CO2
-H2C
1
87
NH2
–
731
745
747
761
763
673
687
689
703
705
730
744
746
760
762
747
763
O
-H2C
2
3
4
58
–
O-
absent
O
-H2C
57
NH2
O
–
–
721
HO
O
5
74
58
13+16
–
-HC
OH
5*
13
747
O
derivatives, which are not observed in TLC plate. The MS
spectra of POPS samples from spots #5 show ions at the
same m/z of ions found in spots #2. These are necessarily
different compounds, since they have different Rf. The
nature of these ions was determined by MS/MS analysis and
will be discussed in another section of this manuscript. In
each spot of PLPS and POPS oxidized samples, additional
ions with plus 14 Da (+O – 2 Da), 16 Da (+O), 30 Da (+2O
– 2 Da), 32 Da (+2O) 46 Da (+3O – 2 Da), and 48 Da (+3O)
were observed. These ions resulted from additional oxida-
tion in unsaturated fatty acyl chain, as is shown in Table 2.
In order to confirm the structure of the oxidation products
found in spots #2 to #5, MS/MS of all the identified
molecular ions (Table 2) were obtained and analyzed. These
spots corresponded to oxidation products resulting from
modification of PS polar head.
The oxidation products with less 29 Da than the
unoxidized PS, found in spots #2, show a neutral loss of
58 Da, instead of the typical 87 Da of PS (Figure 4). The
MS/MS of these ions [M – H – 29]– show the non-modified
carboxylate anions RCOO–, thus confirming that the
structural changes occurred at the polar head and not at the
fatty acyl chains. This main fragmentation pathway (loss of
58 Da) is also observed for the oxidation products with
additional oxidation (+nO) in the unsaturated fatty acyl
chains observed for PLPS and POPS. Tandem mass spectra
of these ions also showed the modified RCOO–. Oxidation
products with less 29 Da were observed during the oxidation
of amino acids and were identified as the result of oxidative
reactions that lead to loss of an amine group and CO2, with
formation of a terminal carboxylic acid group [36]. We
propose that oxidation products observed in spots #2 are
glycerophosphoaceticacid derivatives (Scheme 1).
The new oxidation products identified in spots #3 are due
to oxidation in polar head group of the PS. These oxidative
modifications are not observable when a neutral loss scan of
aziridine-2-carboxylic acid (neutral loss of 87 Da) is
performed to detect oxidized PS from in vivo samples [13].
The MS/MS spectra of molecular ions [M – H – 30]–
found in spots #4 of the three PS show loss of the head
group as loss of 57 Da, but typical loss of 87 Da is not
observed. We propose that these oxidation products resulted
from decarboxylation and generation of a keto moiety, as
observed during amino acid and peptides oxidation
(Scheme 1) [36]. In these species, loss of modified polar
head group (−57 Da) is not the base peak of the spectra, in

1810
E. Maciel, et al.: Oxidation of PS polar head by TLC and MS
PLPS-29 Da
O
PLPS-29 Da+O
O
OH
P
O
O
-58
O
H
O
O
-58
O
O
O
P
O
O
O
O
O-
O
m/z 279
O-
m/z 295
O
O
687.3
O
H
671.3
100
80
60
40
20
0
100
80
391.1
60
391.1
431.1
745.3
40
415.1
473.0
255.1
729.3
255.1
20
0
658.3
642.3
609.3
600
280.1
300
489.2
513.1
200
400
500
m/z
600 700
300
400
500
m/z
700
POPS-29 Da
POPS-29 Da+O
O
O
OH
P
O
O
O
-58
OH
P
O
O
-58
O
O
O
O
O
O
HO
O
O-
m/z 281
O-
m/z 297
O
O
689.3
100
80
673.3
100
50
391.1
60
391.1
417.2
747.4
40
433.1
255.1
20
0
255.1
731.4
700
475.1
500
491.2
282.1
300
644.3
600
309.0
551.5
600
687.6
0
300
400
500
m/z
700
200
400
m/z
dPPS-29 Da
-58
O
OH
P
O
O
O
O
O
O
O-
m/z 255
O
391.1
100
80
647.4
705.4
60
255.1
40
20
0
448.9
256.2
300
553.3
400
500
600
700
m/z
Figure 4. ESI-M/MS spectra of [M – H – 29]– molecular ions that resulted from oxidation of PLPS (m/z 729 and 745), POPS (m/z
731 and 747), and DPPS (m/z 705), found in spots #2. The proposed structures of each molecular ion are also shown. The
structures of the oxidation products PLPS – 29 Da + O and POPS – 29 Da + O illustrate one possible location of the hydroxyl
group in the unsaturated fatty acyl chains
contrast with the typical behavior of PS and the other
identified PS oxidation products. A minor loss of polar head
group resembles the behavior observed in negative mode
tandem mass spectra of PE. Also, as observed in PE tandem
mass spectra, R1COO– ions were found to be more abundant
than R2COO– ions. This is in contrast with the typical
behavior of PS and other oxidation products with a terminal
carboxylic acid function, which show in their MS/MS
spectra that RA of R2COO–9R1COO– [37]. These findings
also contributed to proposing the presence of a free amine
moiety in these oxidation products (M-30 Da). We propose
that oxidation products observed in spots #3 are glycer-
ophosphoacetamide derivatives (Scheme 1) (Figure 5).
The MS/MS spectra of [M – H – 13 Da]– molecular ions
that resulted from oxidation of DPPS and POPS (Figure 6)
and were found in spots #5 (Table 2), are very similar. In
these spectra, we observe a major fragment ion formed by
loss of 32 Da (−O2), indicating the presence of a hydro-
peroxide [28, 38]. Due to the fact that DPPS does not
oxidize in the fatty acyl chain, the hydroperoxide moiety
should be located in polar head group. We propose that these
oxidation products resulted from oxidation in the polar head
group, with formation of terminal hydroperoxydeacetalde-
hyde, as shown in Table 2. The presence of a modified polar
head group is also evidenced by the presence of carboxylate
anions of the non-modified fatty acyl chains in the MS/MS
spectra and the observation of a neutral loss of 74 Da
(hydroperoxydeacetaldehyde). The MS/MS spectra of POPS
(m/z 747) found in spots #5* also shows oleoyl carboxylate
anion + 16 (m/z 297) and loss of 58 Da (CH2(OH)HC=O).
This suggests the presence of an isobar originated by
oxidation of the polar head group, with formation of a

E. Maciel, et al.: Oxidation of PS polar head by TLC and MS
1811
Scheme 1. Oxidation products of PS formed by Fenton reaction with observed modifications on the polar head group:
terminal hydroperoxyacetaldehyde (−13 Da), terminal acetic acid (−29 Da), and terminal acetamide (−30 Da)
PLPS-30 Da
-
O
O
O
O
-57
PLPS-30 Da+O
P
O-
P
O
O
O
O
O
O
-57
O
O
O
N
H2
O
m/z 279
O
O
N
H2
m/z 295
279.1
295.1
100
O
OH
100
80
60
40
20
0
80
60
448.1
255.1
488.1
40
20
0
255.1
472.1
490.2
448.1
431.1
400
744.4
687.4
728.4
700
391.1
400
506.2
279.7
300
594.2
600
310.0
300
595.9
600
200
500
m/z
200
500
m/z
700
-
O
POPS-30 Da
POPS-30 Da+O
O
-
O
O
-57
O
O
O
-57
P
O
O
O
P
O
O
O
O
O
HO
O
H N
2
m/z 281
H N
2
m/z 297
O
O
297.1
100
80
60
40
20
0
281.1
100
50
0
448.1
474.1
255.1
490.2
448.1
255.1
746.4
688.3
730.5
391.1
400
492.2
433.1
400
508.2
295.1
300
694.6
700
585.7
600
200
500
m/z
600
O
300
500
m/z
700
-57
-
dPPS-30 Da
O
O
O
O
P
O
O
O
H2
N
m/z 255
O
448.1
255.1
100
80
60
40
20
0
704.4
466.2
466.8 591.9 668.5
391.1
400
255.7
300
200
500 600 700
m/z
Figure 5. ESI – MS/MS spectra of [M – H – 30]– ions that resulted from oxidation of PLPS, POPS and DPPS, found in spots
#4. The proposed structures of each molecular ion are also shown

1812
E. Maciel, et al.: Oxidation of PS polar head by TLC and MS
(a)
(b)
-74
H
O
O
O
P
O
O
OH
O
O
O
O
m/z 281
O
POPS-13 Da
100
O
POPS-13 Da+O
100
-58
OH
P
O
O
O
H
OH
O
O
O
H
O
O
O
P
O
O
O
O
O
O
HO
O
HO
m/z 297
x20
x20
m/z 297
O
O
731.4
715.3
255.1
281.1
297.1
255.1
50
50
0
-74
689.3
763.3
297.1
689.3
705.3
-74
673.3
391.1
433.1
391.1
400
507.0
687.2
747.3
509.2
375.0
0
300
400
500
m/z
600
700
300
500
m/z
600
700
OH
dPPS-13 Da
O
O
OH
O
O
O
P
O
O
O
m/z 255
O
x20
689.4
100
255.1
50
0
721.4
-74
647.3
391.1
400
433.1
255.8
300
500
m/z
600
700
Figure 6. ESI-M/MS spectra of the [M – H – 13]– ions that resulted from oxidation of POPS (m/z 747 and 763) and DPPS (m/z
721), found in spots #5. The proposed structures of each molecular ion are also shown
species containing a terminal hydroxyacetaldehyde, and a
hydroxylated acyl chain (Figure 6a). The ion at m/z 763,
observed in POPS spots #5, corresponds to a species with a
hydroperoxydeacetaldehyde polar and a hydroxylated acyl
chain, as suggested by the fragment ion at m/z 297 [RCOO +
O]– and a neutral loss 74 Da (Figure 6b).
Phosphatidylserine polar head group has a serine amino
acid moiety linked to the phosphate group It is well known
that amino acids are prone to oxidation, not only in the side
chains but also in the alfa carbon [36, 39, 40]. Oxidative
modification of the PS polar head group has been reported to
occur during oxidation induced by HClO and catalyzed by
myeloperoxidase [41, 42], confirming the reactivity of serine
under oxidative conditions. The oxidation products reported
to be formed in those conditions were not the same as the
ones reported in this work. In those works, the authors
reported the formation of 1,2dipalmitoyl-sn-glycero-3-phos-
phoacetaldehyde and 1,2dipalmitoyl-sn-glycerol-3-phospho-
nitrile derivatives.
to β-scission of an alkoxyl radical at the C terminal alpha
carbon. This reaction is proposed to be initiated from the
abstraction of hydrogen linked to an alfa carbon, generating
a tertiary radical that is stabilized by the amine nitrogen and
carbonyl group [40, 43].
The modification in the PS polar head group leading to
the formation of the oxidation products with less 29 Da
should occur by decarboxylation plus deamination with
formation of a terminal carboxylic acid moiety (oxidation
products with −29 Da). Loss of amine and CO2 resembles
typical modification already observed during amino acid
oxidation, as reviewed elsewhere [36, 39].
Conclusions
Oxidative modifications induced to phosphatidylserine were
identified and characterized by thin layer chromatography
combined with tandem mass spectrometry. Oxidation prod-
ucts were fractionated by TLC and further characterized by
MS/MS. This approach allowed the identification of
hydroxyl, peroxy, and keto derivatives due to oxidation of
unsaturated fatty acyl chains. Additionally, we have identi-
fied for the first time five families of oxidation products,
with lower molecular weight than the non-modified PS,
which resulted from oxidative modifications on the serine
polar head group. They were identified as [M – 29 – H]–
The changes of the serine polar head group induced by
the hydroxyl radical resemble the oxidative modifications of
the amino acid structure, such as the oxidative decarbox-
ylation with formation of an additional keto group (oxidation
products with −30 Da). Decarboxylation of amino acids is a
well-known reaction occurring during amino acid and
peptide oxidation. Loss of CO2 from the C terminal is due

E. Maciel, et al.: Oxidation of PS polar head by TLC and MS
1813
(terminal acetic acid), [M – 30 – H]– (terminal acetamide),
[M – 13 – H]– (terminal hydroperoxideacetaldehyde), and
[M – 29 – H]– (terminal hydroxyacetaldehyde). Phosphatidic
acid derivatives were also formed as result of PE oxidation.
Hydroxy, keto, peroxy, and short chain acyl derivatives of
these new molecules were also identified for PS phospho-
lipids with unsaturated fatty acyl chains.
The results presented here allowed the identification, for
the first time, of modification of PS polar head group
induced by the hydroxyl radical mediated oxidation in vitro
conditions. The identification of oxidation products of PS is
far from being completely explored, although it is essential
to understanding the relation between the PS oxidation
products formed and the specific biological activity that they
mediate.
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Acknowledgments
The authors acknowledge Fundação para a Ciência e a
Tecnologia and COMPTE for funding of projects PTDC/
QUI- BIQ/104968/2008, REDE/1504/REM/2005 (that con-
cerns the Portuguese Mass Spectrometry Network) and Ph.D.
grants to E.M. (SFRH/BD/73203/2010) and to C. S. (SFRH/
BD/46293/2008).
20. Domingues, M.R.M., Reis, A., Domingues: Mass spectrometry analysis
of oxidized phospholipids. Chem. Phys. Lipids 156(1/2), 1–12 (2008)
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phospholipids within and outside of mitochondria. Biochim. Biophys.
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