identification of phospholipid structures in human blood by direct-injection quadrupole-linear...
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RAPID COMMUNICATIONS IN MASS SPECTROMETRY
Rapid Commun. Mass Spectrom. 2005; 19: 2443–2453
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.2080
Identification of phospholipid structures in human
blood by direct-injection quadrupole-linear ion-trap
mass spectrometry
Chang Wang, Jun Yang, Peng Gao, Xin Lu and Guowang Xu*National Chromatographic R&A Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian 116023, P.R. China
Received 23 March 2005; Revised 28 June 2005; Accepted 7 July 2005
Direct-injection electrospray ionization mass spectrometry in combination with information-
dependent data acquisition (IDA), using a triple-quadrupole/linear ion trap combination, allows
high-throughput qualitative analysis of complex phospholipid species from child whole blood.
In the IDA experiments, scans to detect specific head groups (precursor ion or neutral loss scans)
were used as survey scans to detect phospholipid classes. An enhanced resolution scan was then
used to confirm the mass assignments, and the enhanced product ion scan was implemented as a
dependent scan to determine the composition of each phospholipid class. These survey and depen-
dent scans were performed sequentially and repeated for the entire duration of analysis, thus pro-
viding the maximum information from a single injection. In this way, 50 different phospholipids
belonging to the phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphati-
dylcholine and sphingomyelin classes were identified in child whole blood. Copyright# 2005 John
Wiley & Sons, Ltd.
Membrane phospholipids are a complex mixture of molecu-
lar species containing a variety of fatty acyl and head group
moieties. The head group is the functional group that defines
the specific class to which the phospholipid belongs, i.e.,
phosphatidylethanolamine (PE), phosphatidylserine (PS),
phosphatidylinositol (PI), phosphatidylcholine (PC) and
sphingomyelin (SM) classes, while the fatty acyl groups
distinguish the individual phospholipid molecular species
within each class (Scheme 1). In addition to being critical com-
ponents of cellular membranes, phospholipids interact with
all membrane proteins and many non-membrane proteins,
and mediate signal transduction.1 Phospholipids serve as a
reservoir for arachidonic acid (20:4, n–6) and other polyun-
saturated fatty acids that can be metabolized to biologically
active eicosanoids such as prostaglandins, thromboxanes,
leukotrienes and lipoxins.2,3 Due to their structural and func-
tional roles in mammalian cells, the understanding of the
composition, metabolism, and regulation of phospholipids
at the level of molecular species has become increasingly
important.
Analysis of these phospholipids has been performed using
chromatographic techniques such as thin-layer chromato-
graphy (TLC),4,5 and high-performance liquid chromatogra-
phy (HPLC).6–10 However, the identification of the different
phospholipids in most of these approaches is based only on
retention behavior in comparison to known standards.
Several fast and convenient phospholipid analytical methods
have been developed using thermospray or electrospray
mass spectrometry on-line with HPLC (LC/MS),11–15 and
have been employed for the phospholipid analysis of various
biological mixtures. Separation by LC before MS analysis can
clean up samples and distinguish different phospholipid
classes; however, the whole process usually requires more
than 30 min per sample. Short analysis times are critical for
high-throughput analysis.
For the combined purpose of analytical speed and analysis
of individual molecular species of the phospholipid classes,
MS-based methods hold the most promise. Brugger et al.16
took advantage of the fact that a common fragment is formed
from the head-group region of each member of a class of
complex polar lipids upon collisionally activated dissociation
(CAD), using electrospray ionization tandem mass spectro-
metry (ESI-MS/MS). When the head group is lost as a
charged fragment, precursor (Pre) ion scanning is used, and
when the head group is lost as a neutral fragment, neutral loss
(NL) scanning provides the specific detection.38 Speed and
selectivity are the main advantages of this method. Such
selective detection is particularly useful when negatively
charged lipids are being analyzed, since the negative ion
spectrum of lipid extracts is typically highly crowded due to
extensive overlap of species belonging to different head
Copyright # 2005 John Wiley & Sons, Ltd.
*Correspondence to: G. Xu, National Chromatographic R&ACenter, Dalian Institute of Chemical Physics, The ChineseAcademy of Sciences, Dalian 116023, P.R. China.E-mail: [email protected]/grant sponsor: State Ministry of Science and Technologyof China; contract/grant number: High-Tech R & D Plan2003AA223061.Contract/grant sponsor: Foundation for Distinguished YoungScholars from National Natural Science Foundation of China;contract/grant number: 20425516.Contract/grant sponsor: Knowledge Innovation Program of theChinese Academy of Sciences; contract/grant number:K2002A12, K2003A16.
group classes.17 This approach has been used for analysis of
crude lipid extracts;16,18–20 however, the major drawback of
this approach is that the molecular species at the level
of individual fatty acid residues can not be identified.17
Information-dependent data acquisition (IDA) proce-
dures22 are optimized to generate large amounts of useful
MS/MS data, e.g., through the combination of a specific
survey scan, a high-resolution scan and a product ion scan. In
a typical IDA experiment an MS1 survey scan is performed to
generate a peak list (m/z values) of all ions present. The peak
list is subjected to a set of user-defined criteria to filter out
unwanted precursor ions, and the remaining ions are then
subjected to MS/MS analysis. This cycle is repeated through
the duration of the acquisition to generate large amounts of
informative data. In general, an IDA procedure combines two
or more different scan modes in a sequential way within the
same analytical run. In the case of phospholipid analysis, this
approach enables a head-group-specific scan and a product
ion scan to be combined.
In this work we investigated the possibility of analyzing
phospholipid species in human whole blood by combining
direct-injection electrospray ionization mass spectrometry
(DI-ESI-MS) and IDA. The total analysis time was only
2.0 min. Product ion spectra were obtained using CAD of
intact ionized molecular species in a tandem mass spectro-
meter to identify individual molecular species of each
phospholipid class.
EXPERIMENTAL
ChemicalsPhospholipid standards were from Avanti Polar Lipids
(Alabaster, AL, USA) or Sigma (St. Louis, MO, USA). 2,6-
Di-tert-butyl-4-methylphenol was from Aldrich-Chemie
(Steinheim, Germany). Formic acid and all the solvents
were HPLC grade from TEDIA (Fairfield, USA), and ammo-
nia (25%) was analytical grade from Lian-Bang (Shenyang,
China).
Sample preparationHeparinized whole child blood was from Dalian Children’s
Hospital. The lipids in 500mL of blood samples were
extracted essentially as described earlier.21 Briefly, 0.28 mL
of water was added to 500 mL of the blood sample; then
2 mL of methanol with 0.01% (w/v) 2,6-di-tert-butyl-4-
methylphenol and 4 mL of chloroform were added, and the
solution was sonicated for 60 s both before and after adding
the chloroform. The solution was then vortex-mixed for 30 s
and incubated for approximately 1 h at room temperature.
Finally, 2 mL of water were added, the solution was mixed
again, and centrifuged at 2600 g for 10 min. The lower chloro-
form phase was sampled and dried under vacuum. Prior to
analysis, the extracted samples were redissolved in 500 mL
of chloroform/methanol (2:1, v/v) and then diluted 10 times
with methanol.
Direct-injection ESI-MSDI-ESI-MS was performed using a QTRAP LC/MS/MS sys-
tem from Applied Biosystems/MDS Sciex (Concord, ON,
Canada) with a turbo-ionspray source (operated without
hot turbo-gas). Spectra were acquired in the positive or nega-
tive ion mode. This instrument is based on a triple-quadru-
pole ion path in which the final quadrupole can be used as
a linear ion trap mass spectrometer. Thus the QTRAP instru-
ment combines all of the functionality of a classic triple-quad-
rupole mass spectrometer with the capabilities of a very high
sensitivity linear ion trap.22 The combination of highly selec-
tive triple-quadrupole MS/MS scans and high sensitivity ion
H2C
CH
H2C O P O
OH
OR2 C OO
O C
O
R1
CH2CH2NH3+
H2C
CH
H2C O P O
OH
OR2 C OO
O CH2 CH2 R
CH2CH2NH3+
H2C
CH
H2C O P O
OH
OR2 C OO
CH2CHCOOH
NH2
O C
O
R1
H2C
CH
H2C O P O
OH
OR2 C OO
O C
O
R1
HO
HO
OH
OH
OH
H2C
CH
H2C O P O
OH
OR2 C OO
O C
O
R1
CH2CH2N(CH3)3+
CH
H2C O P O
OH
OC
O
CH2CH2N(CH3)3+
R2
CHHO R1
HN
Phosphatidylinositol (PI)Phosphatidylserine (PS)
Phosphatidylethanolamine (PE)
Phosphatidylcholine (PC) Sphingomyelin (SM)
Plasmalogen PE (pPE)
Scheme 1. Structures and abbreviations of common phospholipids. R1 and R2 represent different fatty acyl chains.
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2443–2453
2444 C. Wang et al.
trap product scans on the same instrumental platform turned
out to be ideal to provide rapid identification of phospholi-
pids in child blood extracts.
In this work the survey scan of the phospholipids was
performed using the ‘enhanced MS’ mode (EMS, in which the
first two quadrupoles are operated in funnel (RF-only) mode
and ions are accumulated and then mass-analyzed in the (Q3)
linear ion trap), or using a precursor ion scan (Pre ion scan, in
which Q1 sweeps a given mass range with Q3 fixed to
transmit the diagnostic fragment ion), or using a neutral loss
scan (NL scan, in which both Q1 and Q3 scan a given mass
range together but with a constant mass difference between
the ranges scanned). The structures of the phospholipds were
elucidated using the ‘enhanced’ product ion (EPI) scan mode
in which the product ions are trapped in Q3 (in trap mode)
before mass analysis. With the QTRAP system this approach
can be realized using a single instrument platform with IDA.
The phospholipid extracts from child whole blood samples
were introduced into the mass spectrometer using the
autosampler of an HP 1100 series HPLC system (Agilent
Technologies, Palo Alto, CA, USA). A mobile phase of 100%
methanol was continuously injected into the mass spectro-
meter at a flow rate of 100 mL/min, and aliquots (10mL) of
sample were directly introduced into the carrier flow and
thus on into the ionization source without sample pre-
separation. In order to increase the available IDA cycles per
injection, a homemade union replaced the LC column with
no precautions to avoid dead volume, so the duration of
sample flow into the ionization source was extended to
�1 min. The chloroform in the injection solution may have
increased the peak width to some extent. The different
phospholipid classes were identified using a Pre ion scan or
NL scan as the survey scan (range m/z 650–950, scan time
0.5 s, scan speed 4000 Da s�1, collision energy 40–50 eV); an
enhanced resolution (ER) scan was used to confirm phos-
pholipid mass assignments. An EPI scan was used as the
dependent scan (range m/z 100–950, scan speed 4000 Da s�1,
trap time 100 ms, collision energy 40–50 eV). The survey and
dependent scans were performed sequentially, and repeated
for the entire duration of analysis, thus providing maximum
information from a single injection. The instrument para-
meters were optimized to provide maximum signals from the
(positive or negative) precursor ions of the phospholipid
standards. The nitrogen drying gas and turbo gas were at 45
and 40 psi back-pressure, respectively. The curtain gas that
prevents contamination of the ion optics was set at 30 psi. The
declustering potential (DP) was set at 80 V.
RESULTS AND DISCUSSION
ESI MS1 spectraESI mass spectra (MS1) of the phospholipids in extracts of
child whole blood were obtained in both positive and nega-
tive ion EMS mode (Fig. 1). Many classes of phospholipids
Figure 1. DI-ESI-MS analysis of a child whole blood phospholipid extract: (a) positive ion and
(b) negative ion ESI mass spectra of the same extract.
Phospholipid structures in human blood by DI-ESI-QqLIT-MS 2445
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2443–2453
possess a net negative charge at neutral pH; accordingly,
negative ion ESI mass spectra of such phospholipids (PS, PI
and PE) can be effectively obtained with [M–H]� as the pre-
cursor ions. However, PC, PE and SM are zwitterionic mole-
cules, and therefore either positive or negative ion ESI mass
spectra of these phospholipid classes are readily accessible.
Thus, the positive ion spectrum in Fig. 1(a) contains the
[MþH]þ and [MþNa]þ ions of PE, PS, PC and SM, while
the negative ion spectrum in Fig. 1(b) gives intense signals
for the [M–H]� ions for PS, PI and PE, as well as [M–15]�
(demethylated) ions for PC and SM.
IDA procedureFragmentation of polar head groups of phospholipids allows
the specific detection of phospholipid classes by Pre ion or NL
scanning. However, the presence of alkali cations in the sam-
ple or matrix gives rise to some disadvantageous effects on
the analysis of positive ion spectra of phospholipids. First,
the cationization produces multiple ion signals for any parti-
cular molecular species, thus reducing the intensity of signals
for [MþH]þ ions and producing some degree of ion suppres-
sion.24,25 Second, the alkali-cationized species of some phos-
pholipid classes (e.g., PS and PE) also undergo fragmentation
of the polar head,26 which complicates the head-group-speci-
fic spectra. Furthermore, removal of salts from small amounts
of phospholipid samples can be very difficult. Accordingly,
most of the phospholipid classes (except PC) were analyzed
in the negative ion mode. The head-group-specific scans were
based on the fact that a common fragment is formed from the
head-group region of each member of a given class of polar
lipids upon CAD, when using ESI-MS/MS.16
Child whole blood was analyzed using the IDA sequence:
Pre or NL scan! enhanced resolution scan (ER)! enhanced
product ion scan (EPI).
In such an IDA experiment, the Pre ion scan or the NL scan
is considered as the survey scan, and the EPI scan is the
dependent scan. Phospholipid structural information is
obtained by this technique on two levels. The first level,
referred to as the class mass profile, is provided from the Pre
ion or NL survey scan; the second level, referred to as
providing fatty acyl formula data, is derived from the EPI
scan. As an example, the application of this IDA experiment
to identification of PS species is elucidated in detail in the
following.
Figures 1 and 2 were obtained for the same sample. It can
be observed in Figs. 2(a) and 2(b) that many peaks that
were completely buried in the chemical background of the
conventional mass spectrum (MS1) in Fig. 1(b) have been
revealed using neutral loss scanning for 87 Da16 in the
negative ion mode; this neutral loss is diagnostic for PS.
These overlapping and partially buried peaks can be resolved
by the Pre ion spectra. The resolution of the classical NL or Pre
ion scan modes is not as high as that using the enhanced mode
exploiting the trap functionality; the role of the classical
triple-quadrupole scans is solely the selection of the
precursor ion for the EPI experiment. Furthermore, it is
important to obtain unit mass resolution when the phospho-
lipids differ in mass by 2 Da or less. In the IDA procedure
used here, the resolution is reduced because of fast scan
speed (4000 Da s�1), as exemplified by Fig. 2(a), in which the
peak width is more than 10 Da. To solve this problem, an
enhanced resolution (ER) scan was included between the
initial survey scan (Pre ion or NL) and the EPI experiment. It
was found that the resolution and general performance of the
method were greatly improved by adding an ER scan to the
IDA procedure (Fig. 2(b)).
When a single-step MS/MS method such as Pre ion or NL
scanning is used to characterize phospholipid mixtures, even
though a precursor ion may be observed as a single mass peak
it is possible that several different unresolved (isobaric)
molecules can be dissociated simultaneously and detected.34
Thus, when a single Pre ion or NL scan is used for a head-
group survey (i.e., to survey a selected class), only the
summed numbers of carbon atoms and of double bonds
within individual phospholipid molecules can be obtained;
precise information on the pairs of fatty acyl groups on the sn-
1 and sn-2 positions cannot be elucidated.37 However, in a
typical IDA experiment as used in this work, a Pre ion or NL
survey scan is performed and processed to locate mass peaks
of interest (candidates), and product ion (dependent) scans
are acquired for these precursor ions. This cycle is repeated
through the duration of the acquisition to generate large
amounts of informative data. Thus, head-group-specific
scans, and product ion scans for the species identified,
were acquired sequentially in the same run by the IDA
procedure. Then, even if two (or more) isobaric precursors
are present, the several fatty acyl anions observed in the
overlaid product ion spectrum must be combined pairwise in
order to give the fixed total mass of the precursor, so that
some resolution of isobaric precursors can be obtained. This
approach does not distinguish between positional (sn-1/sn-2)
isomers.
While this IDA procedure is very powerful, improper
selection of the candidate precursor ions may result in the
generation of large amounts of poor or worthless data.
The IDA option in the Analyst software used to control the
QTRAP system includes a group of ion-filtering tools, such as
a time-based windowed exclusion list that allows the user to
specify an exclusion retention time (RT) window width to
indicate ions that should be used as candidates only for
certain time periods during an IDA run. If the precursor ion is
present in the survey scan within its specified time window
and if its intensity is above a specified threshold value, the
system will then acquire the PRI spectrum. The present IDA
procedure provides very good MS/MS spectra when the
precursor ion signal is strong. We have found that the key to
elucidating the structures of phospholipid species of low
abundance is to define an inclusion list indicating precursor
ions that should be preferentially used as candidate pre-
cursor ions during an IDA run. Because of the advantages of
this IDA procedure, enabling head-group-specific scans and
product ion scans to be combined together with this selective
filtering function, the candidate ion selectivity is greatly
improved.
In general, in order to obtain the maximum information,
3–4 injections were needed in view of the limited ‘chromato-
graphic’ peak width available; about 4–5 EPI spectra of
phospholipid species could be obtained per injection in the
current experimental conditions. The entire information on
molecular species within each phospholipid class, together
2446 C. Wang et al.
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2443–2453
with some EPI spectra of precursors with high abundance,
were obtained from the first injection. The other molecular
species can be obtained from the succeeding injections by
properly using inclusion and exclusion lists. It should be
noted that the number of EPI spectra per injection depends on
the ‘chromatographic’ peak width and carrier flow rate; thus
a moderate dead volume can be beneficial in the present
context.
Figure 2. DI-ESI-MS analysis of phospholipids using a negative ion IDA experiment.
(a) Detection of PS by conventional neutral loss (87Da) scanning. (b) Enhanced resolution
(ER) neutral loss scan of the same extract over a limited range. (c) Using IDA to identify a
precursor ion of interest (m/z 810.6, upper spectrum), immediately followed by an
enhanced product ion (EPI) scan to display fragment ions that provide information on
the fatty acyl moieties in the molecule. The chromatograms on the left-hand side of
(c) represent TIC profiles for the two scan modes.
Phospholipid structures in human blood by DI-ESI-QqLIT-MS 2447
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2443–2453
Figure 2(c) displays the IDA results. The top left box shows
the TIC for the NL (87 Da) scan for PS species in the child
blood sample, the top right box is the ER mass spectrum of the
region around m/z 810.6 (a target PS based on Fig. 2(b)) taken
at 0.667 min, the bottom left box shows the extracted ion
chromatogram (XIC) for the [M–H]� ion (m/z 810.6), and the
bottom right box is the EPI spectrum of m/z 810.6 recorded
immediately after the ER spectrum shown in the top right
box. In this EPI spectrum, the fragment ions detected at 283.4
and 303.3 correspond to C18:0 and C20:4 fatty acid residues
(carboxylate anion fragments) of [M–H]�, respectively.
Additionally, the fragment ion at m/z 437.2 corresponds to
C18:0 lysophosphate in the open form, and the abundant ion
at m/z 419.3 corresponds to C18:0 lysophosphate in the cyclic
form. The diagnostic ion [M–87–H]� for PS species is clearly
detected at m/z 723.5.
The positions of the acyl chains on the glycerol backbone of
the phospholipid molecule are believed to be important for
their relative propensity for dissociation. Product ion scan-
ning in negative ion mode can be used to identify the fatty
acyl chains,26 but there is disagreement in the literature on
which carboxylate anion (sn-1 or sn-2) yields the most intense
peak in the product ion spectrum.26,30–33 Hvattum et al.33
reported that the abundance ratio of the carboxylate anions
depends on many factors, such as collision energy, the
phospholipid class, and the nature of the fatty acid attached
to the sn-2 position. Houjou et al.34 and Hsu et al.35 determined
the acyl positions based on relative intensities of lyso-
phospholipid related fragment ions of [MþH]þ or [MþLi]þ,
respectively, in the positive ion mode. In the present work the
acyl positions are tentatively assigned on the basis of the
finding that phospholipids isolated from animals most often
contain a saturated fatty acid in the sn-1 position and an
unsaturated fatty acid in the sn-2 position.36 Therefore, m/z
810.6 is identified as C18:0/C20:4 PS, although the acyl
positions are only inferred. The other PS species identified by
this method are listed in Table 1 and the corresponding EPI
spectra are shown in Fig. 3.
Similarly, Fig. 4 shows the results of ER scans in negative
and positive ESI-MS modes for the detection of other
phospholipid classes. The Pre ion scan form/z 24116 provided
information on the PI molecular species in the child blood
sample (Fig. 4(a)). It was found that multiple molecular
species of phosphatidylinositols containing polyunsaturated
fatty acids (e.g., m/z 833.6, 857.5 and 885.6, corresponding to
C16:0/C18:2 PI, C16:0/C20:4 PI and C18:0/C20:4 PI, respec-
tively) were present (Fig. 4(a)). In addition, PIs containing a
monounsaturated fatty acid, e.g., C18:0/C18:1 PI (m/z 863.6),
was also detected (Fig. 4(a)). The corresponding EPI spectra
are shown in Fig. 5.
By using Pre ion scanning for m/z 196,16 more than ten PE
molecular species were detected (Figs. 4(b) and 6). In addition
to the plasmalogen PE molecular species (e.g., m/z 748.5 and
750.5, corresponding to pC16:0/C22:5 or pC18:1/C20:4 and
to pC18:0/C20:4 or pC16:0/C22:4, respectively), ethanola-
mine glycerophospholipids were predominantly composed
of diacyl molecular species (e.g., m/z 738.5, 766.5 and 790.6,
corresponding to C16:0/C20:4 or C18:2/C18:2 PE, to C18:0/
C20:4 PE or C16:0/C22:4 PE, and to C18:0/C22:6 PE,
respectively).
The EPI spectra of PI and PE were similar to those of the PS
in negative ion mode, revealing the fatty acid residues, since
formation of fatty acid anions represents an effective
fragmentation pathway of negatively charged ions of
phospholipids containing ester-bound fatty acids.26 How-
ever, in the EPI spectra of pPE, only carboxylate anions in the
sn-2 position can be observed because the sn-1 vinyl ether has
relatively higher stability.23,26
SM molecular species were identified by Pre ion scanning
for m/z 168 (dimethylethanolamine phosphate) in the
negative ion mode, as demonstrated in Fig. 4(c). Because
SM molecular species do not contain ester-bound fatty acids,
the ion atm/z 168 is the main fragment observed.27 As PC and
SM both contain a choline phosphate head group, Pre ion
scanning form/z 184,16,18,29 representing the [H2O3PO–CH2–
CH2–N(CH3)3]þ ion, identified [MþH]þ ions for PC and SM
in the positive ion mode. Discrimination between the PC and
SM molecular species was achieved by exploiting the
Table 1. Identification of phospholipid species in child whole
blood using DI-ESI-MS IDA experiments
Class Ion m/zCombinations of molecular
species
PE [M–H]� 714.6 C16:0/C18:2[M–H]� 738.5 C16:0/C20:4 C18:2/C18:2[M–H]� 740.5 C18:1/C18:2 C16:0/C20:3[M–H]� 748.5 pC16:0/C22:5 pC18:1/C20:4[M–H]� 750.5 p C18:0/C20:4 p C16:0/C22:4[M–H]� 762.5 C16:0/C22:6[M–H]� 764.5 C16:0/C22:5 C18:1/C20:4[M–H]� 766.5 C18:0/C20:4 C16:0/C22:4[M–H]� 790.6 C18:0/C22:6
PS [M–H]� 790.7 C18:0/C18:0[M–H]� 808.6 C18:1/C20:4 C18:0/C20:5[M–H]� 810.6 C18:0/C20:4[M–H]� 812.6 C18:0/C20:3[M–H]� 832.6 C18:2/C22:5[M–H]� 834.7 C18:0/C22:6[M–H]� 836.6 C18:0/C22:5
PI [M–H]� 833.5 C16:0/C18:2[M–H]� 857.5 C16:0/C20:4[M–H]� 859.6 C18:1/C18:2[M–H]� 861.6 C18:0/C18:2[M–H]� 863.6 C18:0/C18:1[M–H]� 885.6 C18:0/C20:4[M–H]� 913.7 C18:0/C22:4
SM [M–CH3]� 685.6 34:2[M–CH3]� 687.6 34:1[M–CH3]� 713.5 36:2[M–CH3]� 711.5 36:3[M–CH3]� 745.5 38:0[M–CH3]� 749.5 39:5[M–CH3]� 769.5 40:2[M–CH3]� 771.6 40:1[M–CH3]� 797.7 42:2[M–CH3]� 799.7 42:1[M–CH3]� 829.6 44:0
PC [MþH]þ 758.5 34:2[MþH]þ 760.6 34:1[MþH]þ 762.6 34:0[MþH]þ 780.6 36:5[MþH]þ 782.6 36:4[MþH]þ 784.6 36:3[MþH]þ 786.6 36:2[MþH] 808.6 38:5[MþH] 814.7 38:2
2448 C. Wang et al.
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2443–2453
Figure 3. Enhanced product ion (EPI) spectra of [M–H]� ions of PS species (except m/z 810.6, see Fig. 2(c)) from child blood
sample obtained by DI-ESI-MS with IDA. (a–f) EPI spectra of m/z 790.7, 808.6, 812.6, 832.6, 834.7 and 836.6, respectively.
Fatty acid carboxylate ions are observed as follows: m/z 283, 18:0; m/z 281, 18:1; m/z 301, 20:5; m/z 303, 20:4; m/z 305, 20:3;
m/z 325, 22:7; m/z 327, 22:6; m/z 329, 22:5.
Phospholipid structures in human blood by DI-ESI-QqLIT-MS 2449
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2443–2453
Figure 4. Specific detection of phospholipid classes in an unprocessed lipid extract from child
whole blood by DI-EMS-MS with IDA. (a) Detection of PI by Pre ion scanning form/z 241 (collision
energy 50 eV) in the negative ion mode. (b) Detection of PE by Pre ion scanning for m/z 196
(collision energy 40 eV) in the negative ion mode. (c) Detection of SM by Pre ion scanning for m/z
168 (collision energy 35 eV) in the negative ion mode. (d) Detection of [MþH]þ ions of SM and PC
by Pre ion scanning for m/z 184 (collision energy 40 eV) in the positive ion mode.
2450 C. Wang et al.
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2443–2453
Figure 5. Enhanced product ion (EPI) spectra of [M–H]� ions of PI species from child blood sample obtained by DI-ESI-MS with
IDA. (a–g) EPI spectra of m/z 833.6, 857.5, 859.6, 861.6, 863.6, 885.6 and 913.7, respectively. Fatty acid carboxylate ions are
observed as follows: m/z 255, 16:0; m/z 283, 18:0; m/z 281, 18:1; m/z 279, 18:2; m/z 303, 20:4; m/z 331, 22:4.
Phospholipid structures in human blood by DI-ESI-QqLIT-MS 2451
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2443–2453
Figure 6. Enhanced product ion (EPI) spectra of [M–H]� ions of PE species from
child blood sample obtained by DI-ESI-MS with IDA. (a–i) EPI spectra of m/z 714.6,
738.5, 740.5, 748.5, 750.5, 762.5, 764.5, 766.5 and 790.6, respectively. Fatty acid
carboxylate ions are observed as follows:m/z 255, 16:0;m/z 281, 18:1;m/z 279, 18:2;
m/z 303, 20:4; m/z 305, 20:3; m/z 327, 22:6; m/z 329, 22:5; m/z 331, 22:4.
2452 C. Wang et al.
Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 2443–2453
nitrogen rule; PC (with one nitrogen atom) showed [MþH]þ
signals at even-numbered m/z values, whereas SM (with two
nitrogen atoms) showed [MþH]þ signals at odd-numbered
values. The possible reason why some SM molecular species
detected by Pre ion scanning form/z 168 in negative ion mode
(Fig. 4(c)) could not be detected by Pre ion scanning for m/z
184 in positive ion mode (Fig. 4(d)) is that PC molecular
species in blood samples are much more abundant than SM,
and ionization of the latter could have been suppressed. The
fatty acid residues of PC can be effectively determined by
negative ion EPI scans;23 alternatively, Hsu et al.28 identified
PC species in phospholipid mixtures using Liþ adducts as
precursors in positive mode. Recently, by selecting the [M–
CH3]� ion as the precursor, Houjou et al.34 identified the
fatty acyl chains of PC species, sphingosine or sphinganine
derivatives, and N-acyl species of SM species. These appro-
aches may provide some help to more fully characterize the
PC and SM species.
Table 1 lists all the phospholipid species detected in the
child blood sample using the DI-ESI-MS method with IDA.
Comparing the present approach with HPLC/MS meth-
ods, head-group-specific scans can identify some phospho-
lipid classes as shown here; however, some low abundance
but functionally important phospholipids, such as phospha-
tidic acid, lysobisphosphatidic acid, and cardiolipin, cannot
be analyzed in this way as they produce no diagnostic
fragments (charged or neutral) in MS/MS, so no specific scan
mode is available.17 On the other hand, the advantage of the
present method is obvious; the run time is greatly reduced
(only several minutes) due to no HPLC pre-separation, which
makes the method suitable for high-throughput screening
analysis and rapid identification of particular phospholipid
targets. Some further steps may be implemented to improve
this method, such as use of a short pre-column, slower
flow rate, or combination of a short microcolumn with a
nanospray source.
CONCLUSIONS
DI-ESI-MS in combination with IDA experiments without
prior analyte pre-separation was investigated as a high-
throughput method for the characterization of phospholipids
from child whole blood. Pre ion and NL scan modes are much
less sensitive than the enhanced product ion mode, but they
are used here only to discover the most interesting analytes.
The IDA procedure used here combines two or more different
scan modes in a sequential way within the same run, which
enables head-group-specific scans and product ion scans to
be combined. The use of inclusion and exclusion lists became
mandatory for biological samples to benefit fully from this
approach. In this way phospholipid information, including
mass profiles and fatty acyl formulae, can be obtained in a
single run. Because many biological matrices such as urine
or blood are complex, many peaks of interest that might
have been hidden previously in the chemical background
of the conventional mass spectrum are clearly identified
using head-group-specific scans within the IDA procedure.
This approach is advantageous for targeted analyses because
of the short analysis time (only several minutes). We believe
that this approach has potential application for high-
throughput determinations of some phospholipids in biolo-
gical samples, since it requires little sample preparation, is
rapid, and can be readily automated.
AcknowledgementsThis study was supported by the High-Tech R & D Plan
(2003AA223061) of the State Ministry of Science and Techno-
logy of China, the Foundation (No. 20425516) for Distin-
guished Young Scholars from the National Natural Science
Foundation of China and the Knowledge Innovation Pro-
gram (K2002A12, K2003A16) of the Chinese Academy of
Sciences.
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