CSIRO PUBLISHING

Reproduction, Fertility and Development, 2015, 27, 621–637

Review
http://dx.doi.org/10.1071/RD14310

Ambient ionisation mass spectrometry for lipid profiling

and structural analysis of mammalian oocytes,
preimplantation embryos and stem cells

Christina R. Ferreira A,F, Alan K. Jarmusch A, Valentina Pirro A, Clint M. Alfaro A,

Andres F. González-Serrano B,E, Heiner Niemann B, Matthew B. Wheeler C,
Rathnaweera A. C. Rabel C, Judy E. Hallett D, Rebecca Houser D,
Annemarie Kaufman D and R. Graham Cooks A
A
Department of Chemistry and Center for Analytical Instrumentation Development,
Purdue University, 207 South Martin Jischke Drive, West Lafayette, IN 47907, USA.
B
Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Höltystr. 10,
31535 Neustadt, Germany.
C
Laboratory of Stem Cell Biology and Engineering, Department of Animal Sciences and Institute
for Genomic Biology University, University of Illinois at Urbana-Champaign, 1207 W. Gregory,
Urbana, IL 61801, USA.
D
Purdue Center for Cancer Research Transgenic Mouse Core Facility, Purdue University,
201 S. University Street, West Lafayette, IN 47907, USA.
E
Present address: Research and Development Department, IMV Technologies, ZI N8 1 Est,
61300 L’Aigle, France.
F
Corresponding author. Email: cferrei@purdue.edu

Abstract. Lipids play fundamental roles in mammalian embryo preimplantation development and cell fate. Triacyl-
glycerol accumulates in oocytes and blastomeres as lipid droplets, phospholipids influence membrane functional
properties, and essential fatty acid metabolism is important for maintaining the stemness of cells cultured in vitro. The
growing impact that lipids have in the field of developmental biology makes analytical approaches to analyse structural
information of great interest. This paper describes the concept and presents the results of lipid profiling by mass
spectrometry (MS) of oocytes and preimplantation embryos, with special focus on ambient ionisation. Based on our
previous experience with oocytes and embryos, we aim to convey that ambient MS is also valuable for stem cell
differentiation analysis. Ambient ionisation MS allows the detection of a wide range of lipid classes (e.g. free fatty acids,
cholesterol esters, phospholipids) in single oocytes, embryos and cell pellets, which are informative of in vitro culture
impact, developmental and differentiation stages. Background on MS principles, the importance of underused MS scan
modes for structural analysis of lipids, and statistical approaches used for data analysis are covered. We envisage that MS
alone or in combination with other techniques will have a profound impact on the understanding of lipid metabolism,
particularly in early embryo development and cell differentiation research.

Additional keywords: desorption electrospray ionization, multivariate statistics.

Received 25 August 2014, accepted 13 January 2015, published online 13 March 2015

Introduction
The term ‘lipid’ embraces a wide range of chemically hetero-
geneous molecules having the common property of hydropho-
bicity (insolubility in water, but solubility in non-aqueous
solvents such as chloroform, hydrocarbons and alcohols) or in
some cases amphiphiles. Lipids occur in all cell types and are
well known for their key roles in cellular structure and energy
storage (Gurr et al. 2002). Recent research suggests additional
and emerging roles for lipids, such as their influence on the
transcription of the genetic code via epigenetic mechanisms,
thereby regulating vital metabolic pathways and physiological
processes (Jump et al. 1996; Duplus et al. 2000; Bieberich 2011;
Lu and Thompson 2012). In addition, lipid metabolism is
emerging as a bioenergetic controller of stem cell differentiation
(Monaco et al. 2012; Yusuf and Scadden 2012; Folmes et al.
2013).

Journal compilation Ó CSIRO 2015 www.publish.csiro.au/journals/rfd

622 Reproduction, Fertility and Development
Most mammalian oocytes are rich in lipids and these are vital
during preimplantation development. Interest in lipid analysis of
oocytes and preimplantation embryos was generated by several
observations (Sturmey et al. 2009). The lipid droplets in
the oocytes and embryos of sows, cows and ewes are so great
in number that they appear dark under the microscope (Coull
et al. 1998; Ferguson and Leese 1999; McEvoy et al. 2000).
Intracellular lipids are stored as cytoplasmic droplets, composed
mainly of triacylglycerols (TAGs), which the cells use as an
energy source and fatty acid precursors. The mean lipid content
of a mouse oocyte was reported to be 12% of the total dry
mass, which corresponds to an average of 3.83 ng per oocyte
(Loewenstein and Cohen 1964). TAGs also represent the major
constituents of pig oocytes (Homa et al. 1986). Bovine embryos
produced in vitro have an increased number of lipid droplets,
particularly if cultured with serum-enriched culture medium
(Ferguson and Leese 1999; Abe et al. 2002; Barceló-Fimbres
and Seidel 2011). Furthermore, in vitro supplementation with
unsaturated fatty acids (such as linoleic acid) yielded mostly
positive effects on bovine oocyte maturation, blastocyst devel-
opment rate and tolerance to cryopreservation (Shehab-El-Deen
et al. 2009; Marei et al. 2010; Aardema et al. 2011). Porcine
embryos survived cryopreservation after removal of cyto-
plasmic lipids (Nagashima et al. 1995); lipid metabolism
modifiers such as phenazine ethosulfate and forskolin decreased
the amount of lipid droplets in bovine blastocysts, improved
cryotolerance and increased pregnancy rates (Barcelo-Fimbres
and Seidel 2009; Sanches et al. 2013). It is evident that lipid
content and metabolism are critical factors in embryo handling,
storage and development.
Preimplantation embryos, stem cells and cancer cells have
striking similarities in energy metabolism (Warburg 1956a,
1956b; Krisher and Prather 2012; Pacini and Borziani 2014).
Recent studies in this area indicate that energy metabolism in
highly proliferating or undifferentiated cells is related to lipid
metabolism. As an example, fatty acid synthase (FASN), a key
enzyme of de novo lipogenesis, and the thyroid hormone
responsive SPOT14, a gene previously implicated in lipid
metabolism, have been reported to be very important in adult
neural stem and progenitor cells (Knobloch et al. 2013).
A better understanding of the role of lipids in embryology
and stem cell research is enabled by analytical methodologies
based on mass spectrometry (MS). MS is a powerful analytical
chemical technique broadly applied for the analysis of mole-
cules. Bioanalytical platforms based on MS have multiple
configurations resulting from several possible combinations of
ion sources and mass analysers. The common feature of MS is
the measurement of mass-to-charge ratio (m/z) with correspond-
ing abundance of molecules. Only molecules present in the
sample that have been ionised and transferred to the gas phase
are detected. The output is a mass spectrum, which plots ion
signals as a function of m/z ratio. MS can be used to determine
the elemental or isotopic signature of a sample, the masses of
particles and biomolecules and to elucidate the chemical struc-
tures of molecules by generating and measuring the m/z ratios of
molecule fragments (which comprise the ‘chemical signature’
of each molecule; Siuzdak 1996). This latter experiment is
normally done using mass selection followed by collisional
C. R. Ferreira et al.
activation and a second stage of mass analysis to record a
tandem mass spectrum (the MS/MS experiment; Busch et al.
1989).
Due to the sensitivity and depth of structural information
obtained by MS, studies of lipid composition in oocytes and
embryos have benefited from the introduction of MS lipid
profiling of individual samples, initially by matrix-assisted laser
desorption ionisation (MALDI) and then by desorption electro-
spray ionisation (DESI; Ferreira et al. 2010, 2012a, 2012b;
Apparicio et al. 2012; Sudano et al. 2012; González-Serrano
et al. 2013b; Tata et al. 2013). DESI is an ambient ionisation
technique introduced in 2004 (Takáts et al. 2004). Ambient
ionisation embraces the concept of analysing complex chemical
samples in their native state with minimal sample preparation
(Cooks et al. 2006).
This paper aims to provide an overview of the analysis of
lipids in oocytes and embryos by ambient ionisation MS and
discusses the current status and future direction of its application
in developmental biology. Data on the value of underused MS/
MS scan modes for lipid profiling and the application of ambient
ionisation lipid profiling for stem cell research area are pre-
sented. We take the opportunity to overview basic concepts of
MS, ambient ionisation techniques and lipid structural analysis,
thereby providing context for ease of comprehension. Details
regarding MS lipid analysis have been organised into two topics:
(1) overview of lipid profiling of oocytes and embryos by MS;
and (2) structural characterisation of lipids present in the MS
profiles. Topic 1 is aimed at readers uninitiated in MS analysis,
whereas Topic 2 requires some depth of prior MS knowledge.
Finally, this review discusses strategies of MS data analysis,
particularly multivariate statistics, the use of which is funda-
mental to efficiently exploring and interpreting information-rich
lipid profiles.
Basics of MS
MS is a robust and sensitive technique and, in the forms of
chromatography/MS or MS/MS, allows qualitative and quanti-
tative analysis of complex biological samples (Kondrat and
Cooks 1978; Busch et al. 1989). Although many platforms are
in use worldwide, the ion source, mass analyser, detector and
computer are universal components (Fig. 1a; Glish and Vachet
2003). First, the ion source produces charged gas phase surro-
gates (ions) of the original molecules present in the sample,
through a variety of mechanisms. Negative and positive ions can
be generated, predictably allowing for the establishment of an
initial stage of chemically specific recognition. Next, ions are
transported from the ionisation source into the reduced pressure
regions of the mass analyser where the mass-to-charge ratios
(m/z) of the ionised species are determined. Ions are selectively
transmitted by the mass analyser to the detector, which records
the intensity of ions, usually via measurement of current.
Finally, a computer combines the measured m/z and ion inten-
sities into a mass spectrum that constitutes a chemical finger-
print of the sample (McLafferty and Turecek 1993). The specific
modules (e.g. ion source) of the mass spectrometer determine
the types of molecules that can be analysed, as well as the level
of sensitivity and mass resolution achieved.
Oocyte and embryo MS lipid profiling Reproduction, Fertility and Development 623

(a)

Sample Computer

Ionization Mass
Detector
source analyser

Path of analyte Computer communications

(b) 100 969.59

971.59

80
972.59
60
Theoretical
40
Relative abundance

973.59
20

20 973.59

40

60 Experimental
972.59
80
969.59 971.59
100
960 965 970 975 980 985 990
m/z
(c)
O
O
O

Chemical formula: C59H90O4

Exact mass: 862.68

Fig. 1. (a) Generalised components of a mass spectrometer. (b) Comparison of experimental and
theoretical high mass resolution mass spectrum for the silver adduct of ubiquinone ([MþAg]þ) generated
with DESI-MS using Ag2NO3-doped spray solvent. (c) Chemical structure of ubiquinone and its mono-
isotopic mass. This compound has been detected by desorption electrospray ionisation–mass spectrometry
(DESI-MS) in bovine oocytes and embryos and recently in porcine oocytes (González-Serrano et al. 2013b;
Pirro et al. 2014).

Ionisation techniques such as electrospray ionisation (ESI),
MALDI and ambient ionisation (as detailed below) are well
suited for the analysis of biomolecules, such as lipids, because
they are gentle methods (Karas and Hillenkamp 1988; Fenn
et al. 1989; Cooks et al. 2006; Cole 2010). With these techni-
ques, gas phase ions are produced without significant fragmen-
tation and are generally detected as protonated ([MþH]þ) or
deprotonated ([M-H]) ions in the positive and negative modes,
respectively. ESI is gentle enough to produce gas phase ions of
protein complexes that closely mimic their solution phase
counterparts (Zhang et al. 2013a, 2013b). However, these
techniques require some degree of sample preparation (applica-
tion of matrix for MALDI, usually preseparation and sample
dissolution for ESI), which increases the overall time for
analysis as well as the cost. In the case of ESI, sample dilution
and extraction are not compatible with the tiny amounts of
sample available from single oocytes and embryos. In addition,
it should be noted that direct analysis reduces bias by
624 Reproduction, Fertility and Development
minimising interactions with the sample, although differential
ionisation efficiency and the influence of variations in sample
complexity must be recognised.
Examples of mass analysers include magnetic (B) and
electric (E) sectors, quadrupoles (Q), three-dimensional ion
traps (IT), linear ion traps (LIT), ion cyclotron resonance
(ICR) traps, orbitraps and time-of-flight (TOF) systems. Each
analyser has both advantages and drawbacks. They fall into two
broad categories, low and high mass resolution, with low
resolution analysers such as ion traps and quadrupoles being
the more common because these analysers tend to be less
expensive, more robust, compact, and easier to operate. High
mass resolution analysers, such as the ICR and orbitrap, are also
widely used but require increased maintenance; they are more
expensive and best used by highly qualified personnel. Fre-
quently, mass analysers are combined to compensate for indi-
vidual drawbacks and increase performance for specific
applications. Hybrid instruments combine two analysers using
each member of the pair to compensate for the drawbacks of the
other. The early double-focusing sector instruments used an
electric sector and magnetic sector that give mass resolution
independently (to first order) of ion velocity only when operated
together (Beynon 1982). More recent hybrid MS systems are
capable of providing high mass resolution, accuracy, and
MS/MS capabilities in a single instrument. The most successful
hybrids are the Q-traps (triple quadrupoles with a quadrupole
that can be operated also as an LIT), IT-TOF, Q-TOF, Q-ICR
and LIT-ICR, all of which also confer the advantage of structural
analysis of biomolecules by collision-induced dissociation in
tandem (MS/MS) or MSn.
Tandem MS (MS/MS) and MSn are largely used for structur-
al analysis of molecules. The most common workflow used to
record a product ion MS/MS spectrum involves selecting and
isolating a single m/z value to specifically identify this com-
pound via the reproducible and characteristic fragmentation
patterns of its corresponding ionic forms. Fragmentation of a
precursor ion occurs when excess internal energy is provided to
ions and induces fragmentation (breaking of covalent bonds)
and conformational rearrangements mostly through collisions
with neutral gas molecules (collision-induced dissociation
(CID); Yost and Enke 1978), contact with a specific surface
(surface-induced dissociation (SID); Grill et al. 2001) or irradi-
ance (photon-induced dissociation (PID); Eyler 2009). Further-
more, MS/MS significantly increases the sensitivity of the
analysis primarily by reducing chemical noise (Glish and
Vachet 2003), allowing the detection of minor compounds in
complex matrices that would not otherwise be detected (Cooks
et al. 2014; Jarmusch and Cooks 2014).
High-resolution MS (HRMS) is also advantageous for
structural characterisation of ions. HRMS allows exact mass
measurement from which differentiation of ions with the same
integral mass is possible. The mass defect, which is different
from the integral molecular weight, is fundamentally deter-
mined by the elemental composition of the molecule (12C is
12.000000, 1H is 1.007825; Wu et al. 2004). Exact mass
measurements in the p.p.m. range are routinely achieved using
orbitrap and Fourier-transform ion cyclotron resonance
(FTICR) mass analysers, with the latter having the greatest
C. R. Ferreira et al.
possible resolution (Marshall and Hendrickson 2008; Snijder
and Heck 2014). The isotopic distribution of the elements
can be observed with HRMS instruments and is also useful in
compound identification. The isotopic peaks of lipids reflect
the isotopic abundances of the constituent elements (i.e. C, H, O,
P and N), which is useful for confirmatory identification
(Fig. 1b, c). Even with HRMS data, structural isomers cannot
be differentiated and this is often required in lipid analysis so it
increases the importance of MS/MS measurements. The benefits
of soft ionisation, MS/MS and HRMS in lipid analysis are
founded on the high specificity and sensitivity that MS provides
in the analysis of complex biological mixtures. As discussed in
the next section, ambient MS ionisation sources enable MS data
to be collected from any unmodified samples, in rapid and
informative experiments (Cooks et al. 2006, 2014; Eberlin et al.
2011b; Nemes and Vertes 2012).
Ambient ionisation MS
Ambient ionisation refers to the generation of ions under
ambient conditions (e.g. pressure, temperature, humidity),
requiring little, and in some cases no, sample preparation (Cooks
et al. 2006; Badu-Tawiah et al. 2013). Ambient ionisation
techniques emerged early in this century and have since
expanded into a variety of forms (Venter et al. 2008, 2014). The
ability to examine the sample in its native state and to do so very
rapidly are the primary advantages of ambient MS. DESI, the
first ambient ionisation technique (Takáts et al. 2004), has been
applied in the study of lipids (Eberlin et al. 2011b), metabolites
and large molecules (Ferguson et al. 2011). It is widely imple-
mented and has been reported in more than 500 publications
(Web of Science, January 2015). DESI is ideal for ionising
lipids (Eberlin et al. 2011b), but it is also very suitable for the
ionisation of small molecules in general, such as drugs and
metabolites (Ifa et al. 2008). Isolated proteins in molecular mass
from 12 to 66 kDa can also be ionised by ambient ionisation
(Shin et al. 2007). In a DESI experiment, the sample (e.g. oocyte
or embryo) is placed on an insulating surface (e.g. glass slide)
and, with the aid of inert gas, a solvent stream charged to a high
electrical potential is directed at the sample. Gas phase ions are
produced in the small area that the solvent strikes (Venter et al.
2006) and, by waiting some seconds up to 1 min (in the case of
oocytes and embryos) or by moving the sample in the ‘x’ and ‘y’
directions, mass spectra can be collected and correlated with the
spatial features of the sample (see Fig. S1, available as Sup-
plementary Material to this paper; Nemes and Vertes 2012).
Software can be used to reconstruct a mass spectrometric image
(MSI) of the sample, which can provide valuable biological
information (Eberlin et al. 2011b).
The power of DESI-MS for tissue analysis has gained wide
recognition, especially in the differentiation of healthy and
cancerous cells in tissue sections, including brain, prostate,
breast, kidney and liver tissue (Dill et al. 2010; Perry et al.
2013; Calligaris et al. 2014; Kerian et al. 2014b). The perfor-
mance in this experiment, and especially the correlation of the
data with standard pathology, has been enhanced by the use of
non-destructive solvent systems. The mechanism of non-
destructive DESI-MS imaging is a spot-by-spot microextraction
Oocyte and embryo MS lipid profiling
of molecules from the sample proportionated by specific solvent
combinations. This extraction process is comparable to the
fixative procedures commonly used in histology for morpho-
logical examination: lipids are removed, whereas the intracellu-
lar and extracellular proteins stained in the histochemical
treatment are preserved (Eberlin et al. 2010, 2011a; Santagata
et al. 2014). Data generated using DESI can provide diagnostic
information comparable to techniques that pathologists current-
ly use to define cancer tissue margins (Santagata et al. 2014).
There may also be applications for DESI and other ambient
ionisation techniques in the molecular study of physiology and
development in various biological systems.
The molecular profiles that DESI experiments generate are
largely qualitative, but quantitative determination of the con-
centration of particular compounds is possible. Using an isoto-
pically labelled internal standard, propranolol can be quantitated
from 0.01 to 100 mM, a dynamic range that spans four orders of
magnitude (Ifa et al. 2008). Another advantage of DESI-MS and
other ambient ionisation techniques, such as paper spray (Wang
et al. 2011) and touch spray (Kerian et al. 2014a), is that they can
be operated in a reactive mode. Briefly, reagents added to the
solvent derivatise the analyte (i.e. to transform a compound into
a product of similar chemical structure) in situ during the very
short period of ionisation, increasing ion signal and thereby
allow lower limits of detection (Badu-Tawiah et al. 2013; Espy
et al. 2014). For example, alcohols can be derivatised by betaine
aldehyde to form a permanently charged hemiacetal salt (Espy
et al. 2014). This method was used to improve the ionisation
efficiency of cholesterol in tissues (Wu et al. 2009). A related
process, not strictly a chemical reaction, is the cationisation of
the analyte to generate an ion such as [MþAg]þ. Silver ions
added to the spray solvent form adducts (non-covalent interac-
tions) with alkene functional groups and significantly improve
ionisation efficiency for lipids such as ubiquinone (Fig. 1b, c;
Jackson et al. 2011). Silver ions form cationic adducts with
several functional groups, but they demonstrate high affinity for
olefins (unsaturated hydrocarbon containing one or more pairs
of carbon atoms linked by a double bond), presumably due to
p-back bonding (Nikolova-Damyanova 2009). When small
amounts (1–7 mg mL1) of silver nitrate are added to the DESI
spray solvent, characteristic silver adducts of 26 unsaturated
lipids are generated at up to 50-fold greater abundance com-
pared with the detection of protonated or deprotonated ions
(Jackson et al. 2011). Informative reviews of the many types of
ambient ionisation techniques are available (Nemes and Vertes
2012; Venter et al. 2014).
Overview of lipid profiling of oocytes and embryos by MS
Traditionally, gas chromatography (GC) is the primary analyt-
ical technique used to study lipids present in oocytes and
embryos. In practice, GC requires the pooling of tens to hun-
dreds of oocytes or embryos (Matorras et al. 1998; McEvoy
et al. 2000; Kim et al. 2001), which can be impractical and
compromises the ability to provide individual chemical infor-
mation. Application of MS for direct analysis of unmodified
samples has enabled lipid profiling of single oocytes and
embryos. This analytical development allowed the detection of
Reproduction, Fertility and Development 625
phosphatidylcholines (PCs), sphingomyelins (SMs), TAGs, free
fatty acids (FFAs), phosphatidylethanolamines (PEs), phospha-
tidylinositols (PIs), phosphatidylserines (PSs), diacylglycerols
(DGs), cholesterol precursors, cholesterol derivatives, and ubi-
quinone in individual oocytes or preimplantation embryos with
the additional benefit of obtaining chemical information on lipid
structure by performing MS/MS or HRMS (Table 1; Ferreira
et al. 2012a; González-Serrano et al. 2013b; Tata et al. 2013).
DESI-MS for oocyte and embryo analysis was developed
after the introduction of non-destructive solvent systems, which
allowed signal intensities to be accumulated from microscopic
samples (Eberlin et al. 2011a; Ferreira et al. 2012a; Pirro et al.
2014) without sample destruction. Diverse lipid classes (Table 1)
can be detected with DESI and the structure of the lipids
involved was attributed with the aid of MS/MS and HRMS
(González-Serrano et al. 2013a, 2013b; Pirro et al. 2014). As
detailed in the topic about structural characterization of lipids
present in the MS profiles, the effectiveness of structural
characterisation of lipids in embryos can be extended by the
use of additional MS/MS scanning modes.
Lipid profiling of oocytes and embryos as an analytical tool
presents some peculiarities as an analytical approach. The MS
lipid profile in the full scan mode generates a list of m/z values
and their respective ion intensities. Because of the large
amount of chemical information acquired from each sample,
multivariate statistics are necessary to filter relevant ions for
differentiation among experimental groups. These issues are
detailed in the topic about statistical approaches to tackling the
amount of chemical information present in lipid profiles.
Several publications have described the analysis of single
oocytes and embryos by mass spectrometry (Table 2). Notable
differences are summarised here. Lipid profiles of oocytes and
embryos have so far identified biological differences related to:
(1) species or breed; (2) developmental stage; and (3) environ-
mental growth conditions.
Independent of oocyte and embryo species, there are under-
lying lipid structural similarities evident in the MS profiles, such
as the high abundance of PC 34 : 1 (i.e. containing one 16 : 1 and
one 18 : 1), indicating that this is a major PC for the membrane
bilayer structure in oocytes and preimplantation embryos. Spe-
cies-specific comparisons have been performed among human,
bovine, fish and sheep oocytes, ant and bovine embryos and
among cat, dog and bovine oocytes (Ferreira et al. 2010;
Apparicio et al. 2012). Multivariate analysis segregated samples
according to species. Characteristic examples include the higher
abundance of TAG in bovine compared with human and sheep
oocytes, and the wide range of unsaturated PC in fish eggs
(Ferreira et al. 2010). Regarding cat, dog and bovine oocytes,
the cat oocytes present more abundant and highly unsaturated
TAGs containing 52 and 54 carbon in the fatty acyl residues
compared with the other two species.
Differences related to bovine breeds have been reported
between Simmental and Nelore embryos and between Nelore
and Nelore  Angus breed oocytes. These differences were
related to the number of cytoplasmic droplets in embryos and to
oocyte competence (Sudano et al. 2012; Silva-Santos et al. 2014).
MS lipid profiling also indicated changes related to oocyte
maturation and embryo development (Ferreira et al. 2012a,
626 Reproduction, Fertility and Development C. R. Ferreira et al.

Table 1. Lipid classes characterised in oocytes and preimplantation embryos by mass spectrometry with representative species
FFA, free fatty acid; PA, phosphatidic acid; PC, phosphatidylcholine; SM, sphingomyelin; PI, phosphatidylinositol; PS, phosphatidylserine; PE,
phosphatidylethanolamine; DAG, diacylglycerol; TAG, triacylglycerol; PG, phosphatidylglycerol; DESI, desorption electrospray ionisation; MALDI,
matrix-assisted laser desorption

Lipid class Source Samples References

FFAs or FFA DESI () Porcine oocytes, mouse oocytes and Ferreira et al. (2012a, 2012b), González-Serrano et al. (2013a),
dimers embryos, bovine embryos Pirro et al. (2014)
PA MALDI (þ) Bovine embryos Ferreira et al. (2014)
PC MALDI (þ), DESI (), Bovine, human, sheep, fish, dog and cat Ferreira et al. (2010, 2012a, 2012b), Apparicio et al. (2012), Sudano
DESI (þ) oocytes; bovine and ant embryos et al. (2012), González-Serrano et al. (2013b), Tata et al. (2013)
SM MALDI (þ) Bovine, human, sheep and fish oocytes; Ferreira et al. (2010), Sudano et al. (2012), Silva-Santos et al. (2014)
bovine and ant embryos
PI DESI (), MALDI () Porcine and Drosphila oocytes, mouse Urban et al. (2011), Ferreira et al. (2012a), González-Serrano et al.
oocytes and embryos, bovine embryos (2013b), Tata et al. (2013), Pirro et al. (2014)
PS DESI (), MALDI () Porcine oocytes, mouse oocytes and Ferreira et al. (2012b), Tata et al. (2013), Pirro et al. (2014)
embryos, bovine embryos
PE DESI (), MALDI () Mouse oocytes and embryos Ferreira et al. (2012a, 2014), González-Serrano et al. (2013b),
Tata et al. (2013)
Squalene, DESI (þ) þ AgNO3 Porcine oocytes, bovine oocytes and González-Serrano et al. (2013b), Pirro et al. (2014)
cholesterol esters embryos
and ubiquinone
Cholesterol sulfate DESI () Bovine oocytes and embryos González-Serrano et al. (2013b)
DAG DESI (þ) þ AgNO3 Porcine oocytes Pirro et al. (2014)
TAG MALDI (þ), DESI (þ) Bovine, human, sheep, fish, dog and cat Ferreira et al. (2010), Apparicio et al. (2012), González-Serrano
þ AgNO3 oocytes; bovine and ant embryos et al. (2013b), Silva-Santos et al. (2014)
PG DESI (), MALDI () Porcine oocytes, mouse oocytes and Ferreira et al. (2012a, 2012b), González-Serrano et al. (2013b),
embryos, bovine embryos Pirro et al. (2014)
Ceramides MALDI () Bovine embryos Ferreira et al. (2014)

2012b; González-Serrano et al. 2013b; Pirro et al. 2014). These
changes include increased TAG metabolism, especially during
maturation, and membrane specialisation in blastocysts due to
the increased complexity of their phospholipid (PL) profiles.
Specifically for mouse embryos analysed in the negative ion
mode by DESI-MS, unfertilised oocytes were mainly charac-
terised by m/z 794, the chlorine adduct of PC 34 : 1 (Fig. 2a),
whereas 2- and 4-cell embryos were found to contain abundant
FFAs, detected as dimers in the m/z range 500–600 (Fig. 2b, c).
Blastocysts have a higher contribution of m/z 820 and 844,
which correspond to chlorine adducts of PC species with 36 and
38 carbon and higher unsaturation levels (Fig. 2d). These
observations have been attributed to the highly active fatty acid
metabolism during preimplantation development, and to mem-
brane structural and functional specialisation in blastocysts.
In addition, significant changes in MS lipid profile have been
observed in response to the culture environment (Ferreira et al.
2010; González-Serrano et al. 2013b). Combinations of culture
medium supplement (bovine serum albumin (BSA) or fetal calf
serum (FCS)) and incubator atmosphere (high or low oxygen
concentration) impacted SMs, PCs and TAGs from single
bovine embryos. Low incubator oxygen concentration and
BSA supplementation in the culture medium favoured PCs
containing 18 : 1 (PC 36 : 1) and not 16 : 0 (PC 34 : 1 and SM
16 : 0; Ferreira et al. 2010). Cholesterol esters and FFAs are
more abundant in bovine blastocysts collected in vivo (Gonzá-
lez-Serrano et al. 2013b) compared with those cultured in vitro.
In addition to the small molecule profiling already proposed for
quality control of in vitro culture media (Ferreira et al. 2009),
knowledge from MS lipid profiling is likely to be of value for
understanding cryopreservation.
Furthermore, the gamete and embryo cryopreservation area
can particularly benefit from MS lipid profiling. During cryo-
preservation, the plasma membranes of oocytes and embryos
are damaged by thermotropic phase transition during cooling
(temperatures just below 158C), causing irreversible damage to
the oocyte or embryo, and it is well documented that the lipid
content of bovine embryos and the use of lipid metabolism
modifiers can influence post-thaw survival and pregnancy
rate (Abe et al. 2002; Barcelo-Fimbres and Seidel 2009;
Barceló-Fimbres et al. 2010; Sanches et al. 2010, 2013).
A genetic approach to study PL metabolism in vivo has
limitations because mutations in PL-related genes are likely to
affect several cell functions or compromise cell integrity. In
addition, gene mutations usually do not affect PL structure but
rather the biosynthetic pathways involving PLs. Gene expres-
sion analysis is more specific, but it may include pathways such
as fatty acid synthase (FAS gene) or formation of acetoacyl-CoA
from two molecules of acetyl-CoA (ACAT1 gene), which will
involve many PL rather than one particular PL structure.
Fortunately, lipid profiling by MS can provide additional
information on lipid specificity and has been shown to be a
valuable tool in combination with quantitative gene expression
analysis in bovine oocyte and embryo studies. DESI-MS with
Oocyte and embryo MS lipid profiling Reproduction, Fertility and Development 627

Table 2. Summary of main results of lipid profile mass spectrometry in oocytes and embryos, ionisation source, species and references
PC, phosphatidylcholine; TAG, triacylglycerol; BSA, bovine serum albumin; FCS, fetal calf serum; FFA, free fatty acid; PL, phospholipid; SM,
sphyngomyelin; MALDI, matrix-assisted laser desorption; PBS, phosphate-buffered saline; MS, mass spectrometry; DAG, diacylglycerol; DESI, desorption
electrospray ionisation

Results Source Species Reference

Lipid profiles are species specific MALDI (þ) Human, bovine, sheep, fish and ant Ferreira et al. (2010)
Oocytes of queen and bitches differentiated by PC and TAG lipids. Cat MALDI (þ) Cat, dog and cattle oocytes Apparicio et al. (2012)
oocytes were richer in TAG containing 52 and 54 carbon and these lipids
were unsaturated compared with bitch and cow oocytes.
5% oxygen concentration in the incubator and BSA favoured unsaturated MALDI (þ) Bovine oocytes and embryos Ferreira et al. (2010)
PC compared with 20% oxygen and FCS.
Main FFAs detected in bovine embryos produced in vitro were palmitic, DESI (–) Mouse oocytes and embryos, Ferreira et al. (2010)
stearic, eicosanoic and docosanoic. Mouse oocytes and embryos presented bovine embryos
abundant palmitic, linoleic, arachidonic and docosahexanoenic acid ions.
Unfertilised oocytes presented less complex PL compositions compared DESI (–) Mouse oocytes, 2- and 4-cell Ferreira et al. (2012b)
with blastocysts, embryos cultured in vitro exhibited more homogeneous embryos, blastocysts
lipid profiles compared with in vivo
Bovine blastocyst PC and SM profiles differ with regard to unsaturation MALDI (þ) Bovine blastocysts (Nellore and Sudano et al. (2012)
level and carbon chain composition due to subspecies and in vitro culture Simmental)
conditions. PC 32 : 0, 34 : 1, 34 : 2 and 36 : 5 suggested as potential markers
of post-cryopreservation embryonic survival.
MALDI (þ) and MALDI (–) performed on a single embryo with binary MALDI (þ) Bovine embryos Tata et al. (2013)
matrix showed that lipid degradation or selective extraction during long and
embryo storage at 808C can be avoided by storage in PBS only. MALDI (–)
Saturated FFAs present in higher relative abundance within in vitro- than DESI (–) and Bovine oocytes and embryos González-Serrano et al.
in vivo-produced blastocysts. In vivo-produced blastocysts showed higher DESI (þ) (2013b)
concentrations of neutral lipid species, including cholesterol sulfate, with
cholesteryl esters of palmitic and eicosatrienoic acids, TAG 52 : 2, AgNO3
squalene and oleic acid. Gene expression and MS results match.
DAG 36 : 2 and 38 : 4 more abundant in immature oocytes. Specific TAG DESI (þ) þ Porcine oocytes Pirro et al. (2014)
changes during oocyte maturation and increased abundance of FFAs by AgNO3
the end of in vitro maturation.
PC P-38 : 5, PC P-36 : 2, PC 38 : 2, PC 38 : 5 and TAG 60 : 8 were more MALDI (þ) Bovine oocytes Silva-Santos et al.
abundant in Bos indicus than 1/2 B. indicus  taurus oocytes obtained (2014)
by ovum pick-up, independent of high or low oocyte yields.

silver nitrate containing solvent in the positive ion mode detects
cholesterol-related molecules and TAGs with great sensitivity.
The presence of squalene (a cholesterol precursor) in immature
bovine oocytes and the accumulation of cholesterol esters in
bovine blastocysts collected in vivo indicate the importance of
cholesterol metabolism during bovine preimplantation develop-
ment. Analysis of endpoint metabolism by DESI was confirmed
upstream in metabolism by reverse transcription quantitative
polymerase chain reaction (RT-qPCR) of sterol regulatory
element-binding protein cleavage-activating protein (SCAP)
and sterol regulatory element-binding protein 1 (SREBP1)
genes, which encode key functional enzymes for regulation of
cholesterol levels. These enzymes were downregulated after in
vitro maturation and in in vivo-derived blastocysts (Fig. 3;
González-Serrano et al. 2013b).
Structural characterisation of lipids present in
the MS profiles
Structural characterisation is a critical step in determining which
specific lipids are associated with changing environmental
conditions or biological state. In this pursuit, there are two main
questions to be addressed: what are the atoms that comprise the
molecule; and what is the structural arrangement of such atoms?
Both questions can be answered by MS with high reliability. The
first question is addressed via HRMS, which provides a set of
molecular formulas, as discussed above. Molecular formulas
can be generated for all peaks in HRMS data, and the set of
allowed formulas requires intellectual trimming based on
additional evidence (e.g. biological information or experimental
data such as MS/MS). For example, a molecule containing two
acyl fatty acid chains that share 36 carbons and 2 units of
unsaturated bonds has many possible combinations (i.e. 18 : 0/
18 : 2, 18 : 1/18 : 1 or 18 : 2/18 : 0). As mentioned earlier in the
text, the use of MS/MS can resolve structural characteristics,
such as the (major) lipid composition of the molecule and, in
some cases, it can provide the location of subunits (i.e. 18 : 0/
18 : 2 vs 18 : 2/18 : 0).
The usefulness of single-stage MS (MS1) for detecting lipids
present in biological samples is evident from the lipid profiling
studies not only in oocytes and embryos, but also in tissue in
general. The detection of ionised lipid molecules allows differ-
entiation of complex biological state differences (e.g. disease,
metabolic and developmental states). MSn, where n $ 2,
628 Reproduction, Fertility and Development C. R. Ferreira et al.

794.5
(a) 100 (c) 100 537.5

563.5 794.5
80 80
585.5
613.5
60 60

820.5 511.5 635.5

40 40
768.5
663.5 768.5 816.5
737.5
20 737.5 20 844.5
836.5
Relative abundance

400 600 800 1000 400 600 800 1000

(b) 100 563.5 (d ) 100 820.5

794.5

537.5 80
80 613.5

60 60
563.5
537.5 844.5
635.5 794.5
40 40
766.5
737.5
659.5 511.5
20 511.5
687.5 778.5 820.5 20

400 600 800 1000 400 600 800 1000

m /z

Fig. 2. Representative desorption electrospray ionisation–mass spectrometry (DESI-MS) negative ion mode mass spectra (accumulated over 2–3 min of
analysis for each sample) of individual oocytes and embryos from each of four mouse preimplantation developmental stages: (a) unfertilised oocyte, (b) 2-cell
embryo, (c) 4-cell embryo and (d) blastocyst. Reproduced from Ferreira et al. (2012b).

provides the ability to determine the ion’s structure and there-
fore that of the original molecule. This is the principal use of
MS/MS. The first stage of mass analysis is performed on the
precursor ions (historically known as parent ions), which
can undergo fragmentation and then a second stage of mass
analysis, MS2. Multiple fragmentation methods exist for defin-
ing the chemical structure of an ion, such as CID, electron-
capture dissociation (ECD), electron-transfer dissociation
(ETD) and SID, among others. Each technique generates
interpretable information from the distribution of fragment
ions. Fragments include losses (e.g. neutral loss scan) and
retentions (e.g. precursor ion scan) of neutral and charged
species. MS2 can be performed in either time (trap MS) or
space (TOF and beam-type instruments).
The value of MS/MS scan modes for lipid profiling of
embryos is demonstrated in Fig. 4 (ionisation by nanoESI
(nESI)). The shared structural motifs of lipids make them suited
to analysis by the less used MS/MS scan modes. There are, in
principle, a total of five MS/MS scan modes (Fig. 4a). The two-
dimensional MS2 data domain contains the masses and intensi-
ties of all precursor and product ions (Fig. 4a). Given that a fixed
mass is represented by a single filled circle () and a single
unfilled circle (J) represents a scanned or variable mass, the
scan modes available to explore this data domain, namely
(J-J), include: a product ion scan (-J), a precursor ion
scan (J-), a neutral loss scan (J.J) and a reaction-
monitoring scan (.) (Schwartz et al. 1990; Jarmusch and
Cooks 2014). Note that the heavy arrow (.) indicates a defined
relationship and the light arrow (-) indicates an unspecified
relationship. Also note that it is possible to generalise the neutral
loss scan to include any specified relationship between parent
and product, the so-called functional relationship scan of which
the constant neutral loss scan is a particular example. Many mass
analysers such as three-dimensional traps, two-dimensional
traps and triple quadrupoles are capable of some or all such
scans, but the design of some mass analysers (e.g. TOF)
precludes particular scans.
The characterisation of lipids is most commonly performed
using the product ion scan (Manicke et al. 2008), providing
detailed information about a single ion and the neutral molecule
for which it is a surrogate. For example, m/z 885 was detected
from bovine embryos with DESI-MS in full scan and subse-
quently analysed using the product ion MS/MS scan, as shown in
Fig. 4c. Major structural components are seen with an initial loss
of m/z 304, suggesting the presence of arachidonic acid, fatty
acid (FA) 20 : 4. A further loss of inositol indicates that the
precursor ion (original lipid) belongs to the phosphatidylinositol
class of lipids. The presence of m/z 283.4 (FA 18 : 0, stearic acid)
Oocyte and embryo MS lipid profiling Reproduction, Fertility and Development 629

(a) 100 688.18 Squalene

80

60

TAG (52 : 3)
40
TAG (50 : 2) 963.66 TAG (54 : 4)
20
937.63 989.67 TAG (56 : 4)
1013.67
0
600 700 800 900 1000 1100 1200
(b) 100 963.65

80
937.62
60 688.19

40
989.67
Relative abundance

20 1013.67

0
600 700 800 900 1000 1100 1200
(c) 100 967.67 TAG (52 : 1)

80 TAG (54 : 1)
Ubiquinone
1140.48
995.69
60
937.62
40 TAG (56 : 2)

20 1017.69

0
600 700 800 900 1000 1100 1200
(d) 100 965.65 TAG (52 : 2)

80

688.19 18 : 1 Chol ester

60
20 : 3 Chol ester TAG (54 : 3)
937.62
40 991.69
757.50 TAG (48 : 1) TAG (56 : 1)
20
781.50 911.62 1019.71 1140.48

0
600 700 800 900 1000 1100 1200

Fig. 3. Representative high mass resolution desorption electrospray ionisation–mass spectrometry

(DESI-MS) mass spectra in the positive ion mode showing adducts of silver (DESI solvent doping with
AgNO3) in (a) an immature oocyte, (b) an in vitro-matured oocyte, (c) a blastocyst produced in vitro and
(d) a blastocyst produced in vivo. Reproduced from González-Serrano et al. (2013b).
630 Reproduction, Fertility and Development C. R. Ferreira et al.

(a) (c) 350 m /z 885 581.3 885.5

(b) (Defined) Neutral
loss scan [M-H]⫺
MS1
300
283.4

Absolute abundance
FA(18 : 0) 419.3
250 -FA(20 : 4)

MS2

Product ion
200
-inositol
Product ion scan 150
303.3
FA(20 : 4)
100

Precursor ion scan 50

0
200 300 400 500 600 700 800 900
Precursor ion
(d ) 891.4 (f ) 500 747.7
m /z 184 m /z 281
400
400
-C5H14NO4P FA(18 : 1)
300 869.2 300
867.4 773.8 775.9
200 200
Absolute abundance

805.3
100 810.5 841.3 100
863.8

0 0
(e) 500 (g)
m /z 59 m /z 279 885.7
782.5 804.7
400
-N(CH3)3 200 861.7
FA(18 : 2)
300 775.9 863.8

200 100 773.8

726.7 780.4 866.5
810.7 892.6
100 754.6 890.2 749.8

0 0
700 720 740 760 780 800 820 840 860 880 900 700 720 740 760 780 800 820 840 860 880 900
m/z

Fig. 4. (a) All possible single stage (MS1) and two stage (MS2) mass spectrometry scans (closed circles and bold arrows indicate fixed values; open circles and
regular arrows indicate scanned values) obtained from mouse 2-cell embryos by nESI. (b) MS2 data space with three major scan types each shown in relation to
the two-dimensional data space. (c) Product ion scan, negative mode, of m/z 885 detected from bovine embryos with characteristics neutral losses and major
fragment ions. (d) Neutral loss scan for 184 (neutral phosphocholine), positive mode, from bovine embryos. (e) Neutral loss scan for 59 (neutral choline),
positive mode, from bovine embryos. (f) Precursor ion scan of m/z 281 (fatty acid (FA) 18 : 1), negative mode from bovine embryos, indicating complex lipids
containing the FA 18 : 1 acyl chain. (g) Precursor ion scan of m/z 279 (FA 18 : 2), negative mode from bovine embryos, indicating complex lipids containing the
FA 18 : 2 acyl chain.

completes the identification of the acyl chain (arachidonic acid
is further supported by the ion of m/z 303.3), yielding the
tentative identification as PI 38 : 4 or, more specifically, PI 20 :
4/18 : 0 and/or PI 18 : 0/20 : 4.
Lipids can be analysed for their class based on neutral loss
scan for characteristic losses associated with their common
structural motifs. In practice this is accomplished by scanning
with both MS1 and MS2 and correlating the two based on a
defined, fixed neutral loss. The loss of 59 (Fig. 4e) corresponds
to the neutral loss of trimethylamine from choline head groups in
the PC and SM lipid structures. Similarly, the loss of 184
corresponds to a phosphocholine (Fig. 4d). When both lipid
classes share the choline motif, the relative abundance of the two
losses differentiates the two based on the ease of loss. For
example, PCs lose 59 more abundantly than 184, as is shown in
Fig. 4e. The same logic applies to the SMs, which are repre-
sented in Fig. 4d. The reason for this difference is the availability
of charged sites in the two structures, with SMs containing an
additional amide residue to sustain charge after loss of the
charged head group.
Precursor ion scans (Fig. 4f–g) are equally informative and
are particularly well suited for lipid structural studies. The
product ion can be selected and all the precursor ions from
which it originated are inherently identified. Therefore, a
fragment ion can be defined and all precursor ions producing
that ion are reported. For example, in Fig. 4f, a precursor ion scan
for all lipids containing an acyl chain 18 : 1 (oleic acid) is
recorded. The lipids displayed are from multiple lipid classes
Oocyte and embryo MS lipid profiling
(m/z 747 is due to a phosphatidylglycerol and m/z 863 is due to a
PI) allowing one to track the common structural elements.
Similarly, by selecting the characteristic ion of FA 18 : 2 in
Fig. 4g, a different ratio of lipids containing the acyl chain is
detected. This spectrum includes an ion at m/z 885 that is not the
same as the ion analysed in Fig. 4c, because that ion was found
not to contain the defined acyl fatty acid. In this way, isobaric
peaks associated with isomeric lipids can be deconvoluted and
processed separately.
Statistical approaches to tackling the amount of chemical
information present in lipid profiles
Performing lipid profiling analysis involves not only profi-
ciency in MS, but also requires the appropriate use of statistics in
order to reduce the dimensionality of the data, observe innate
variation and perform biological interpretation. Experimental
data have been processed by using the entire MS profile rather
than applying peak-picking strategies. The entire MS profile can
be considered a chemical fingerprint and it is usually normalised
to most easily investigate the qualitative rather than the quan-
titative information due to instability of the absolute signal
and the difficulty of adding internal standards (Eberlin et al.
2011b).
An inherent feature of MS full scan profiles and MS images is
the large amount of information contained in each dataset. The
chemical information can be explored and interpreted in a
deeper and more efficient way by using multivariate data
analysis. For lipid profiling, MS spectra acquired over a defined
span of time or representing regions of interest (ROI) of MS
images are averaged and converted into two-dimensional data
matrices that list, for each sample (i.e. row), the ion abundances
of all the m/z values (i.e. columns) acquired. Data dimensionali-
ty depends on the ion acquisition step (i.e. mass resolution) and
the m/z range of interest, but it can easily reach tens of thousands
of columns even for low resolution MS data. Thus, data reduc-
tion via consecutive-window averaging algorithms has been
reported in some cases to facilitate data handling (Pirro et al.
2012). Spectra are usually normalised to the total ion current
(TIC), rather than the base peak, so that the entire spectrum
counts in the normalisation process, rather than a single ion (as
in the latter case) that may come from background contribution.
In some experimental studies, normalisation by the standard
normal variate (SNV) transform has also been applied in order to
correct for both baseline shifts and global intensity variations
(Oliveri et al. 2010).
When dealing with MS imaging of tissue sections or cells,
spectral data are rearranged into the corresponding hyperspec-
tral datacubes (X  Y  MS), where ‘X’ and ‘Y’ are spatial
domains and the ‘MS’ domain contains the m/z variables
and corresponding intensity (i.e. an entire mass spectrum).
Multivariate statistics applied on such datacubes can tackle
the chemical and spatial information simultaneously (Pirro
et al. 2012).
The hallmark of multivariate statistics is the capability to
efficiently compress a multidimensional dataset and to extract
from it informative chemical features with easy visualisation
and interpretation. Many techniques are available for pattern
Reproduction, Fertility and Development 631
recognition analysis. They tackle the full data matrix to com-
prehensively consider the spectral information enclosed within
it, taking into account the intercorrelation among all variables (e.
g. ion abundances), on the basis of which characterisation and/or
discrimination of the samples is determined. These techniques
can be roughly divided between unsupervised and supervised
methods. The first provides information about the presence of
structures (i.e. relationships) among the objects and/or the
variables studied, whereas the latter uses mathematical models
for the prediction of qualitative or quantitative performance in
the case of unknowns. Additional tools, including pretreatment
processes and variable selection techniques, complete the che-
mometric pattern recognition arsenal (Oliveri et al. 2010;
Marini 2013).
The chemical characterisation of cells and cell compartments
by lipid profiling has focused on diverse studies using unsuper-
vised techniques (i.e. the exploration of the data not guided by a
priori categorisation of samples). These include the use of
principal component analysis (PCA) to extract informative
chemical features and describe dynamic lipid changes during
oocyte and embryo development.
By means of PCA, the data information content is reorga-
nised and compacted into a few principal components (linear
combinations of the original variables), which are orthogonal (i.
e. uncorrelated) and describe independent contributions to part
of the variance in the data. The differences observed between
samples are dependent on variations between them and PCA is
used to search for maximum variance directions in the multidi-
mensional space of the original variables, preferably passing
through the data centroid, meaning that mean-centering should
be at least performed as column preprocessing, whereas the
well-known column autoscaling pretreatment is generally
avoided to process spectral data, otherwise the same weight
will be given to both noise and informative features (Oliveri
et al. 2010). Occasionally, Pareto scaling has been used as a
halfway pretreatment between mean-centering and autoscaling
(Ivosev et al. 2008).
The first principal component is the direction of the maxi-
mum variance. The second principal component is in the
direction of the maximum remaining variance, orthogonal to
the first one, and so on. The projections of the data objects onto
the principal components are called scores, whereas the impor-
tance of each original variable in defining a certain principal
component is given by the loading coefficient.
DESI-MS lipid profiling in cultured cells in the negative ion
mode has been recently described and reported for the detection
of FFAs and PLs after induced cellular stress (Bodzon-
Kulakowska et al. 2014a, 2014b). The detection of squalene,
cholesterol esters and TAGs by the addition of silver nitrate in
the DESI spray has been reported for the analysis of bovine
oocytes and embryos and for porcine (González-Serrano et al.
2013b; Pirro et al. 2014). Similarly, cell pellets of swine stem
cell adipocytes have been analysed (three replicates) after
differentiation was induced in vitro on Days 0, 2, 7 and 21 of
culture using experimental conditions reported previously
(Monaco et al. 2012; Bionaz et al. 2013). Representative mass
spectra are shown in Fig. 5a–d. Data were acquired at high mass
resolution using an orbitrap mass spectrometer (Exactive;
632 Reproduction, Fertility and Development C. R. Ferreira et al.

Cholesterol esters

(a) 100 757.50 (b) 100 688.18

Squalene 18 : 2 cholesteryl ester

80 80
757.50 TAG (52 : 4)
688.18 701.42
60 1140.47
60
969.58
22 : 6 cholesteryl ester
40 729.47 40
Ubiquinine 727.44 803.54
Triacylglyerols
Relative abundance

20 1140.47
20
969.58

0 0
600 700 800 900 1000 1100 1200 600 700 800 900 1000 1100 1200

(c) 100 755.48 (d ) 100 1140.47

80 80 969.58

60 60
701.42

40 40 727.44
803.54
TAG (52 : 2) 688.18
771.48 TAG (50 : 1)
20 965.67 20 939.65
729.47 803.47 TAG (54 : 3) 757.50 991.68
911.62
688.18 939.65 991.68
0 0
600 700 800 900 1000 1100 1200 600 700 800 900 1000 1100 1200
m/z

Fig. 5. Representative desorption electrospray ionisation–mass spectrometry (DESI-MS) mass spectra obtained from swine adipose stem cell pellets.
Lipids were ionised as silver adducts in the positive ion mode with high mass resolution. (a) Newly isolated swine adipocytes (Day 0); (b) adipose stem
cells cultured for 2 days in vitro differentiation (Day 2); (c) mass spectra of adipose stem cells after 7 days of in vitro culture (Day 7); and (d ) fully
differentiated adipocytes after 21 days of in vitro culture (Day 21). Lipid attribution was performed based on high mass resolution measurements as
reported previously (González-Serrano et al. 2013b; Pirro et al. 2014).

⫻ 104 Score plot Loading plot

0.4 18 : 1 and 18 : 2
4 cholesterol esters
D7
Loadings on PC2 (21.2%)

0.3
Scores on PC2 (21.2%)

2 Ubiquinone
D21 0.2
D0 and D2
0
0.1

⫺2
0

⫺4 ⫺0.1

Squalene
⫺0.2
⫺6

⫺1 ⫺0.5 0 0.5 1 ⫺0.3 ⫺0.2 ⫺0.1 0 0.1 0.2 0.3 0.4

⫻ 105
Scores on PC1 (58.7%) Loadings on PC1 (58.7%)

Fig. 6. Principal components analysis score plot (left; dashed lines–circles for display; not related to confidence intervals) and corresponding
loading plot (right) showing discrimination of adipose stem cells after isolation (Day 0) and after in vitro culture for 7 and 21 days, during which
differentiation occurred. Undifferentiated cells exhibited higher abundances of oleic and linoleic cholesterol esters, whereas squalene was most
prominent at Day 7 and ubiquinone (indicative of mitochondrial activity) at Day 21.

Thermo Scientific, San Jose, CA, USA). Changes in the lipid indicated by PCA (Fig. 6). In the PCA score plots it is possible to
profile, especially in the abundance of the silver adducts of visualise groupings (colour labelled) that indicate similarities
squalene (m/z 688.18), cholesterol esters of oleic and linoleic among objects, based on the information derived from the mass
acids (m/z 755.48 and 757.50) and ubiquinone (m/z 1140.47) are spectra, and these can be associated with particular
Oocyte and embryo MS lipid profiling
Score plot
3
2
Scores on PC2 (14.7%)
Loadings on PC2 (14.7%)
1
⫺1
⫺2
⫺3
⫺3 ⫺2 ⫺1 0 1 2 3
Scores on PC1 (16.0%)
Fig. 7. Principal components analysis of the fused datasets from positive and negative ion mode mass spectra of pig oocytes at different times of in vitro
maturation using a mid-level fusion approach. Score plot of principal component (PC) 1 vs PC2 (left) and corresponding loading plot (right) labelled in
terms of m/z ratio. Green circles, immature oocytes (n ¼ 31); red diamonds, 24-h in vitro-matured oocytes (n ¼ 25); blue triangles, 44-h in vitro-matured
oocytes (n ¼ 40). Negative ions are green, positive ions are violet. Figure reproduced from Pirro et al. (2014).
characteristics of the samples analysed. Simultaneous examina-
tion of the score plot and loading plot allows observation of the
chemical variation among individuals and experimental groups.
Interpretation of the relationship between scores and loadings is
done by means of vectors representing the relative direction and
magnitude. Indeed, squalene, a cholesterol precursor, is most
abundant (in terms of relative intensity in the entire spectrum) in
recently isolated (Day 0) swine adipose stem cells (ASC). As
differentiation progresses, at Day 2 and Day 7 (red dots, Fig. 6),
cholesterol esters of oleic and linoleic acid become the most
characteristic lipids of these cells profiles. Fully differentiated
adipocytes at Day 21 exhibit greater ion intensities for ubiqui-
none compared with Days 0, 2 and 7 (Fig. 5).
When a single MS profile is not enough to see clear
differences among a set of samples, multiple sources of
chemical information can be used together to improve char-
acterisation via data fusion strategies. For example, positive
and negative ion modes from the same embryos can be
merged, after positive and negative blocks of data were scaled
to the unit variance, and PCA performed on all data simulta-
neously (Fig. 7; González-Serrano et al. 2013b; Pirro et al.
2014). DESI-MS profiling in the negative ion mode (for
detection of FFs and complex PLs) was sufficient to show
separation between immature mice oocytes and diverse
embryo developmental stages (Ferreira et al. 2012b). Con-
versely, data fusion of positive and negative ions was needed
to better characterise chemical differences between bovine
in vivo oocytes and their in vitro counterparts, and to detect
dynamic changes during in vitro maturation of pig oocytes
(González-Serrano et al. 2013b; Pirro et al. 2014). Remark-
ably, DESI-MS analysis followed by multivariate statistics has
proven sensitive to even minor changes in the lipid content,
and thus it may be feasible to reveal dynamic differences in
single cells.
Reproduction, Fertility and Development 633
Original loading plot
0.10
0.05
0
⫺0.05
⫺0.10
⫺0.10 ⫺0.08 ⫺0.06 ⫺0.04 ⫺0.02 0 0.02 0.04 0.06 0.08 0.10
Loadings on PC1 (16.0%)
Using similar multivariate approaches, lipid characterisation
of embryos was achieved on analysing 2- and 8-cell bovine
embryos by silica plate laser desorption ionisation MSI
(SP-LDI-MSI; Ferreira et al. 2014). MALDI-MS followed by
PCA was also used to explore the lipid profiles of single
embryos and oocytes from various species (human, bovine,
sheep and fish; Ferreira et al. 2010). As is the case for DESI,
MALDI showed the ability to generate characteristic profiles
and respond to chemical changes related to developmental
stages and culture conditions (Sudano et al. 2012; Tata et al.
2013). Supervised discriminant classification methods, such as
partial squares discriminant analysis (PLS-DA), have also been
used to separate cow oocytes based on their lipid content as
detected by MALDI-MS (Silva-Santos et al. 2014).
Summary, perspectives and conclusions
Much remains to be understood about lipid metabolism in
oocytes and embryos. Endogenous fatty acid composition
(i.e. combination of FFAs and fatty acyl residues in lipids such
as TAG and PL) has been studied frequently in the oocytes and
embryos of humans and domestic mammals. Results indicate
that specific ratios or combinations of fatty acids are important
for proper embryonic and even fetal development and viability
(McKeegan and Sturmey 2012). In addition, unsaturated fatty
acyl PL residues are important for metabolic properties of the
membrane and for cryopreservation. And now, as exemplified
above, the role of fatty acids in the structure of complex lipids
such as TAG and PLs is starting to be unravelled with the aid
of MS.
The use of DESI-MS has expanded the capabilities of direct
lipid profiling on oocytes and embryos to include FFAs, choles-
terol esters, squalene, ubiquinone and a wide range of PLs. In all
cases, statistical analysis of the data was vital and it guided
634 Reproduction, Fertility and Development
interpretation and understanding of relevant differences related
to maturation, preimplantation developmental stages and the
impact of in vitro culture.
Lipid profiling is usually complemented by product ion scans
(MS/MS) or HRMS for structural identification. We exemplify
in this review the value of scan modes such as neutral loss and
precursor ion scan using bovine embryos. These scan modes
provide information on lipids containing specific fatty acyl
residues (such as palmitic, linoleic and arachidonic acids) and
specific PL classes and can expand the biological significance
and interpretation of lipid profiling information. As already
pointed out, the structure of the MS data changes according to
the ion acquisition modes used, but multivariate data analysis
offers a wide portfolio of methods for pattern recognition that
can follow the characteristics of the acquired data.
Lipid profiling by MS is becoming important in this area of
research as an analytical approach per se or combined with other
techniques such as RT-qPCR (González-Serrano et al. 2013a)
and has been applied in diverse studies in the reproductive field,
such as with embryo culture media for quality control and
prediction of viability (Ferreira et al. 2009; Cortezzi et al.
2013) and for follicular fluid (Montani et al. 2012; Cataldi
et al. 2013). Lipid profiling can also be coupled to chemical
imaging for the study of ovarian metabolism (Campbell et al.
2012), follicle, preimplantation, embryo and fetal development
(Burnum et al. 2009; Hayasaka et al. 2009; Urban et al. 2011;
Eberlin et al. 2012; Bionaz et al. 2013; Ferreira et al. 2014; Tian
et al. 2014). Therefore, this paper has highlighted recent pub-
lications using MS profiling in the reproductive field and has
introduced the use of DESI-MS lipid profiling for stem cell
differentiation studies. The information presented is of general
interest for a broad range of lipid and small molecule profiling
experiments in any other biological sample and using ionisation
sources other than ambient ionisation.
Acknowledgements
CRF is supported by the Purdue University Center for Cancer Research
Small Grants and the Brazilian National Council for Scientific and
Technological Development (CNPq; process 237237/2012–1). RGC is
supported by National Science Foundation, Division of Chemistry
(NSF CHE) 1307264. AFGS is supported by the German Academic
Exchange Service (DAAD) and the Fundación Gran Mariscal Ayacucho
(FUNDAYACUCHO). MBW is supported by the Illinois Regenerative
Medicine Institute through grant #63080017.
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