Produção Nacional
Revisado por pares
Single embryo and oocyte lipid fingerprinting by mass spectrometry
2009; Elsevier BV; Volume: 51; Issue: 5 Linguagem: Inglês
10.1194/jlr.d001768
ISSN1539-7262
AutoresChristina R. Ferreira, Sérgio A. Saraiva, Rodrigo Ramos Catharino, Jerusa S. Garcia, Fábio C. Gozzo, Gustavo B. Sanvido, Luiz F. A. Santos, E.G. Lo Turco, J. H. F. Pontes, Andréa Cristina Basso, Ricardo Pimenta Bertolla, Roberto Sartori, M. M. Guardieiro, Felipe Perecin, Flávio Vieira Meirelles, Juliano Rodrigues Sangalli, Marcos N. Eberlin,
Tópico(s)Lipid metabolism and biosynthesis
ResumoMethods used for lipid analysis in embryos and oocytes usually involve selective lipid extraction from a pool of many samples followed by chemical manipulation, separation and characterization of individual components by chromatographic techniques. Herein we report direct analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of single and intact embryos or oocytes from various species. Biological samples were simply moisturized with the matrix solution and characteristic lipid (represented by phosphatidylcholines, sphingomyelins and triacylglycerols) profiles were obtained via MALDI-MS. As representative examples, human, bovine, sheep and fish oocytes, as well as bovine and insect embryos were analyzed. MALDI-MS is shown to be capable of providing characteristic lipid profiles of gametes and embryos and also to respond to modifications due to developmental stages and in vitro culture conditions of bovine embryos. Investigation in developmental biology of the biological roles of structural and reserve lipids in embryos and oocytes should therefore benefit from these rapid MALDI-MS profiles from single and intact species. Methods used for lipid analysis in embryos and oocytes usually involve selective lipid extraction from a pool of many samples followed by chemical manipulation, separation and characterization of individual components by chromatographic techniques. Herein we report direct analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of single and intact embryos or oocytes from various species. Biological samples were simply moisturized with the matrix solution and characteristic lipid (represented by phosphatidylcholines, sphingomyelins and triacylglycerols) profiles were obtained via MALDI-MS. As representative examples, human, bovine, sheep and fish oocytes, as well as bovine and insect embryos were analyzed. MALDI-MS is shown to be capable of providing characteristic lipid profiles of gametes and embryos and also to respond to modifications due to developmental stages and in vitro culture conditions of bovine embryos. Investigation in developmental biology of the biological roles of structural and reserve lipids in embryos and oocytes should therefore benefit from these rapid MALDI-MS profiles from single and intact species. The double molecular layer of polar lipids is a marvelous architectural feature of exquisite biological engineering in cell membranes. Specific functions and variations of the various phospholipids (PL), the most abundant lipids in eukaryotic cell membranes, are, however, still poorly understood (1van Meer G. Voelker D.R. Feigenson G.W. Membrane lipids: where they are and how they behave.Nat. Rev. Mol. Cell Biol. 2008; 9: 112-124Crossref PubMed Scopus (4234) Google Scholar). A diversity of PL in a finely balanced equilibrium is used by cells to construct stable and functional membranes, and PL composition determines most of the physico-chemical cell membrane properties such as fluidity, permeability and thermal phase behavior (2Edidin M. Lipids on the frontier: a century of cell-membrane bilayers.Nat. Rev. Mol. Cell Biol. 2003; 4: 414-418Crossref PubMed Scopus (389) Google Scholar). Knowledge of the function of lipids within the cell has benefited from the development of increasingly sensitive and selective analytical techniques, particularly those based on mass spectrometry (3Roberts L.D. McCombie G. Titman C.M. Griffin J.L. A matter of fat: an introduction to lipidomic profiling methods.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2008; 871: 174-181Crossref PubMed Scopus (98) Google Scholar, 4Ejsing C.S. Sampaio J.L. Surendranath V. Duchoslav E. Ekroos K. Klemm R.W. Simons K. Shevchenko A. Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry.Proc. Natl. Acad. Sci. USA. 2009; 106: 2136-2141Crossref PubMed Scopus (684) Google Scholar). Among these techniques, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) (5Karas M. Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons.Anal. Chem. 1988; 60: 2299-2301Crossref PubMed Scopus (4764) Google Scholar) has been very successful in studies of the compositions of lipids and other crucial biological molecules. MALDI-MS has allowed direct analysis of complex and unfractionated samples, such as the study of peptide profiles below the level of a single cell (6Li L. Garden R.W. Sweedler J.V. Single-cell MALDI: a new tool for direct peptide profiling.Trends Biotechnol. 2000; 18: 151-160Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). In lipidomics, MALDI-MS has provided fast and simple acquisition of mass spectra with lipid profiles of cells, tissues and body fluids (7Fuchs B. Schiller J. MALDI-TOF MS analysis of lipids from cells, tissues and body fluids.Subcell. Biochem. 2008; 49: 541-565Crossref PubMed Scopus (45) Google Scholar). MALDI-MS lipid fingerprinting can, for example, help studies aimed at understanding the effect of membrane lipid composition on cell membrane behavior after temperature changes. This knowledge is essential for cryopreservation studies of a variety of cells, including oocytes from patients undergoing cancer treatment (8Tao T. Del Valle A. Human oocyte and ovarian tissue cryopreservation and its application.J. Assist. Reprod. Genet. 2008; 25: 287-296Crossref PubMed Scopus (63) Google Scholar); gametes and embryos for conservation of animal genetic resources; and to facilitate the international transit of genetic material (9Pereira R.M. Marques C.C. Animal oocyte and embryo cryopreservation.Cell Tissue Bank. 2008; 9: 267-277Crossref PubMed Scopus (121) Google Scholar). Cytoplasm lipid accumulation has been shown to affect the rate of post-thaw development of bovine embryos (10Abe H. Yamashita S. Satoh T. Hoshi H. Accumulation of cytoplasmic lipid droplets in bovine embryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media.Mol. Reprod. Dev. 2002; 61: 57-66Crossref PubMed Scopus (248) Google Scholar, 11Barcelo-Fimbres M. Seidel Jr., G.E. Effects of either glucose or fructose and metabolic regulators on bovine embryo development and lipid accumulation in vitro.Mol. Reprod. Dev. 2007; 74: 1406-1418Crossref PubMed Scopus (66) Google Scholar, 12George F. Daniaux C. Genicot G. Verhaeghe B. Lambert P. Donnay I. Set up of a serum-free culture system for bovine embryos: embryo development and quality before and after transient transfer.Theriogenology. 2008; 69: 612-623Crossref PubMed Scopus (50) Google Scholar), which suggests that the embryo culture process causes lipid metabolism changes that affect the properties and stability of cell membranes (13Dinnyes A. Nedambale T.L. Cryopreservation of manipulated embryos: tackling the double jeopardy.Reprod. Fertil. Dev. 2009; 21: 45-59Crossref PubMed Scopus (12) Google Scholar). In laboratories for assisted reproduction for humans, embryos and oocytes are routinely cryopreserved, but there is still considerable need of research aiming to improve knowledge of the role of lipids and the efficiency of protocols used (14Desai N. Blackmon H. Szeptycki J. Goldfarb J. Cryoloop vitrification of human day 3 cleavage-stage embryos: post-vitrification development, pregnancy outcomes and live births.Reprod. Biomed. Online. 2007; 14: 208-213Abstract Full Text PDF PubMed Scopus (80) Google Scholar, 15Cobo A. Kuwayama M. Perez S. Ruiz A. Pellicer A. Remohi J. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method.Fertil. Steril. 2008; 89: 1657-1664Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 16Hiraoka K. Fuchiwaki M. Horiuchi T. Okano S. Kinutani M. Kinutani K. Vitrified human day-7 blastocyst transfer: 11 cases.Reprod. Biomed. Online. 2008; 17: 689-694Abstract Full Text PDF PubMed Scopus (15) Google Scholar). To date, changes in PL composition profiles of mammalian embryos and oocytes are poorly understood. Gas chromatography (GC) has been applied to study triacylglycerol (TAG) composition in human and bovine oocytes and embryos (17Matorras R. Ruiz J.I. Mendoza R. Ruiz N. Sanjurjo P. Rodriguez-Escudero F.J. Fatty acid composition of fertilization-failed human oocytes.Hum. Reprod. 1998; 13: 2227-2230Crossref PubMed Scopus (66) Google Scholar, 18Kim J.Y. Kinoshita M. Ohnishi M. Fukui Y. Lipid and fatty acid analysis of fresh and frozen-thawed immature and in vitro matured bovine oocytes.Reproduction. 2001; 122: 131-138Crossref PubMed Scopus (220) Google Scholar, 19Haggarty P. Wood M. Ferguson E. Hoad G. Srikantharajah A. Milne E. Hamilton M. Bhattacharya S. Fatty acid metabolism in human preimplantation embryos.Hum. Reprod. 2006; 21: 766-773Crossref PubMed Scopus (100) Google Scholar), but this approach requires chemical transformation via saponification and derivatization, providing free fatty acyl residue profiles but not TAG or PL identification. A main drawback for GC analyses is the need of a large pool of samples (more than 10 oocytes or embryos for each lipid analysis), especially when human embryos or oocytes are investigated (19Haggarty P. Wood M. Ferguson E. Hoad G. Srikantharajah A. Milne E. Hamilton M. Bhattacharya S. Fatty acid metabolism in human preimplantation embryos.Hum. Reprod. 2006; 21: 766-773Crossref PubMed Scopus (100) Google Scholar). MALDI-MS offers, however, the advantage of detecting intact lipids, and due to its unmatched high spatial resolution, allows targeting single species of microscopic dimensions (100–200 μm) such as mammalian oocytes and embryos. Herein we show that direct analysis by MALDI-MS can contribute significantly to embryo and oocyte lipidomic studies. With no solvent extraction and no chemical manipulation, direct MALDI-MS of single and intact embryos or oocytes of different species (from invertebrates to mammalian) is shown to provide reproducible lipid profiles. Besides species recognition, the method described herein is also shown to be sensitive and, hence, to reveal important changes in lipid profiles during bovine preimplantation development and in bovine embryo in vitro culture conditions. Unless mentioned, chemicals and culture media for handling bovine and ovine oocytes were purchased from Sigma (St. Louis, USA). Media used for human oocytes handling (modified Human Tubal Fluid [HTF]) was purchased from Irvine Scientific (Santa Ana, CA, USA). Phosphate buffer saline (PBS) solution was supplied by Nutricell (Campinas, SP, Brazil). Methanol (ACS/HPLC) grade was purchased from Burdick and Jackson (Muskegon, MI, USA) and 2,5-dihydroxybenzoic acid (DHB) was purchased from ICN Biomedicals (Aurora, OH, USA). Ultrapure water, purified by a Direct-Q water system (Millipore, Bedford, MA, USA) was used for the preparation of solvents. The care and use of animal samples (bovine, sheep, fish and fire ant) was approved by the Institutional Committee for Ethics in Animal Research of the State University of Campinas (UNICAMP), which follows the Ethical Principles of Animal Research established by the Brazilian College for Animal Experimentation (COBEA) under protocol number 1752-1. The use of human unfertilized oocytes was approved by the São Paulo Federal University (UNIFESP) Institutional Committee for Ethics under the protocol number 0411/07. Immature bovine (Bos taurus) and sheep (Ovis Aries) oocytes were obtained by post mortem follicular aspiration of ovaries from cows and ewes slaughtered at commercial slaughterhouses. Ovaries were transported in 0.9% (w/v) saline solution at 25–30°C to the laboratory and follicles were aspirated using an 18-gauge needle attached to a 20-ml syringe. Cumulus oocyte complexes with at least three layers of cumulus cells and homogeneous cytoplasm were denuded of cumulus cells by gentle pipetting in 0.5% hyaluronidase. Bovine in vivo–derived embryos were obtained from superovulated heifers kept in pasture (Brachiaria decumbens). The emergence of the animal's follicular wave was synchronized by one intramuscular (i.m.) injection of 2.0 mg estradiol benzoate (Estrogin, Farmavet, São Paulo, Brazil) and insertion of an intravaginal controlled internal drug release device (CIDR), containing 1.9 g progesterone (Pfizer, Hamilton, New Zealand) on day 0. On day 4.5, the superstimulatory treatments were initiated (follicle stimulating hormone [FSH]; Folltropin-V, Bioniche Animal Health, Belleville, Canada) and given in decreasing doses of 28, 21, 14 and 7 mg FSH twice daily, over a 4-day period, for a total dose of 70 mg. At the time of the fifth and sixth injections of FSH, 25 mg of dinoprost tromethamine (Lutalyse, Pfizer, Paulinia, Brazil) was injected intramuscularly. The CIDR was removed at the time of the seventh superstimulatory injection. Ovulation was induced with an i.m. injection of 0.05 mg gonadotrophin releasing hormone (GnRH; Gestran Plus; ARSA S.R.L., Buenos Aires, Argentina) 12 h after the last superstimulatory injection. All heifers were artificially inseminated (AI) with frozen/thawed semen from the same bull 12 and 24 h after GnRH injection. Seven days after the first AI, embryos/ova were recovered using a nonsurgical uterine flushing technique (20Neto A.S.C. Sanches B.V. Binelli M. Seneda M.M. Perri S.H. Garcia J.F. Improvement in embryo recovery using double uterine flushing.Theriogenology. 2005; 63: 1249-1255Crossref PubMed Scopus (30) Google Scholar). For bovine in vitro embryo production, oocytes obtained by post mortem follicular aspiration were in vitro fertilized and cultured as described previously (21Ferreira C.R. Souza G.H.M.F. Riccio M.F. Catharino R.R. Pontes J.H.F. Basso A.C. Junior J.C.E. Perecin F. Eberlin M.N. Mass spectrometry fingerprinting of media used for in vitro production of bovine embryos.Rapid Commun. Mass Spectrom. 2009; 23: 1313-1320Crossref PubMed Scopus (16) Google Scholar), but with changes in culture medium supplementation and incubator atmosphere. After IVF, embryos were cultured in four different in vitro conditions: 5% O2 and BSA (Group 1); 20% O2 and BSA (Group 2); 20% O2 and FCS (Group 3); and 5% O2 and FCS (Group 4). Human unfertilized oocytes from women submitted to transvaginal oocyte retrieval were provided by the Human Reproduction Service of São Paulo Federal University (UNIFESP) in Brazil. These samples would normally be discarded. Mullet ova (Mugil spp.) were collected from fresh fish in the City Fish Market of Santos-SP, Brazil, and transported at 4°C to the laboratory. Fire ant (Solenopsis spp.) eggs were collected in the field after species identification and were immediately transported to the laboratory. Oocyte and embryo samples were stored in microtubes containing 100 μl of a 50% aqueous methanol solution at -80°C until analysis. Sample preparation involved placing each oocyte, egg or embryo in a given spot of the target plate under the stereomicroscope. Samples were allowed to dry at room temperature, and their location was recorded in order to place the laser at the correct location during analysis. Just before analysis, 1 μl of 1.0 mol/l 2,5-dihydroxybenzoic acid (DHB) in methanol was placed in each target spot and allowed to dry at room temperature. MALDI-MS and MALDI-MS/MS spectra were acquired in the positive ion and reflectron modes using a Q-ToF Premier mass spectrometer (Waters, Manchester, UK) equipped with a 200-Hz solid-state laser in the m/z range of 700–950. The principal operating condition used was 10 V (sample plate), and laser irradiation consisted of diverse shots during the time of 60–90 s in the region were the sample had been placed on the target plate, until signals in the region of interest were observed and disappeared due to the consumption of the microscopic sample (Fig. 1). MALDI-MS/MS were manually acquired by increasing the collision energy until extensive dissociation of the precursor ion was observed. Argon was used as the collision gas. Spectra were centered and aligned using the MassLynx 4.0 software (Waters, Manchester, UK). From each spectrum, after the exclusion of isotopic peaks, the fifty most intense ions were considered as the starting point for searching m/z values corresponding to lipids. After attribution, only the m/z values which were clearly distinct from noise level in the spectra were included in the principal component analysis (PCA), which was performed using Pirouette v.3.11 (Infometrix Inc., Woodinville, WA, USA). As proof-of-principle cases, we investigated the potential of MALDI-MS fingerprinting to provide characteristic lipid profiles from a representative set of single embryos and oocytes from mammals and from two distantly related species: insect and fish. We then evaluated the ability of the technique to reveal changes in the lipid profiles during embryo development and under different in vitro culture conditions for bovines as a representative biological model. MALDI-MS analysis and sample preparation (described in detail in the "Material and Methods" section) involved no lipid extraction and no chemical manipulation which, as already mentioned, would require substantial sample pooling of these highly valuable and sometimes unavailable (particularly in large numbers) microscopic structures. An intact single embryo or oocyte was collected and then placed in one of the many spots of the MALDI target plate placed under a stereomicroscope (Fig. 1A). Then, the target plate spot was moistened with the MALDI matrix before spectra acquisition (Fig. 1B). Fig. 2 displays representative MALDI-MS spectra from single embryos or oocytes from the five species investigated in this work. The spectra were acquired in the m/z 700–950 range, where most PL and TAG should be detected by MALDI(+)-MS mainly as either their protonated [M + H]+ or sodiated molecules [M + Na]+, or both (22Schiller J. Arnhold J. Benard S. Muller M. Reichl S. Arnold K. Lipid analysis by matrix-assisted laser desorption and ionization mass spectrometry: a methodological approach.Anal. Biochem. 1999; 267: 46-56Crossref PubMed Scopus (266) Google Scholar). Note that the MALDI-MS profiles are rather characteristic, and in some cases, very distinctive with unique PC, SM and TAG ions (see below) with contrasting abundances or ratios. Group comparison by principal component analysis (PCA; see below) was also found to allow their prompt characterization. The PL species were first attributed (Table 1) based on previous lipid profile studies by MALDI-MS and are described by the class abbreviation followed by the total number of carbons and double bounds in the acyl residues attached to the glycerol backbone (in parenthesis). The MALDI-MS of the human oocyte is characterized mainly by two clusters of ions, in which those of m/z 760.6, assigned as [PC (34:1) + H]+, and m/z 782.6, [PC (36:4) + H]+ or [PC (34:1) + Na]+ or both, predominate. These are also seen as major ions in the other mammalian samples: bovine oocytes (Fig. 2B), in vivo–produced bovine embryos (Fig. 2C) and sheep oocytes (Fig. 2D).TABLE 1Phospholipids (PL) and triacylglycerols (TAG) identified via MALDI(+)-MS of single human oocytes, bovine oocytes, bovine embryos, sheep oocytes, fish oocyte or ant eggsm/zLipid Ion (carbons:unsaturations)Reference703.5[SM (16:0) + H]+26Kawooya J.K. Osir E.O. Law J.H. Uptake of the major hemolymph lipoprotein and its transformation in the insect egg.J. Biol. Chem. 1988; 263: 8740-8747Abstract Full Text PDF PubMed Google Scholar, 30Fuchs B. Jakop U. Goritz F. Hermes R. Hildebrandt T. Schiller J. Muller K. MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa.Theriogenology. 2009; 71: 568-575Crossref PubMed Scopus (37) Google Scholar723.5[PC (34:1) + Na]+ and loss of N(CH3)329Petkovic M. Schiller J. Muller M. Benard S. Reichl S. Arnold K. Arnhold J. Detection of individual phospholipids in lipid mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: phosphatidylcholine prevents the detection of further species.Anal. Biochem. 2001; 289: 202-216Crossref PubMed Scopus (258) Google Scholar725.5[SM (16:0) + Na]+28Brugger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Crossref PubMed Scopus (719) Google Scholar, 30Fuchs B. Jakop U. Goritz F. Hermes R. Hildebrandt T. Schiller J. Muller K. MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa.Theriogenology. 2009; 71: 568-575Crossref PubMed Scopus (37) Google Scholar731.5[SM (18:0) + H]+26Kawooya J.K. Osir E.O. Law J.H. Uptake of the major hemolymph lipoprotein and its transformation in the insect egg.J. Biol. Chem. 1988; 263: 8740-8747Abstract Full Text PDF PubMed Google Scholar732.5[PC (32:1) + H]+26Kawooya J.K. Osir E.O. Law J.H. Uptake of the major hemolymph lipoprotein and its transformation in the insect egg.J. Biol. Chem. 1988; 263: 8740-8747Abstract Full Text PDF PubMed Google Scholar734.6[PC(32:0) + H]+26Kawooya J.K. Osir E.O. Law J.H. Uptake of the major hemolymph lipoprotein and its transformation in the insect egg.J. Biol. Chem. 1988; 263: 8740-8747Abstract Full Text PDF PubMed Google Scholar753.6[SM (18:0) + Na]+26Kawooya J.K. Osir E.O. Law J.H. Uptake of the major hemolymph lipoprotein and its transformation in the insect egg.J. Biol. Chem. 1988; 263: 8740-8747Abstract Full Text PDF PubMed Google Scholar754.6[PC (32:1) + Na]+26Kawooya J.K. Osir E.O. Law J.H. Uptake of the major hemolymph lipoprotein and its transformation in the insect egg.J. Biol. Chem. 1988; 263: 8740-8747Abstract Full Text PDF PubMed Google Scholar756.6[PC (32:0) + Na]+29Petkovic M. Schiller J. Muller M. Benard S. Reichl S. Arnold K. Arnhold J. Detection of individual phospholipids in lipid mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: phosphatidylcholine prevents the detection of further species.Anal. Biochem. 2001; 289: 202-216Crossref PubMed Scopus (258) Google Scholar, 30Fuchs B. Jakop U. Goritz F. Hermes R. Hildebrandt T. Schiller J. Muller K. MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa.Theriogenology. 2009; 71: 568-575Crossref PubMed Scopus (37) Google Scholar758.6[PC (34:2) + H]+26Kawooya J.K. Osir E.O. Law J.H. Uptake of the major hemolymph lipoprotein and its transformation in the insect egg.J. Biol. Chem. 1988; 263: 8740-8747Abstract Full Text PDF PubMed Google Scholar, 27McEvoy T.G. Coull G.D. Broadbent P.J. Hutchinson J.S. Speake B.K. Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida.J. Reprod. Fertil. 2000; 118: 163-170Crossref PubMed Google Scholar, 30Fuchs B. Jakop U. Goritz F. Hermes R. Hildebrandt T. Schiller J. Muller K. MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa.Theriogenology. 2009; 71: 568-575Crossref PubMed Scopus (37) Google Scholar760.6[PC (34:1) + H]+28Brugger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Crossref PubMed Scopus (719) Google Scholar762.6[PC (34:0) + H]+27McEvoy T.G. Coull G.D. Broadbent P.J. Hutchinson J.S. Speake B.K. Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida.J. Reprod. Fertil. 2000; 118: 163-170Crossref PubMed Google Scholar780.6[PC (34:2) + Na]+, [PC (36:5) + H]+26Kawooya J.K. Osir E.O. Law J.H. Uptake of the major hemolymph lipoprotein and its transformation in the insect egg.J. Biol. Chem. 1988; 263: 8740-8747Abstract Full Text PDF PubMed Google Scholar, 27McEvoy T.G. Coull G.D. Broadbent P.J. Hutchinson J.S. Speake B.K. Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida.J. Reprod. Fertil. 2000; 118: 163-170Crossref PubMed Google Scholar, 30Fuchs B. Jakop U. Goritz F. Hermes R. Hildebrandt T. Schiller J. Muller K. MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa.Theriogenology. 2009; 71: 568-575Crossref PubMed Scopus (37) Google Scholar782.6[PC (36:4) + H]+, [PC (34:1) + Na]+27McEvoy T.G. Coull G.D. Broadbent P.J. Hutchinson J.S. Speake B.K. Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida.J. Reprod. Fertil. 2000; 118: 163-170Crossref PubMed Google Scholar, 28Brugger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Crossref PubMed Scopus (719) Google Scholar, 29Petkovic M. Schiller J. Muller M. Benard S. Reichl S. Arnold K. Arnhold J. Detection of individual phospholipids in lipid mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: phosphatidylcholine prevents the detection of further species.Anal. Biochem. 2001; 289: 202-216Crossref PubMed Scopus (258) Google Scholar, 30Fuchs B. Jakop U. Goritz F. Hermes R. Hildebrandt T. Schiller J. Muller K. MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa.Theriogenology. 2009; 71: 568-575Crossref PubMed Scopus (37) Google Scholar784.6[PC (34:0) + Na]+30Fuchs B. Jakop U. Goritz F. Hermes R. Hildebrandt T. Schiller J. Muller K. MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa.Theriogenology. 2009; 71: 568-575Crossref PubMed Scopus (37) Google Scholar786.6[PC (36:2) + H]+26Kawooya J.K. Osir E.O. Law J.H. Uptake of the major hemolymph lipoprotein and its transformation in the insect egg.J. Biol. Chem. 1988; 263: 8740-8747Abstract Full Text PDF PubMed Google Scholar, 28Brugger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Crossref PubMed Scopus (719) Google Scholar, 30Fuchs B. Jakop U. Goritz F. Hermes R. Hildebrandt T. Schiller J. Muller K. MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa.Theriogenology. 2009; 71: 568-575Crossref PubMed Scopus (37) Google Scholar788.6[PC (36:1) + H]+27McEvoy T.G. Coull G.D. Broadbent P.J. Hutchinson J.S. Speake B.K. Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida.J. Reprod. Fertil. 2000; 118: 163-170Crossref PubMed Google Scholar, 28Brugger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Crossref PubMed Scopus (719) Google Scholar802.6[PC 36:5 + Na]+aAttribution performed in this work.804.6[PC (38:7) + H]+, [PC36:4 + Na]+30Fuchs B. Jakop U. Goritz F. Hermes R. Hildebrandt T. Schiller J. Muller K. MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa.Theriogenology. 2009; 71: 568-575Crossref PubMed Scopus (37) Google Scholar, aAttribution performed in this work.806.6[PC (38:6) + H]+, [PC36:3 + Na]+aAttribution performed in this work.808.6[PC (38:5) + H]+, [PC (36:2) + Na]+26Kawooya J.K. Osir E.O. Law J.H. Uptake of the major hemolymph lipoprotein and its transformation in the insect egg.J. Biol. Chem. 1988; 263: 8740-8747Abstract Full Text PDF PubMed Google Scholar, 30Fuchs B. Jakop U. Goritz F. Hermes R. Hildebrandt T. Schiller J. Muller K. MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa.Theriogenology. 2009; 71: 568-575Crossref PubMed Scopus (37) Google Scholar810.6[PC (38:4) + H]+, [PC (36:1) + Na]+26Kawooya J.K. Osir E.O. Law J.H. Uptake of the major hemolymph lipoprotein and its transformation in the insect egg.J. Biol. Chem. 1988; 263: 8740-8747Abstract Full Text PDF PubMed Google Scholar, 27McEvoy T.G. Coull G.D. Broadbent P.J. Hutchinson J.S. Speake B.K. Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida.J. Reprod. Fertil. 2000; 118: 163-170Crossref PubMed Google Scholar, 28Brugger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Crossref PubMed Scopus (719) Google Scholar, 30Fuchs B. Jakop U. Goritz F. Hermes R. Hildebrandt T. Schiller J. Muller K. MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa.Theriogenology. 2009; 71: 568-575Crossref PubMed Scopus (37) Google Scholar828.6[PC (38:6) + Na]+aAttribution performed in this work.830.6[PC (38:5) + Na]+28Brugger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Crossref PubMed Scopus (719) Google Scholar832.6[PC (38:4) + Na]+26Kawooya J.K. Osir E.O. Law J.H. Uptake of the major hemolymph lipoprotein and its transformation in the insect egg.J. Biol. Chem. 1988; 263: 8740-8747Abstract
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