Oocyte freezing confers thermal and chemical stress upon the oolemma and various other intracellular structures due to the formation of ice crystals. The lipid profiles of oocytes and embryos are closely associated with both, the degrees of their membrane fluidity, as well as the degree of chilling and freezing injuries that may occur during cryopreservation. In spite of the importance of lipids in the process of cryopreservation, the phospholipid status in oocytes and embryos before and after freezing has not been investigated. In this study, we employed mass spectrometric analysis to examine if vitrification has an effect on the phospholipid profiles of mouse oocytes. Freshly prepared metaphase II mouse oocytes were vitrified using copper grids and stored in liquid nitrogen for 2 weeks. Fresh and vitrified-warmed oocytes were subjected to phospholipid extraction procedure. Mass spectrometric analyses revealed that multiple species of phospholipids are reduced in vitrified-warmed oocytes. LIFT analyses identified 31 underexpressed and 5 overexpressed phospholipids in vitrified mouse oocytes. The intensities of phosphatidylinositol (PI) {18∶2/16∶0} [M−H]− and phosphatidylglycerol (PG) {14∶0/18∶2} [M−H]− were decreased the most with fold changes of 30.5 and 19.1 in negative ion mode, respectively. Several sphingomyelins (SM) including SM {d38∶3} [M+H]+ and SM {d34∶0} [M+K]+ were decreased significantly in positive ion mode. Overall, the declining trend of multiple phospholipids demonstrates that vitrification has a marked effect on phospholipid profiles of oocytes. These results show that the identified phospholipids can be used as potential biomarkers of oocyte undergoing vitrification and will allow for the development of strategies to preserve phospholipids during oocyte cryopreservation.
The deuterium, a frequently used
stable isotope in isotopic labeling
for quantitative proteomics, could deteriorate the accuracy and precision
of proteome quantification owing to the retention time shift of deuterated
peptides from the hydrogenated counterpart. We introduce a novel three-plexed
peptide “diethylation” using only 13C isotopologues
of acetaldehyde and demonstrate that the accuracy and precision of
our method in proteome quantification are significantly superior to
the conventional deuterium-based dimethylation labeling in both a
single-shot and multidimensional LC–MS/MS analysis of the HeLa
proteome. Furthermore, in time-resolved profiling of Xenopus
laevis early embryogenesis, our 3-plexed diethylation
outperformed isobaric labeling approaches in terms of the quantification
accuracy or the number of protein identifications, generating more
than two times more differentially expressed proteins. Our cost-effective
and highly accurate 3-plexed diethylation method could contribute
to various types of quantitative proteomics applications in which
three of multiplexity would be sufficient.
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