Exploring the phosphoproteome profiles duringXenopusegg activation by calcium stimulation using a fully automated phosphopeptide purification system

Abstract

To explore the phosphoproteome profiles during Xenopus egg activation by Ca 2+ -stimulation, an automated phosphopeptide purification system involving a titania column was improved by introducing 4-step elution with phosphate buffers. The number of detected phosphopeptides in the tryptic digest of a Xenopus egg cytosol fraction on mass spectrometry (MS) was increased 1.5-fold and the percentage of multiply phosphorylated peptides increased from 17 to 24% with introduction of the 4-step elution method. Phosphopeptides were purified by the improved method from tryptic digests of cytosol fractions of Xenopus eggs without and with a Ca 2+ -stimulus, and then, analysed by MS. One thousand three hundred and seventy-five and 994 phosphopeptides were reproducibly detected on duplicate MS, respectively. They included 818 and 437 phosphopeptides specific to each digest, respectively. A method involving isobaric tags for relative and absolute quantitation (iTRAQ) was also applied to compare the phosphorylation levels in Xenopus eggs without and with a Ca 2+ -stimulus, the ratios for 112 phosphopeptides in tryptic digests of these egg cytosol fractions being obtained. It was suggested from all the results that the phosphorylation sites and levels change during Xenopus egg activation for many known and unknown sites on structural proteins, signalling related proteins, cell cyclerelated proteins and others.Keywords: egg/phosphoproteome/proteome/titania/ Xenopus.Abbreviations: 4EBP, eukaryotic translation initiation factor 4E binding protein; iTRAQ, isobaric tags for relative and absolute quantitation; LC MS/MS, nano-flow liquid chromatography-linear ion trap/ Orbitrap tandem mass spectrometry; MAPK, mitogen-activated protein kinase; MS, mass spectrometry; mTOR, mammalian target of rapamycin; m/z, massto-charge ratio; ODS, octadecylsilica; TFA, trifluoroacetic acid.Xenopus eggs have been used in studies on egg maturation, egg activation and other mechanisms, which facilitated the progress of developmental biology (1, 2). It has been shown that signalling pathways including protein phosphorylation and dephosphorylation participate greatly in these regulation mechanisms (1, 3, 4). To clarify the complicated regulation mechanism of egg activation, comprehensive analysis of protein phosphorylation during egg activation is a powerful means. The cell cycle of Xenopus eggs is arrested at the second meiotic metaphase. So, Xenopus eggs without Ca 2+ -stimulation show meiotic metaphase properties. When eggs are activated by Ca 2+ -stimulation to mimic fertilization, they show synthetic phase properties. So, we can analyse the changes of protein phosphorylation during activation of Xenopus eggs by comparison of the phosphorylation in Ca 2+ -unstimulated and Ca 2+ -stimulated eggs (5). In this study, therefore, we obtained phosphoproteome profiles of Xenopus egg cytosol fractions without and with Ca 2+ -stimulation as a basis for understanding the egg activation mechanism.To obtain high quality phosphoproteome data, i) determination of a ...