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Phaeocystis globosa is an important unicellular eukaryotic alga that can also form colonies. P. globosa can cause massive harmful algal blooms and plays an important role in the global carbon or sulfur cycling. Thus far, the ecophysiology of P. globosa has been investigated by numerous studies. However, the proteomic response of P. globosa to nitrogen depletion remains largely unknown. We compared four protein preparation methods of P. globosa for two-dimensional electrophoresis (2-DE) (Urea/Triton X-100 with trichloroacetic acid (TCA)/acetone precipitation; TCA/acetone precipitation; Radio Immuno Precipitation Assay (RIPA) with TCA/acetone precipitation; and Tris buffer). Results show that the combination of RIPA with TCA/acetone precipitation had a clear gel background and showed the best protein spot separation effect, based on which the proteomic response to nitrogen depletion was studied using 2-DE. In addition, we identified six differentially expressed proteins whose relative abundance increased or decreased more than 1.5-fold (P<0.05). Most proteins could not be identified, which might be attributed to the lack of genomic sequences of P. globosa. Under nitrogen limitation, replication protein-like, RNA ligase, and sn-glycerol-3-phosphate dehydrogenase were reduced, which may decrease the DNA replication level and ATP production in P. globosa cells. The increase of endonuclease III and transcriptional regulator enzyme may affect the metabolic and antioxidant function of P. globosa cells and induce cell apoptosis. These findings provide a basis for further proteomic study of P. globosa and the optimization of protein preparation methods of marine microalgae. [ABSTRACT FROM AUTHOR]