Proteomics and phosphoproteomics analysis of Haematococcus pluvialis: An improved method to generate and interpret proteomics and phosphoproteomics data of a non-model species

Haematococcus pluvialis is a green microalga of commercial interests due to its ability to produce a high value ketocarotenoid, astaxanthin. As a non-model species that lacks a well annotated genome, omics analyses such as transcriptomics and proteomics analysis have often been used together with physiological and biochemical analysis to explore pathways of interest. However, interpretation of these datasets remains challenging. In this work, TMT-based proteomics and phosphoproteomics analyses were conducted on Haematococcus cells grown under favorable conditions (green stage biomass) and high-light stress conditions (red stage biomass). Phosphoproteins were enriched using titanium dioxide before LC-MS/MS analysis. Our proteomics and phosphoproteomics analyses identified 1394 proteins and 569 phosphosites on 366 phosphoproteins, respectively. Of these, 1315 proteins and 396 phosphosites on 314 phosphoproteins were quantifiable, among which 370 proteins and 121 phosphosites on 94 phosphoproteins were differentially expressed. Using an improved analysis pipeline that combines Blast2GO, KEGG, and DAVID to analyze differentially expressed proteins and phosphoproteins, total identified proteins increased from 255 to 322 and total identified phosphoproteins increased from 59 to 70, which were 26.28% and 18.64%, respectively, higher than with the UniProt analysis alone. Using this pipeline, a previously uncharacterized protein and phosphoprotein were identified as an ATPase subunit B and a phosphofructokinase, respectively, and further confirmed with translated genomic and transcriptomic data. This work provides the first example of phosphoproteomics analysis in H. pluvialis, while the proteomics and phosphoproteomics analysis pipelines described here may be useful to analyze omics data from other non-model algal species.

雨生红球藻（Haematococcus pluvialis）是一种兼具商业价值的绿色微藻，因其可合成高价值酮类类胡萝卜素虾青素（astaxanthin）而备受关注。作为缺乏完善注释基因组的非模式物种，转录组学（transcriptomics）、蛋白质组学（proteomics）等组学分析常与生理生化分析结合，用于探究目标代谢通路。然而，此类组学数据集的解读仍存在诸多挑战。
本研究针对在适宜培养条件（绿色生长期生物量）与高光胁迫条件（红色生长期生物量）下培养的雨生红球藻细胞，开展了基于串联质量标签（Tandem Mass Tag, TMT）的蛋白质组学与磷酸化蛋白质组学（phosphoproteomics）分析。磷酸化蛋白经二氧化钛富集后，进行液相色谱-串联质谱（Liquid Chromatography-Tandem Mass Spectrometry, LC-MS/MS）检测。
本研究的蛋白质组学与磷酸化蛋白质组学分析分别鉴定得到1394个蛋白质，以及366个磷酸化蛋白上的569个磷酸化位点。其中，可定量的蛋白质共1315个，314个磷酸化蛋白上的396个磷酸化位点；差异表达的蛋白质共370个，94个磷酸化蛋白上的121个磷酸化位点。
本研究采用整合了Blast2GO、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）与数据库注释可视化与整合发现工具（Database for Annotation, Visualization and Integrated Discovery, DAVID）的优化分析流程，对差异表达蛋白质和磷酸化蛋白质进行注释分析。经该流程处理后，总鉴定蛋白质数从仅使用通用蛋白质资源（Universal Protein Resource, UniProt）分析时的255提升至322，总鉴定磷酸化蛋白质数从59提升至70，分别较单一UniProt分析提升了26.28%与18.64%。
通过该分析流程，本研究分别将一个此前未被注释的蛋白质与磷酸化蛋白鉴定为ATP酶亚基B与磷酸果糖激酶，并通过翻译得到的基因组和转录组数据完成了验证。
本研究首次在雨生红球藻中开展磷酸化蛋白质组学分析，所建立的蛋白质组学与磷酸化蛋白质组学分析流程，可为其他非模式藻类物种的组学数据分析提供参考范式。