## 4 Materials and Methods
### Proteome Sampling, Sequencing, and Bioinformatics  
The microalgal cells of _H. pluvialis_ were added with SDT lysate, homogenized, and broken, and boiled in hot water at 100 \({}^{\circ}\)C for 10 min. The samples were centrifuged for 15 min at 14,000 g with the supernatant collected. BCA method was used for protein quantification [89]. Filter aided proteome preparation (FASP) was used for enzymatic hydrolysis of proteins [90]. The peptides were desalted by C18 cartridge. After freeze-drying, 0.1% formic acid solution was added for re-dissolution, and the peptides were quantified. The peptide mixture was graded by Agilent 1260 infinity II HPLC system (Agilent Technologies, Santa Clara, CA, USA).  
The proteomic sequencing was performed by Hangzhou Lianchuan Biotechnology Co., Ltd. (LC Sciences, Houston, TX, USA). The original mass spectrometry data were combined and analyzed by Spectronaut Pulsar X (version 12, Biognosys AG, Schlieren, Switzerland). TransDecoder ([[https://github.com/TransDecoder/TransDecoder/](https://github.com/TransDecoder/TransDecoder/)]([https://github.com/TransDecoder/TransDecoder/](https://github.com/TransDecoder/TransDecoder/)), accessed on 16 March 2020) was used to predict the transcript of unigenes to obtain the protein sequences and to establish the spectrum database by Spectronaut Pulsar X (version 12, Biognosys AG, Schlieren, Switzerland). Trypsin enzymolysis was set to allow two missing sites, with the following searching parameters included: (1) fixed modification: carbamidomethyl (c); (2) variable modification: oxidation (m) oxidation; and (3) N-terminal acetylation of acetyl (protein N-term) protein. A total of 1 ug of peptides from each sample was added with iRT peptides to mix and then to inject for separation using Nano-LC. Data were analyzed by online electrospray tandem mass spectrometry. The complete liquid-mass tandem system contained both the liquid system based on the Easy nLC system (Thermo Fisher Scientific, Waltham, MA, USA) and the mass spectrometry system using the Orbitrap Exploris 480 (Thermo Fisher Scientific, Waltham, MA, USA). Buffer solution A solution was 0.1% formic acid aqueous solution and B solution was 0.1% formic acid acetonitrile aqueous solution (80% acetonitrile). The sample was separated by a non-linearly increasing gradient in an analytical column (Nano Technology Column, 18 cm C18 column, with 1.9 \(\upmu\)m C18 Resin, Catalog Number 26350-3) at a flow rate of 300 nL/min: 0-1 min, 2% B to 8% B; 1-99 min, 8% B to 27% B; 99-113 min, 27% B to 35% B; 113-117 min, 35% B to 100% B; and 117-120 min, 100% B. The electrospray voltage was set to 2056 V.  
Raw data of DIA were processed and analyzed by Spectronaut 14.6 (Biognosys AG, Schlieren, Switzerland) with default settings with the Retention time prediction type set to dynamic iRT. Data extraction was determined by Spectronaut Pulsar X based on the extensive mass calibration. Spectronaut 14.6 was used to determine the ideal extraction window dynamically depending on iRT calibration and gradient stability. Q-value (FDR) cutoff on precursor and protein level was applied at 1%. Decoy generation was set to mutated which was similar to scrambled but only applied a random number of AA position swamps (min = 2, max = length/2). All selected precursors passing the filters were used for quantification. MS2 interference was used to remove most interfering fragment ions except for the 3 least interfering ones. The average top 3 filtered peptides which passed the 1% Q-value cutoff were used to calculate the major group quantities. Differentially expressed proteins were filtered based on Student's \(t\)-test (\(p<0.05\) and fold change \(>1.5\)).  
GO annotation based on unigenes was performed using Blast2 GO [91]. KEGG enrichment analysis was performed based on the KEGG automatic annotation server (KAAS) [92].