Identification of Reference and Biomarker Proteins in Chlamydomonas reinhardtii Cultured under Different Stress Conditions

Abstract

Reference proteins and biomarkers are important for the quantitative evaluation of protein abundance. Chlamydomonas reinhardtii was grown under five stress conditions (dark, cold, heat, salt, and glucose supplementation), and the OD750 and total protein contents were evaluated on days 0, 1, 2, 4, and 6 of culture. Antibodies for 20 candidate proteins were generated, and the protein expression patterns were examined by western blotting. Reference protein(s) for each treatment were identified by calculating the Pearson’s correlation coefficient (PCC) between target protein abundance and total protein content. Histone H3, beta tubulin 1 (TUB-1), ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RBCL), and mitochondrial F1F0 ATP synthase subunit 6 (ATPs-6) were the top reference proteins, because they were expressed stably under multiple stress conditions. The average relative-fold change (ARF) value of each protein was calculated to identify biomarkers. Heat shock protein 90B (HSP90B), flagellar associated protein (FAP127) and ATP synthase CF0 A subunit (ATPs-A) were suitable biomarkers for multiple treatments, while receptor of activated protein kinase C1 (RCK1), biotin carboxylase (BCR1), mitochondrial phosphate carrier protein (MPC1), and rubisco large subunit N-methyltransferase (RMT1) were suitable biomarkers for the dark, cold, heat, and glucose treatments, respectively.

Mutation of negative regulatory geneCEHC1encoding an FBXO3 protein results in normoxic expression ofHYDAgenes inChlamydomonas reinhardtii

“…The greatest differences in transcript levels observed between the CEHC1 and cehc1-1 strains were in the set of genes upregulated in the mutant; 89 genes were upregulated between 4- and >1×10 4 -fold in the mutant strain, whereas 37 were down-regulated, between 4- and 42-fold. Controls for the experiment included transcript levels for genes previously shown to have stable expression levels (Shi et al 2017; Zones et al 2015) (Supplementary Table 10). For six such genes, the cehc1-1 / CEHC1 ratio ranged from 1.5 to 0.7-fold.…”

Identifying new sperm Western blot loading controls

“…GAPDH, TUB, ACTB and Histone H3 have been used as reference proteins in many studies with sperm samples (Brohi et al., 2017; Govin et al., 2007; Hou et al., 2019; Lee et al., 2013), while these classical housekeeping proteins may be involved in cellular activities mentioned above. Consequently, it is noteworthy that these internal standards might not be suitable to be applied as normalisation standards, just as suggested in the same studies (Catalan et al., 2007; Gabrielsson et al., 2005; Perez‐Perez et al., 2012; Shi et al., 2017). Besides, preservation conditions might also affect RNA and protein levels in sperm samples (Mao et al., 2013).…”

“…Consequently, it is noteworthy that these internal standards might not be suitable to be applied as normalisation standards, just as suggested in the same studies (Catalan et al, 2007;Gabrielsson et al, 2005;Perez-Perez et al, 2012;Shi et al, 2017). Besides, preservation conditions might also affect RNA and protein levels in sperm samples (Mao et al, 2013).…”

“…Therefore, the consistency of the housekeeping protein is essential for accurate quantification of the target protein levels in WB. It is a consensus that an internal control is expressed at constant levels across samples within a certain study (Perez-Perez et al, 2012;Shi et al, 2017).…”

“…Therefore, the consistency of the housekeeping protein is essential for accurate quantification of the target protein levels in WB. It is a consensus that an internal control is expressed at constant levels across samples within a certain study (Perez‐Perez et al., 2012; Shi et al., 2017). The ubiquitous loading controls mainly include, but not limited to, the following proteins: glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, which plays a role in glycolysis and nuclear functions), β‐tubulin (TUB, a major constituent of microtubules), β‐actin (ACTB, which is involved in cell motility, structure, integrity and intercellular signalling) and Histone H3 (a core component of nucleosome), which are encoded by housekeeping genes.…”