Abstract
The unicellular alga Chlamydomonas reinhardtii is a model photosynthetic organism for the study of microalgal processes. Along with genomic and transcriptomic studies, proteomic analysis of Chlamydomonas has led to an increased understanding of its metabolic signaling as well as a growing interest in the elucidation of its phosphorylation networks. To this end, mass spectrometry-based proteomics has made great strides in large-scale protein quantitation as well as analysis of posttranslational modifications (PTMs) in a high-throughput manner. An accurate quantification of dynamic PTMs, such as phosphorylation, requires high reproducibility and sensitivity due to the substoichiometric levels of modified peptides, which can make depth of coverage challenging. Here we present a method using TiO2-based phosphopeptide enrichment paired with label-free LC-MS/MS for phosphoproteome quantification. Three technical replicate samples in Chlamydomonas were processed and analyzed using this approach, quantifying a total of 1775 phosphoproteins with a total of 3595 phosphosites. With a median CV of 21% across quantified phosphopeptides, implementation of this method for differential studies provides highly reproducible analysis of phosphorylation events. While the culturing and extraction methods used are specific to facilitate coverage in algal species, this approach is widely applicable and can easily extend beyond algae to other photosynthetic organisms with minor modifications.
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References
Harris EH (2001) Chlamydomonas as a model organism. Annu Rev Plant Physiol Plant Mol Biol 52:363–406
Hu Q, Sommerfeld M, Jarvis E et al (2008) Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J 54(4):621–639
Merchant SS, Prochnik SE, Vallon O et al (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318:245–250
Zones JM, Blaby IK, Merchant SS et al (2015) High-resolution profiling of a synchronized diurnal transcriptome from Chlamydomonas reinhardtii reveals continuous cell and metabolic differentiation. Plant Cell 27:2743–2769
Miller R, Wu G, Deshpande RR et al (2010) Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism. Plant Physiol 154:1737–1752
Wang H, Alvarez S, Hicks LM (2012) Comprehensive comparison of iTRAQ and label-free LC-based quantitative proteomics approaches using two Chlamydomonas reinhardtii strains of interest for biofuels engineering. J Proteome Res 11:487–501
Roustan V, Bakhtiari S, Roustan P-J et al (2017) Quantitative in vivo phosphoproteomics reveals reversible signaling processes during nitrogen starvation and recovery in the biofuel model organism Chlamydomonas reinhardtii. Biotechnol Biofuels 10:280. https://doi.org/10.1186/s13068-017-0949-z
Krebs EG, Fischer EH (1955) Phosphorylase activity of skeletal muscle extracts. J Biol Chem 216:113–120
Fischer EH, Krebs EG (1955) Conversion of phosphorylase b to phosphorylase a in muscle extracts. J Biol Chem 216:121–132
Eriksson J, Fenyö D (2010) Modeling experimental design for proteomics. Methods Mol Biol 673:223–230
Blackburn K, Goshe MB (2009) Challenges and strategies for targeted phosphorylation site identification and quantification using mass spectrometry analysis. Brief Funct Genomic Proteomic 8:90–103
Dunn JD, Reid GE, Bruening ML (2010) Techniques for phosphopeptide enrichment prior to analysis by mass spectrometry. Mass Spectrom Rev 29:29–54
Kokubu M, Ishihama Y, Sato T et al (2005) Specificity of immobilized metal affinity-based IMAC/C18 tip enrichment of phosphopeptides for protein phosphorylation analysis. Anal Chem 77:5144–5154
Ruprecht B, Koch H, Medard G et al (2015) Comprehensive and reproducible phosphopeptide enrichment using iron immobilized metal ion affinity chromatography (Fe-IMAC) columns. Mol Cell Proteomics 14:205–215
Larsen MR, Thingholm TE, Jensen ON et al (2005) Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics 4:873–886
Tsai C-F, Wang Y-T, Chen Y-R et al (2008) Immobilized metal affinity chromatography revisited: pH/acid control toward high selectivity in phosphoproteomics. J Proteome Res 7:4058–4069
Ye J, Zhang X, Young C et al (2010) Optimized IMAC protocol for phosphopeptide recovery from complex biological samples. J Proteome Res 9:3561–3573
Aryal UK, Ross ARS (2010) Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry. Rapid Commun Mass Spectrom 24:219–231
Werth EG, McConnell EW, Lianez IC et al (2019) Investigating the effect of target of rapamycin kinase inhibition on the Chlamydomonas reinhardtii phosphoproteome: from known homologs to new targets. New Phytol 221:247–260
Neilson KA, Ali NA (2011) Less label, more free: approaches in label-free quantitative mass spectrometry. Proteomics 11:535–553
Bantscheff M, Schirle M, Sweetman G et al (2007) Quantitative mass spectrometry in proteomics: a critical review. Anal Bioanal Chem 389:1017–1031
Werth EG, McConnell EW, Gilbert TSK et al (2017) Probing the global kinome and phosphoproteome in Chlamydomonas reinhardtii via sequential enrichment and quantitative proteomics. Plant J 89:416–426
Wang H, Gau B, Slade WO et al (2014) The global phosphoproteome of Chlamydomonas reinhardtii reveals complex organellar phosphorylation in the flagella and thylakoid membrane. Mol Cell Proteomics 13:2337–2353
Hutner SH, Provasoli L, Schatz A et al (1950) Some approaches to the study of the role of metals in the metabolism of microorganisms. Proc Am Philos Soc 94:152–170
VizcaÃno JA, Côté RG, Csordas A et al (2013) The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res 41:D1063–D1069
Acknowledgments
This research was supported by a National Science Foundation CAREER award (MCB-1552522) awarded to L.M.H. NSF MRI (CHE-1726291) supported the purchase of the Q-Exactive HF-X mass spectrometer, and we thank Dr. Brandie Ehrmann for training on the HF-X instrument.
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Ford, M.M., Lawrence, S.R., Werth, E.G., McConnell, E.W., Hicks, L.M. (2020). Label-Free Quantitative Phosphoproteomics for Algae. In: Jorrin-Novo, J., Valledor, L., Castillejo, M., Rey, MD. (eds) Plant Proteomics. Methods in Molecular Biology, vol 2139. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0528-8_15
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DOI: https://doi.org/10.1007/978-1-0716-0528-8_15
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