Updated project metadata. The pyrenoid, a single microcompartment of the chloroplasts of most algae and hornworts, is the major site of CO2 fixation. To compensate the limited availability of CO2 in aquatic environments due to slower CO2 diffusion in water than air, the RubisCO-containing pyrenoid is involved in a carbon concentrating mechanism (CCM) and promotes the carboxylase activity rather than the oxygenase activity of RubisCO, the Calvin-Benson cycle enzyme catalyzing the first step of CO2 fixation. Data in the literature suggest that pyrenoid can have other functions in addition to CO2 fixation. To decipher its functions, the characterization of the pyrenoid proteome in the microalga Chlamydomonas reinhardtii was undertaken. Pyrenoid-enriched fractions from two independent biological cultures (sample1 and sample2) were obtained from cells (WT-cell) or isolated chloroplasts (WT-cp) either from the wild-type (WT) strain or from pyrenoid-deficient Chlamydomonas strains (MX-CW15 and SS-AT) and analyzed by nLC-MS/MS. Substractive proteomic analysis allowed the identification of contaminant proteins and the characterization of the pyrenoid proteome.