For modeling approaches in systems biology, knowledge of the absolute abundances of cellular proteins is essential. One way to gain this knowledge is the use of quantification concatamers (QconCATs), which are synthetic proteins consisting of proteotypic peptides derived from the target proteins to be quantified. The QconCAT protein is labeled with a heavy isotope upon expression in E. coli and known amounts of the purified protein are spiked into a whole cell protein extract. Upon tryptic digestion, labeled and unlabeled peptides are released from the QconCAT protein and the native proteins, respectively, and both are quantified by LC-MS/MS. The labeled Q-peptides then serve as standards for determining the absolute quantity of the native peptides/proteins. Here, we have applied the QconCAT approach to Chlamydomonas reinhardtii for the absolute quantification of the major proteins and protein complexes driving photosynthetic light reactions in the thylakoid membranes and carbon fixation in the pyrenoid. We found that with 25.2 attomol/cell the Rubisco large subunit makes up 6.6% of all proteins in a Chlamydomonas cell and with this exceeds the amount of the small subunit by a factor of 1.56. EPYC1, which links Rubisco to form the pyrenoid, is eight times less abundant than RBCS, and Rubisco activase is 32-times less abundant than RBCS. With 5.2 attomol/cell, photosystem II is the most abundant complex involved in the photosynthetic light reactions, followed by plastocyanin, photosystem I and the cytochrome b6/f complex, which range between 2.9 and 3.5 attomol/cell. The least abundant complex is the ATP synthase with 2 attomol/cell. While applying the QconCAT approach, we have been able to identify many potential pitfalls associated with this technique. We analyze and discuss these pitfalls in detail and provide an optimized workflow for future applications of this technique.

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11The productivity of plants and microalgae needs to be increased to feed the growing world population 12 and to promote the development of a low-carbon economy. This goal can be achieved by improving 13 photosynthesis via genetic engineering. In this study, we have employed the Modular Cloning strategy 14to overexpress the Calvin-Benson cycle (CBC) enzyme sedoheptulose-1,7 bisphosphatase (SBP1) up 15 to 3-fold in the unicellular green alga Chlamydomonas reinhardtii. The protein derived from the 16 nuclear transgene represented ~0.3% of total cell protein. Photosynthetic rate and growth were 17 significantly increased in SBP1-overexpressing lines under high-light and high-CO2 conditions. 18Absolute quantification of the abundance of all other CBC enzymes by the QconCAT approach 19 revealed no consistent differences between SBP1-overexpressing lines and the recipient strain. This 20 analysis also revealed that the eleven CBC enzymes represent 11.9% of total cell protein in 21Chlamydomonas. Here the range of concentrations of CBC enzymes turned out to be much larger than 22 estimated earlier, with a 128-fold difference between the most abundant CBC protein (rbcL) and the 23 least abundant (triose phosphate isomerase). Accordingly, the concentrations of the CBC intermediates 24 are often but not always higher than the binding site concentrations of the enzymes for which they act 25 as substrates. The enzymes with highest substrate to binding site ratios might represent good candidates 26 for overexpression in subsequent engineering steps. 27 28 29 30 31 Overexpression of SBP1 in Chlamydomonas 2