Isolute c18 specartridges

PSII-I complexes were purified and desalted using Isolute C18 SPE cartridges (Biotage, Sweden). The columns were first washed and equilibrated, the sample diluted in 0,1% trifluoroacetic acid (TFA) and loaded onto the column. After washing with 2 ml 0.1% TFA, the proteins were eluted with 500 µl 80% acetonitrile (ACN), 20% water. The organic fraction was lyophilized in a vacuum concentrator (Eppendorf, Germany), reconstituted in 0.1% TFA and mixed in a 1:1 ratio with HCCA matrix solution (HCCA (alpha-cyano-4-hydroxycinnamic acid) saturated in 50% ACN, 50% water and supplemented with 0.1% TFA). Subsequently, 1 µl aliquots of the mixture were deposited on a ground steel MALDI target and allowed to dry and crystallize at ambient conditions. MS and MS/MS spectra were acquired on a prototype rapifleX MALDI-TOF/TOF (Bruker Daltonics, Germany) in positive ion mode. The Compass 2.0 (Bruker Daltonics, Germany) software suite was used for spectra acquisition and processing (baseline subtraction, smoothing, peak picking), a local Mascot server (version 2.3, Matrixscience, UK) was used for database searches against the T. elongatus proteome (UniProt, retrieved 4/2019) and BioTools 3.2 (Bruker Daltonics) was used for manual spectrum interpretation, de novo sequencing and peak annotation.