In plants chloroplasts, the lumen of the thylakoid membrane houses proteins that are vital for photosynthetic electron transport, including water-splitting at photosystem (PS) II and shuttling of electrons from cytochrome b6f to PSI. Other lumen proteins maintain photosynthetic activity through biogenesis and turnover of PSII complexes. Although all lumen proteins are soluble, these known details have highlighted interactions of some lumen proteins with thylakoid membranes or thylakoid-intrinsic proteins. Meanwhile, the functional details of most lumen proteins, as well as their distribution between the soluble and membrane-associated lumen fractions, remain unknown. The current study isolated the soluble free lumen (FL) and membrane-associated lumen (MAL) fractions from Arabidopsis thaliana, and used mass spectrometry-based proteomics methods to analyse the contents of each proteome. These results identified lumenal proteins, and clearly distinguished the difference between the FL and MAL proteomes. The most abundant proteins in the FL fraction were involved in PSII assembly and repair, while the MAL proteome was enriched in proteins that support of the oxygen-evolving complex (OEC). Selected Reaction Monitoring (SRM) was employed to assess the ratio bewteen PsbO1 and PsbO2 in FL and MAL proteomes. Novel proteins, including a new PsbP domain-containing isoform, as well as several novel post-translational modifications and N-termini, are reported, and bi-dimensional separation of the lumen proteome identified several potential multi-protein oligomers in the thylakoid lumen.

For analysis in mass spectrometry (MS), lumen proteins have been analyzed by SDS-PAGE. Gels were then stained with silver nitrate. Then bands containing PsbO proteins were excised and subjected to in-gel tryptic digestion. The excised gel pieces (~ 1 mm3) were shrunk completely by incubating 3 times for 10 min in 100 µl of 100% acetonitrile (ACN), incubated in 25 mM Dithiothreitol (DTT)/100 mM NH4HCO3 for 30 min at 56°C in a Thermomixer (Eppendorf) and, after shrinking as above, alkylated for 20 min with 55 mM of iodacetamide (IAA)/ 100 mM NH4HCO3 at 22°C in the dark. After two consecutive washes alternating 100% ACN and 100 mM NH4HCO3, the gel pieces were completely shrunk as described above. For protein digestion, the gel pieces were completely re-hydrated in a solution of 40 mM NH4HCO3/10% ACN with either of 0.01 (10 µl) of trypsin (Sequencing Grade Modified Trypsin V5111, Promega, Madison) at 4°C. After adding 20 µl of 40 mM NH4HCO3/10% ACN, the gel pieces were incubated at 37°C in the Thermomixer (Eppendorf) shaking at 600 rpm for 13-14 hours. The tryptic peptides have been extracted by an equal volume of 100% ACN and incubating for 15 min at 37°C. The supernatant has been collected in a new Eppendorf and the gel pieces have been incubated two more times for 15 min with 50% ACN/5% formic acid (FA) to collect the remaining peptides. The supernatants have been pooled and dried in a speed-vac for 3 hours, and dissolved in 2% FA to be analyzed by nLC-ESI MS/MS. For mass spectrometry analysis, the peptides samples were separated on a C18 precolumn (5 × 0.3 mm, PepMap C18, LC Packings) and a C18 nanocolumn (15 cm × 75 μm, Magic 5 μm, 200 Å C18, Michrom BioResources, Sacramento, CA) with a flow rate of 300 nL/min using 0.1% FA, 2% ACN as a buffer A and 0.1% FA, 80% ACN as a buffer B. Gradients comprised 3 % to 43 % solvent B over 13 min, followed by 43 % to 100 % solvent B for 2 min, and finally 100 % solvent B for 5 min. The mass spectrometer used was a Q-Exactive (Thermo Fisher Scientific), with resolution set to 140000, scan range 300 to 1800 m/z for MS1, and 17500 with scan range 200 to 2000 m/z for MS2. Dynamic exclusion was set to 20 s and up to 20 masses with m/z ≥2+ were selected for MS2 for every MS1 scan. The obtained spectra have been analyzed via Mascot (v.2.6.1) using Proteome Discover 2.2 (Thermo Scientific) and the embedded ptmRS algorithm to validate the identified phosphorylations. The variable modifications allowed were Met oxidation, protein N-term acetylation, Ser, Thr and Tyr phosphorylation. Cys carbamidomethylation was set as fixed modification. Posterior error probabilities was evaluated by validating PSMs with Mascot identity threshold for strict and relaxed significance and allowing only PSMs with medium or high confidence. The MS/MS spectra were matched against a non-redundant Arabidopsis proteome database (TAIR10; https://www.arabidopsis.org/) supplemented with most common contaminants (35,502 entries in total). Quantification of PsbO1 and PsbO2 isoforms was performed via selected reaction monitoring (SRM). The MS/MS spectra of unique (proteotypic) PsbO1 and PsbO2 peptides were used to generate a spectral library and then the transitions to be used to detect proteotypic PsbO1 and PsbO2 peptides in SRM and the transitions used for quantification. The PsbO1/PsbO2 ratio was calculated as the relative percentage of each isoform to the sum of the integrated peak area of the three most intense transitions of every proteotypic PsbO1 or PsbO2 peptide

Fifty rosettes of six weeks old Arabidopsis thaliana (L.) Heynh. Col-0 plants grown under 100 µmoles photons m-2 s-1 in a phytotron (8 h light/16 h darkness) were harvested and, under dim light in a cold room, plant tissue was ground in ice-cold grinding buffer containing 330 mM sorbitol, 50 mM Hepes-KOH (pH 7.8), 10 mM KCl, 10 mM EDTA and 10 mM NaF (Buffer A), followed by filtration through Miracloth (Millipore) mesh. The filtrate was centrifuged twice at 1000 × g at 4 °C for 1 min, the supernatant discarded and the pellet re-suspended in buffer A with 2.5 mM EDTA and 5 mM NaCl. The suspension was diluted to a chlorophyll concentration of 0.2 mg ml-1 in shock buffer containing 10 mM Na4P2O7 (pH 7.8) and 10 mM NaF, and homogenised thoroughly with a glass homogeniser, before centrifugation at 7500 × g at 4 °C for 5 min. The resulting thylakoid membrane pellet was washed once with shock buffer, twice with wash buffer (2 mM Tricine pH 7.8, 300 mM sucrose), and once with fragmentation buffer (30 mM NaH2PO4 pH 7.8, 50 mM NaCl, 5 mM MgCl2, 100 mM sucrose) by repeated centrifugations at 7500 × g at 4 °C for 5 min, and then re-suspended in fragmentation buffer with 10 mM NaF and EDTA-free protease inhibitor cocktail (Merck). The solution was passed slowly through a cold Yeda press twice at N2 pressure of 10 MPa to invert the membranes, centrifuged twice at 200 000 × g at 2 °C for 60 min, and the supernatant (free lumen-FL-proteome) was removed and stored. The membrane pellet was washed in fragmentation buffer and centrifuged at 200 000 × g at 2 °C for 60 min, re-suspended to 1.0 mg ml-1 chlorophyll in stripping buffer (2.6 M urea, 0.2 M NaCl, 50 mM CaCl2, 20 mM MES pH 6.0, 0.3 M sucrose), incubated on ice for 60 min, and then centrifuged twice at 200 000 × g at 2 °C for 60 min, to obtain the ‘membrane-associated lumen’ (MAL) proteome supernatant.