Overview of the Generation of the Three Thylakoid Subproteomes and Their Protein Separation and Identification by MS.
(A) Sequential extraction of thylakoid membrane proteins based on membrane affinity and solubility in organic solvents. Yeda press–treated purified thylakoid membranes were incubated with Na₂CO₃, and released proteins were collected. Residual peripheral thylakoid proteins in these Na₂CO₃-stripped thylakoid membranes were extracted by incubation with Triton X-114, followed by two-phase separation. The water-soluble proteins in the upper phase were collected. These two water-soluble proteomes were separated on 2-DE gels, with IPG strips in the first dimension, followed by SDS-PAGE in the second dimension. To analyze the hydrophobic thylakoid proteins, the Na₂CO₃-stripped thylakoids were solubilized in SDS and sequentially extracted with organic solvents, as explained in (B). The different fractions were separated by RP-HPLC or by SDS-PAGE.
(B) Scheme of fractionation and identification of the hydrophobic membrane proteome by sequential organic solvent extraction, followed by 1-DE PAGE or RP-HPLC and identification by MS analysis. Thylakoid membranes were stripped of lumenal and peripheral proteins with chaotropic agents and sonication and dissolved in Laemmli buffer before organic solvent extraction. Membranes first were dissolved in Laemmli solubilization buffer, followed by extraction in 100% acetone to remove the large amount of chlorophylls and carotenoids. These chromophores would otherwise interfere with further analysis. The remaining protein population, mostly devoid of pigments, was further fractionated by extraction with a mixture of C/M in a ratio of 9:1. This resulted in a three-phase separation with one subset of proteins fractionating to the upper, methanol-enriched phase, one set of proteins dissolving in the lower, chloroform-enriched phase, and a fraction of proteins accumulating in the interphase. The interphase was collected and further extracted by a C/M mixture in a ratio of 1:1, which yielded a subset of C/M 1:1 soluble proteins and a C/M 1:1 insoluble pellet (rest).
(C) Protein gel blot analysis. Partitioning of the lumenal peripheral protein OEC23 was assessed by protein gel blot analysis. The presence of chloroplast envelopes in the starting material for the proteome analysis in this study was determined by blotting with Toc75, a well-known envelope protein. Lane 1, intact chloroplasts; lane 2, Yeda press–treated thylakoid membranes; lane 3, Na₂CO₃-stripped, Yeda press–treated thylakoids; lane 4, Na₂CO₃-extracted thylakoid proteins. Thirty micrograms of chlorophyll was loaded for lanes 1, 2, and 3. Lane 4 contained Na₂CO₃-extracted proteins from Yeda press–treated thylakoids equivalent to 30 μg of chlorophyll.

Distribution of Identified Proteins Over the Different Fractions.
(A) Distribution of the identified proteins over the different fractions identified from 2-DE gels. Overlap of the different protein sets is shown.
(B) Stacked bar diagram with the distribution of identified proteins (separated in soluble and TM proteins) over the Yeda press fraction (lumenal enriched), the Na₂CO₃ fraction, the Triton X-114 fraction, and the eight organic solvent fractions. 1, Lumenal-enriched (Yeda press); 2, Triton X-114; 3, Na₂CO₃ fraction; 4, acetone; 5, C/M 9:1 lower phase; 6, C/M 9:1 upper phase; 7, C/M 1:1; 8, residual insoluble pellet; 9, C/M 9:1 total; 10, C/M 1:1 RP-HPLC. Membrane proteins are shown in black.
(C) Distribution of the identified proteins over the 2-DE gels and organic solvent fractions. Overlap between the proteins identified from the 2-DE gels and organic solvents is shown.

Identification of Paralogues of the LHCII Family.
Alignment and identification of processed LHCII-2.1 (At2g05100), LHCII-2.2 (At2g05070), and LHCII-2.4 (At3g27690) (predicted cTPs were removed). One unique peptide was identified for LHCII-2.2 (boxed in green) and one for LHCII-2.4 (boxed in blue). Many other peptides were identified that matched to all three LHC proteins, some are indicated (boxed in black). Arrows indicate amino acid residues that help to identify the different paralogues.

Functional Classification of the Identified Proteins.
(A) Functional classification of all 198 identified proteins. 1, nonchloroplast; 2, translation (ribosomal subunits, RNA binding proteins, and PSRPs); 3, chaperones, protein insertion and assembly, proteases, and protein isomerases; 4, primary and secondary metabolism; 5, thylakoid photosynthetic apparatus (including FNR and PC); 6, alternative electron flow; 7, oxidative stress (Fe-SOD, peroxyredoxins, thioredoxins, ascorbate peroxidase, fibrillins, and Ohp1 and Ohp2); 8, new chloroplast proteins without obvious function; 9, other lumenal proteins without obvious function.
(B) Identified proteins of the photosynthetic machinery, as compared with all predicted proteins and grouped with (black) or without (blue) TM, for PSII (including LHCII), PSI (including LHCI), cytochrome b6f complex, and ATP synthase. The striped areas represent the unidentified proteins.

2-DE Gels of the Na₂CO₃ and Triton X-114–Extracted Thylakoid Proteomes, Stripped by Yeda Press.
Data from the gel identifications are summarized in Table 1 and in Table 1 in the supplemental data online. Details of experimental coordinates and MS identification can be found in Tables 2 and 3 in the supplemental data online.
(A) Analysis of the Na₂CO₃-extracted thylakoid proteome stripped of lumenal proteins by three cycles of Yeda press. Sypro Ruby–stained 2-DE gel (pI range 3 to 10 and inset 6 to 11) of peripheral proteins extracted by Na₂CO₃.
(B) and (C) Comparison between the Na₂CO₃ (B) and Triton X-114 (C)–extracted proteomes and the identified proteins in the pI range of 4 to 6.
(B) and (C) Comparison between the Na₂CO₃ (B) and Triton X-114 (C)–extracted proteomes and the identified proteins in the pI range of 3 to 6.
(D) and (E) Close-ups of the region around the OEC33 cluster of gels stained by Sypro Ruby (D) or by silver nitrate (E). The region around this cluster contains a number of fibrillins and they are partially masked by OEC33 and more clearly visible in the Triton X-114 map. Sypro Ruby staining gives superior detection because saturation of staining, as seen in the silver stained maps, is avoided.

Separation and Identification of the Acetone and C/M-Soluble Thylakoid Proteins.
(A) High-resolution gradient 1-DE Tricine-PAGE of the five protein fractions from sequential organic solvent fractionation of Na₂CO₃-stripped thylakoid membranes. The five fractions are acetone-soluble proteins (panel at left), C/M 9:1 lower phase (9:1 L), C/M 9:1 upper phase (9:1 U), C/M 1:1 soluble proteins (1:1), and the residual insoluble pellet (R). Gels were stained by silver nitrate in the acetone fraction and by Coomassie blue for the other fractions. Proteins identified in the acetone fraction are cytb559β (Atcg00570), psbL (Atcg00560), psbW (At2g30570), psbX (At2g06520), psbZ/ycf9 (Atcg00300), psaI/subunit VIII (Atcg00530), and CFo-III (Atcg00140).
(B) Proteins extracted by C/M 1:1 were separated using RP-HPLC, and a representative chromatogram is shown. Fraction number and the percentage of solvent B are indicated. Protein identities of a number of proteins are indicated. The absorbance was monitored at 280 nm (rather than 214 nm) because isopropanol absorbs strongly at 205 nm; thus, the peaks are not quantitative.

Linking the Physical-Chemical Properties of the Identified Processed (Mature) Proteins to the Sequential Fractionation.
Cleavable presequences were removed.
(A) Scatter plot of GRAVY index and number of TMs per amino acid for all identified proteins with predicted or identified TMs.
(B) Frequency distribution of the GRAVY index for proteins identified in four organic solvent fractions: acetone, C/M 1:9 U+L, C/M 1:1, and the rest fraction.
(C) Correlation between protein length (in amino acids) and GRAVY index for those photosynthetic proteins that were identified (open squares) and those that were not identified (closed squares).