{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Tabular":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD069946/sdrf-plants_BP.tsv"],"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD069946/checksum.txt"],"Raw":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD069946/20251009_003_S1025743_Fraction6.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD069946/20251009_002_S1025742_Fraction5.raw"],"Fasta":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD069946/2023-06-19-decoys-uniprot-compressed_true_download_true_format_fasta_query__28_28prote-2023.06.19-10.39.21.40.fasta"],"Other":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/02/PXD069946/FragPipe_output_DIA_seqlib_quant.7z"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["szeeman@ethz.ch"],"submitter":["Barbara Pfister"],"technology_type":["Mass Spectrometry","Data-independent acquisition","Bottom-up proteomics"],"software":[""],"submitter_keywords":["Tobacco","Lc-ms/ms","Maturase k","Size-exclusion chromatography","Dia","Chloroplast"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD069946"],"tissue":["Leaf"],"sample_protocol":["Homoplastomic Nicotiana tabacum (tobacco) lines modified to express NtMatK fused to a C-terminal 3x HA tag (line NtMatK C+; Zoschke et al. 2010; PNAS, 107, 3245–3250. https://doi.org/10.1073/pnas.0909400107) were grown for 24 days under 12-h light and 12-h dark cycles. Approximately 60 g of leaves were homogenized in 500 ml homogenization buffer (50 mM HEPES-KOH, pH 7.8, 330 mM sorbitol, 2 mM EDTA, 1 mM MgCl₂, 50 mM sodium ascorbate, 0.25% [w/v] BSA) using a blender. The homogenate was filtered through two layers of Miracloth (Calbiochem) and centrifuged at 2,000 g for 5 min at 4 °C to enrich for chloroplasts. The pellet was resuspended in 2.5 ml lysis buffer (50 mM HEPES-KOH, pH 7.8, 1 mM MgCl₂) and incubated on ice for 1 h to lyse the chloroplasts, followed by three rounds of grinding with a glass homogenizer. The homogenate was centrifuged at 100,000 g for 20 min at 4 °C, and the resulting supernatant was further concentrated using an Amicon Ultra-4 (10 kDa molecular weight cut off) filter column by centrifugation at 4,000 g at 4 °C until the volume was reduced to 3 ml. The proteins present in the concentrated soluble chloroplast extract were fractionated by size on a Superose 6 Increase 10/300 column on an ÄKTA Pure 25 system. Elution was performed with chloroplast lysis buffer at a flow rate of 0.25 ml min-1 and 1 ml subfractions were collected. Subfractions 1–6 and 7–11 were pooled as sample fractions 1 and 2, respectively, and reduced to 1 ml using an Amicon Ultra-4 (10 kDa molecular weight cut off) columns as above. Other subfractions were kept individually. Protein standards of the gel filtration calibration kit (Cytiva) were prepared and concentrated according to the manufacturer’s instructions. They were fractionated and eluted as the chloroplast extracts. For MS/MS analysis of, 300 µl of sample fractions 5 and 6 were precipitated in 80% (v/v) acetone. The protein pellet was resuspended in 45 µl 10 mM Tris (pH 8.0), 2 mM CaCl2, and the concentration was determined using the Lunatic UV/Vis polychromatic spectrophotometer (Unchained Labs). Proteins were reduced with 5 mM tris(2-carboxyethyl)phosphine and alkylated with 15 mM chloroacetamide at 30°C for 30 min, then digested using 250 ng Sequencing Grade Trypsin (Promega) overnight at 37°C. After drying the peptides, they were re-solubilized in 20 µL 3% (v/v) acetonitrile, 0.1% (v/v) formic acid and spiked in with  indexed Retention Time (iRT) peptides (Biognosys). Mass spectrometry analysis was performed on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) equipped with a Nanospray Flex Ion Source (Thermo Fisher Scientific) and coupled to an M-Class UPLC (Waters). Solvent composition at the two channels was 0.1% formic acid for channel A and 0.1% formic acid, 99.9% acetonitrile for channel B. Column temperature was 50°C. For each sample 200 ng of peptides were loaded on a commercial nanoEase MZ Symmetry C18 Trap Column (100Å, 5 µm, 180 µm x 20 mm, Waters) followed by a nanoEase MZ C18 HSS T3 Column (100Å, 1.8 µm, 75 µm x 250 mm, Waters). The peptides were eluted at a flow rate of 300 nL/min. After a 3 min initial hold at 5% B, a gradient from 5 to 22 % B in 40 min and 22 to 32% B in additional 10 min was applied. The column was cleaned after the run by increasing to 95 % B and holding 95 % B for 10 min prior to re-establishing loading condition for another 10 minutes.  For the analysis of the individual samples, the mass spectrometer was operated in data-independent mode (DIA). DIA scans covered a range from 400 to 960 m/z in windows of 8 m/z. The resolution of the DIA windows was set to 30’000, with a normalized AGC target value of 1’000%, the maximum injection time set to auto and a fixed normalized collision energy (NCE) of 30 %. Each instrument cycle was completed by a full MS scan monitoring 396 to 1’000 m/z at a resolution of 60’000."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["DIA-MS raw files were converted into mzML files using MSConvert (v3.0.2472-63d00b1). The MS data were further processed using MSFragger-DIA implemented in the computational platform FragPipe (v23.1) with DIA-NN (v2.1.0). Data was analysed using the DIA_SpecLib_Quant workflow, including spectral library built from the primary DIA data. Spectra were searched with MSFragger(v4.3) against the N. tabacum UniProt reference proteome (UP000084051) concatenated to its reversed decoyed database and supplemented with MKIP1 proteins annotated in N. sylvestris [A0A1U7XZG9_NICSY, A0A1U7Y1B8_NICSY] and common contaminants using default settings, i.e. allowing for one missed tryptic cleavage and setting carbamidomethylation of cysteine as fixed modification and methionine oxidation and acetylation of the protein N-terminus after methionine removal as variable modifications."],"omics_type":["Proteomics"],"labhead":["Samuel C. Zeeman"],"instrument_platform":[""],"labhead_affiliation":["Institute of Molecular Plant Biology, Department of Biology, ETH Zurich, Switzerland"],"submission_type":["PARTIAL"],"species":["Nicotiana Tabacum (common Tobacco)"],"publication":["Not available"],"submitter_mail":["bpfister@ethz.ch"],"submitter_affiliation":["ETH Zurich"],"submitter_country":["Switzerland"],"additional_accession":[]},"is_claimable":false,"name":"Size-exclusion chromatography of soluble chloroplast proteins from Nicotiana tabacum (tobacco)","description":"Maturase K (MatK) is the only splicing factor encoded in the chloroplast genomes of land plants. Here we used a transplastomic Nicotiana tabacum line, in which the plastidial MatK gene is C-terminally tagged with a 3x-HA tag, to conduct size-exclusion chromatography of soluble protein extracts enriched for chloroplast proteins. Fractions 5 and 6, in which MatK-HA was found most abundant by western blot analysis, were subjected to mass spectrometry analysis.","dates":{"publication":"2026-02-10","submission":"2025-10-27"},"accession":"PXD069946","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:2042546","NEWT:259447","NEWT:295546","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:1073882","NEWT:6819","NEWT:475174","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:135622","NEWT:672127","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:1584","NEWT:36630","NEWT:1585","NEWT:383379","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:7719","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:3347","NEWT:160621","NEWT:212790","NEWT:110368","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:44685","NEWT:161934","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:410661","NEWT:1140","NCBITaxon:2157","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:3714","NCBITaxon:37162","NEWT:2065263","NEWT:177437","NEWT:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:485","NEWT:56636","NEWT:2499525","NEWT:2853422","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:1502","NEWT:128017","NEWT:376686","NEWT:237610","NEWT:95486","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:491","NEWT:460523","NEWT:45954","NEWT:235443","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:108458","NEWT:5936","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:1255228","NEWT:410289","NEWT:373153","NEWT:1640278","NEWT:472","NEWT:940825","NEWT:529507","NEWT:1071661","NEWT:28128","NEWT:470","NEWT:5911","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:906914","NEWT:28141","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:3728","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:2903","NEWT:185579","NEWT:13076","NEWT:207340","NEWT:1006581","NEWT:33940","NEWT:550","NEWT:554","NEWT:451516","NEWT:1325291","NEWT:42410","NEWT:36185","NEWT:1054211","NEWT:1225786","NEWT:771870","NEWT:51338","NEWT:575412","NEWT:28112","NEWT:49012","NEWT:6493","NEWT:10832","NEWT:6494","NEWT:6491","NEWT:507601","NEWT:520","NEWT:186441","NEWT:643680","NEWT:214092","NCBITaxon:6157","NEWT:13095","NEWT:162425","NEWT:104105","NEWT:216257","NEWT:9986","NEWT:8654","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:5147","NEWT:28108","NEWT:28104","NEWT:407821","NEWT:568703","NCBITaxon:2","NEWT:568708","NEWT:1613","NEWT:986","NEWT:52641","NEWT:28532","NEWT:353152","NEWT:298176","NEWT:353153","NEWT:227377","NEWT:1774284","NEWT:40674","NEWT:1194669","NEWT:443906","NEWT:13037","NEWT:519","NEWT:2510939","NEWT:7394","NEWT:6063","NEWT:1328388","NEWT:105487","NEWT:1548728","NEWT:667127","NEWT:9557","NEWT:377586","NEWT:300641","NEWT:39655","NEWT:9554","NEWT:38323","NEWT:475932","NEWT:256318","NEWT:206411","NCBITaxon:6191","NEWT:229533","NEWT:214053","NEWT:28550","NEWT:80863","NEWT:90675","NEWT:52639","NEWT:52638","NEWT:57075","NEWT:1666905","NEWT:8697","NEWT:861109","NEWT:8695","NEW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