Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/msms.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F14_R1_T1_MS3TMT001_001358_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F4_R1_T1_MS3TMT001_001347_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F10_R1_T1_MS3TMT001_001354_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F18_R1_T1_MS3TMT001_001362_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F8_R1_T1_MS3TMT001_001351_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F1_R1_T3_MS3TMT001_001344_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F11_R1_T1_MS3TMT001_001355_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F3_R1_T1_MS3TMT001_001346_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F15_R1_T1_MS3TMT001_001359_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F17_R1_T1_MS3TMT001_001361_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F7_R1_T1_MS3TMT001_001350_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F12_R1_T1_MS3TMT001_001356_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F6_R1_T1_MS3TMT001_001349_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F2_R1_T1_MS3TMT001_001345_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F1_R1_T1_MS3TMT001_001342_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F16_R1_T1_MS3TMT001_001360_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F9_R1_T1_MS3TMT001_001353_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F13_R1_T1_MS3TMT001_001357_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F5_R1_T1_MS3TMT001_001348_JASK011_FL2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/03/PXD075635/ForkHeadCellSize_Std_F1_R1_T2_MS3TMT001_001343_JASK011_FL2.rawprimaryOK200skotheim@stanford.eduMichael LanzData-dependent acquisitionMass SpectrometryWhi5Size scalingCell sizehttps://www.ebi.ac.uk/pride/archive/projects/PXD075635The pellets were then resuspended in a denaturation/reduction buffer (0.07M Tris-Cl, 5% (v/v) mercaptoethanol, 0.6% (w/v) SDS and 15% (v/v) glycerol) by boiling for 10 min at 95 °C with intermittent vortexing. Debris was pelleted by centrifugation for 10 minutes (10,000 ×g). Cleared supernatants were alkylated with 5mM iodoacetamide, and then precipitated with three volumes of a solution containing 50% acetone and 50% ethanol. Proteins were re-solubilized in 2 M urea, 50 mM Tris-HCl, pH 8.0, and 150 mM NaCl, and then digested with TPCK-treated trypsin (50:1) overnight at 37 °C. Trifluoroacetic acid and formic acid were added to the digested peptides for a final concentration of 0.2%. Peptides were desalted with a Sep-Pak 50mg C18 column (Waters). The C18 column was conditioned with 500 µL of 80% acetonitrile and 0.1% acetic acid and then washed with 1000 µL of 0.1% trifluoroacetic acid. After samples were loaded, the column was washed with 2000 µL of 0.1% acetic acid followed by elution with 400 µL of 80% acetonitrile and 0.1% acetic acid. The elution was dried in a Concentrator at 45 °C. De-salted peptides were resuspended in 0.1% Formic acid. For each sample, 25 µg of desalted peptide samples were resuspended in 20 µL of 100 mM Triethylammonium bicarbonate solution and labeled with 16-plex TMTpro at a ratio 4:1 (TMT:peptide). Total reaction volume was less than 25 µL. The labeling reaction was quenched with a final concentration of 0.5% hydroxylamine for 15 min. Labeled peptides were pooled and acidified to pH ∼ 2 using drops of 10% TFA. Excess TMT label was removed by re-running the pooled sample through a Sep-Pak 50-mg C18 column (as described above). TMT-labeled peptides were resuspended in 0.1% formic acid analyzed on a Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with a Thermo EASY-nLC 1200 LC system (Thermo Fisher Scientific, San Jose, CA). Peptides were separated by capillary reverse phase chromatography on a 25 cm column (75 µm inner diameter, packed with 1.6 µm C18 resin, AUR2-25075C18A, Ionopticks, Victoria Australia). Peptides were introduced into the Fusion Lumos mass spectrometer using a 180-min stepped linear gradient at a flow rate of 300 nL / min. The steps of the gradient are as follows: 6–33% buffer B (0.1% (v:v) formic acid in 80% acetonitrile) for 145 min, 33-45% buffer B for 15min, 40–95% buffer B for 5 min and maintain at 90% buffer B for 5 min. The column temperature was maintained at 50°C throughout the procedure. Xcalibur software (v.4.4.16.14) was used for the data acquisition and the instrument was operated in data-dependent mode. Advanced peak detection was disabled. Survey scans were acquired in the Orbitrap mass analyzer (centroid mode) over the range 380–1,400 m/z with a mass resolution of 120,000 (at m/z 200). For MS1, the normalized AGC target (%) was set at 250 and maximum injection time was set to 100 ms. Selected ions were fragmented by collision-induced dissociation (CID) with normalized collision energies of 34 and the tandem mass spectra were acquired in the ion trap mass analyzer with the scan rate set to ‘Rapid’. The isolation window was set to the 0.7 m/z window. For MS2, the normalized AGC target (%) was set to ‘Standard’ and maximum injection time to 35 ms. Repeated sequencing of peptides was kept to a minimum by dynamic exclusion of the sequenced peptides for 30 s. The maximum duty cycle length was set to 3 s. Relative changes in peptide concentration were determined at the MS3 level by isolating and fragmenting the five most dominant MS2 ion peaks.PrideAll raw files were searched using the Andromeda engine embedded in MaxQuant (v2). Reporter ion MS3 search was conducted using TMTpro (16-plex) isobaric labels. Variable modifications included oxidation (M) and protein N-terminal acetylation. Carbamidomthyl (C) was a fixed modification. The number of modifications per peptide was capped at five. Digestion was set to tryptic (proline-blocked). Database search was conducted using the UniProt proteome - Ecoli_UP000000625_83333. The minimum peptide length was 7 amino acids. 1% FDR was determined using a reverse decoy proteome.ProteomicsJan SkotheimPARTIALDepartment of Biology, Stanford UniversitySaccharomyces Cerevisiae (baker's Yeast)Not availablemikelanz@stanford.eduStanford UniversityUnited StatesfalseA Fkh1/2 binding site array in the WHI5 promoter drives sub-scaling transcriptionCells typically regulate their size within a relatively tight range by coupling growth to the cell division cycle using a dedicated set of molecular mechanisms. In budding yeast, cells are born with a similar amount of the G1/S inhibitor protein Whi5 that is then diluted by growth throughout G1. As cells grow, Whi5 concentration decreases and cells become more likely to enter the cell cycle. Cells are born in G1 with similar amounts of Whi5 because of the size-independent (sub-scaling) expression of WHI5 mRNA during S/G2/M phases and the equal partitioning of Whi5 protein at division. While the latter is known to be achieved by association with chromatin before anaphase, the mechanism for the former is poorly understood. Through systematic mutations of the WHI5 promoter, we discovered that WHI5’s core promoter region located -126 to -75 base pairs upstream of the start codon is responsible for sub-scaling expression. This sequence contains a repeating array of binding sites for the transcription factors Fkh1 and Fkh2. Mutation of any of these sites, deletion of either FKH1 or FKH2, or preventing Fkh1 or Fkh2 dimerization weakens the sub-scaling of WHI5 transcription. Taken together with structural predictions and a mathematical model of cooperative Fkh-DNA binding, we conclude that WHI5’s sub-scaling transcription is regulated by a Fkh1/2 heteropolymer binding an array of sites in its core promoter.2026-03-312026-03-14PXD075635NEWT:1773NEWT:3555NEWT:1182590NEWT:10090NEWT:749200NEWT:35554NEWT:4120NEWT:5693NEWT:347515NEWT:1216979NEWT:307972NEWT:92867NEWT:990346NEWT:544496NEWT:5334NEWT:145953NEWT:257309NEWT:284812NEWT:115104NEWT:43330NEWT:67825NEWT:44544NEWT:13076NEWT:544404NEWT:3702NEWT:8839NEWT:4232NEWT:1736309NEWT:4113NEWT:7227NEWT:11298NEWT:885318NEWT:4081NEWT:876138NEWT:554NEWT:5691NEWT:260710NEWT:106592NEWT:237561NEWT:9913NEWT:10036NEWT:4100NEWT:7574NEWT:1351NEWT:1076NEWT:6763NEWT:7215NEWT:8030NEWT:380394NEWT:272563NEWT:507601NEWT:1639NEWT:188229NCBITaxon:79857NEWT:746360NEWT:6239NEWT:135588NEWT:135622NEWT:6915NEWT:9986NEWT:101510NEWT:95486NEWT:3880NEWT:58002NEWT:9103NEWT:4577NEWT:146479NEWT:1000589NEWT:145943NEWT:85962NEWT:160488NEWT:317447NEWT:3635NEWT:7955NCBITaxon:2NEWT:7959NEWT:2261NEWT:3197NEWT:9615NEWT:884019NEWT:4565NEWT:1264690NEWT:169963NCBITaxon:38727NEWT:36329NEWT:34305NEWT:59729NCBITaxon:183674NEWT:626528NEWT:139927NEWT:4558NEWT:9606NEWT:367830NEWT:243230NEWT:931281NEWT:7029NEWT:1283300NEWT:334747NEWT:470NCBITaxon:79824NCBITaxon:4563NEWT:3218NEWT:5759NEWT:9838NCBITaxon:9615NEWT:1736231NEWT:1193501NEWT:6287NEWT:6326NEWT:9796NEWT:2762NEWT:5476NEWT:562NEWT:260707NEWT:287NEWT:10117NEWT:10116NEWT:1280NEWT:1836NEWT:29760NEWT:260705NEWT:1148NEWT:4932NEWT:70448NEWT:9825NEWT:3603NEWT:698936NEWT:39946NEWT:11676NEWT:9823NEWT:100226NCBITaxon:6073NEWT:4896NEWT:6279NEWT:7370NEWT:573NEWT:6282NEWT:7091