Pulsar

Each distinct protein spot was excised from at least three 2-DE gels in order to digest them separately with trypsin, thermolysin and chymotrypsin. MS/MS analysis of enzymatic digests of protein spots was carried out with a QSTAR Pulsar i quadrupole time-of-flight mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada) that was equipped with a nano-electrospray source and nano-flow liquid chromatograph [27 (link)]. Automatic determination of the appropriate collision energy (relative to m/z) was carried out by the Analyst QS 1.1 software. When analyzing samples digested either with chymotrypsin or thermolysin the intercept of the collision energy values was decreased by eight units relative to that used with trypsin. The spectra from each digest were used to interrogate a "SuperWheat" database (Version # 100211) as described in [59 (link)]. For this study, the "SuperWheat" database was constructed by concatenating the following publicly available databases; NCBI non-redundant green plant protein sequences (download date: 2/11/2010) [84 ], nucleotide sequences translated in all six reading frames of contigs from TaGI Releases 10.0 and 11.0 [63 ], US Wheat Genome Project [85 ], HarvEST 1.14 (WI all NSF "stringent" assembly from 05/08/04) [86 ], NCBI Unigene Build #55 [84 ], and all ESTs from Butte 86 developing grain, as well as translated sequences (reading frame only) of 94 Butte 86 contigs (Additional file 2), including those for alpha-gliadins and gamma-gliadins [12 (link),13 (link)]. Additionally the database contained a list of proteins known to be common laboratory contaminants [87 ] and sequences for thermolysin. The "SuperWheat" database contained 2,094,746 protein sequences. Two search engines, X!Tandem [57 (link),87 ] and Mascot version 2.1 (Matrix Science, London, UK) [88 ,89 (link)] were used to match the peptide mass spectra to spectra generated in silico from database peptides. It has been demonstrated that Mascot and X!tandem yield somewhat different results [59 (link),70 (link)]. Mascot identified significantly more tryptic peptides than did X!tandem, while X!tandem identified more peptides from proteins digested with thermolysin or chymotrypsin. Scaffold Version 2_02_04 [62 ] was used to assemble and visualize MS/MS derived peptide and protein identifications. A "subset" database was generated from the initial search of the SuperWheat database by exporting from Scaffold all protein sequences that had a 20% or greater probability of being a match. Appended to these 2,134 sequences was an equal number of decoy protein sequences from the archaeobacter Jannaschia sp, translated sequences from the set of Butte 86 contigs not already included in the subset database, and the set of common protein contaminants. A "second pass" search [90 (link),91 (link)] was conducted with both search engines and the results assembled and validated with Scaffold. Identifications of proteins were required to meet the following criteria: at least two peptides having a parent mass tolerance threshold of less than or equal to 100 ppm and a greater than 90% peptide probability as specified by the Peptide Prophet algorithm [92 ]. Scaffold Version 3.00.03 was used to compile the final set of MS/MS based peptide and protein identifications, using the MUDPIT algorithm to independently analyze the data for each spot. The false discovery rate was generally found to be 0.0% under the filter settings used. The data associated with this manuscript may be downloaded from ProteomeCommons.org Tranche using the following hash:
hCc5INiKGH0m4DEfxLbShm1F+us+JyZ/HENjkOTlGcni8NmnyoEwU5i7Onf/Po2kNtnP10SCdgODD6Swo0hgF69d3dIAAAAAAAB6hg==

A spectral library was built in Spectronaut v16 (Biognosys Schlieren, Switzerland) using the Pulsar search algorithm. Specific trypsin digestion was used for the enzyme setting. A peptide length of 7–52 was used and 2 missed cleavages per peptide were allowed. Carbamidomethylation was added as a fixed modification, N-terminal acetylation and methionine oxidation were added as variable modifications. A Swissprot Human FASTA file (downloaded on 12 June 2021) including common contaminating proteins was used as the search database. For DIA analysis, the standard identification and quantification settings were used for data processing except for data filtering which was set at q-value percentile (0.5 fraction) without imputation (i.e., precursors need to be identified in at least 50% of runs to be included in the analysis). A q-value ≤ 0.05 cut-off was applied at the precursor peptide and protein levels. Quantification was performed at the MS2 level. Label-free cross-run normalization was employed using a global normalization strategy.

Samples were resuspended in 11 µL of 2 % acetonitrile (ACN) and 1 % FA containing 0.5X of iRT standard peptides (Biognosys, Ki-3002-1), of which, 2 µL of each sample was further diluted 1/5 in the same buffer. Diluted samples were loaded (2.5 µL each) at 450 nL/min on a 17 cm × 75 µm i.d. PicoFrit fused silica capillary column (New Objective), packed in-house with Jupiter 5 µm C18 300 Å (Phenomenex). The column was mounted in an Easy-nLC II system (Proxeon Biosystems) and coupled to an Orbitrap Fusion mass spectrometer (ThermoFisher Scientific) equipped with a Nanospray Flex Ion source (Proxeon Biosystems). Peptides were eluted at a flow rate of 250 nL/min on 2-slope gradient made with 0.2% FA in water (buffer A) and 0.2% FA in 100% ACN (buffer B). Concentration of buffer B first increased from 2% to 36% over 105 minutes, and from 36 % to 80 % over 12 minutes. The mass spectrometer was operated in data-independent acquisition mode. Full MS scans in the range of 400-1000 m/z range were acquired in the Orbitrap at a resolution of 120 K. Each full scan was followed by 25 MS2 acquisition windows with 24 m/z increments and 0.5 m/z overlaps, covering each full scan. Matching MS2 m/z window precursor ions were fragmented by HCD at a 30% collision energy and acquired in the Orbitrap at a 30 K resolution. Both MS and MS2 AGC target were set to 4e5 with maximum fill times of 60 ms. The lock mass option (lock mass: m/z 371.101233) was used for internal calibration. MS RAW files were analyzed with Spectronaut v15.1.210713 (Biognosys) by directDIA. The Fasta database was downloaded from Uniprot and consisted of all Saccharomyces cerevisiae S288c verified nuclear proteins entries (1749). Considered peptide length for Pulsar search was 6-52 amino acids with two missed cleavages tolerance, using the semi-specific Trypsin/P rules. Carbamidomethylation of cysteines was set as fixed modifications while oxidation of methionines, protein N-termini acetylation, biotinylation of lysines and protein N-termini as well as phosphorylation (STY) were set as variable modifications, with a maximum of five. Peptide spectrum match, peptides, and protein group FDR were set at 0.01. Machine learning was applied per run and both precursor and protein were identified at a Qvalue cutoff of 0.01. Proteins were quantified at the MS2 level on stripped peptide median quantities. Unique peptides were used for quantification. Only proteins for which values from all biological triplicates were measured in the sgRNA-BirA*-dCas9 were kept for quantification, and no values were imputed during the analysis. The resulting protein datasets were crosschecked with a list of chromatin and transcription related proteins obtained from Uniprot (Supplementary Data 5). The protein list and associated ontologies was built from searches using the keyword chromatin (461 reviewed entries), and transcription (1211 reviewed entries) in S. cerevisiae. After merging both lists and removing duplicates, 1320 entries remained (Supplementary Table 6). A two-sided unpaired Student’s t-test with equal variance was used to help in the evaluation of significant protein enrichment from sgRNA-BirA*-dCas9 triplicates. Analyses of evidence for association of chromatin remodeling subunits with nuclear protein complexes was performed for all significant candidates with definitions of subunit compositions of protein complexes taken from the Saccharomyces Genome Database (SGD)^{81 (link)} and UniProt^{82 (link)}, and protein-protein interactions were identified in The Biological General Repository for Interaction Datasets (BioGrid)^{83 (link)}. For representation clarity, proteins having a log2 fold change above 2.5 were limited at the 2.5 value on MA plots (Fig. 5). We removed two sgRNA-BirA*-dCas9 samples out of a total of 4 because they did not reveal significant fold changes for many or most of the candidates that were identified in the samples kept for further analysis. Both MA and box plots were made in RStudio (v2022.07.2) using the ggplot2 package (v3.3.6). Data are available via ProteomeXchange with identifier PXD029913.