{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Xlsx":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD074603/Pivot_report.xlsx","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD074603/Files_overview.xlsx","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD074603/HTT_peptides.xlsx"],"Tabular":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD074603/Gene_ontology_annotation.tsv"],"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD074603/checksum.txt"],"Pdf":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD074603/HTT_alignment.pdf"],"Fasta":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD074603/OnePerGene_DB.fasta"],"Other":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD074603/Raw_DDA_data.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD074603/Sus_scrofa_library.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD074603/Export_schemes.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD074603/Abundances.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD074603/DIALibQC.zip","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD074603/Raw_DIA_data.zip"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["vodicka@iapg.cas.cz"],"submitter":["Jakub Cervenka"],"technology_type":["Data-dependent acquisition","Mass Spectrometry","Bottom-up proteomics","Data-independent acquisition","SWATH MS"],"disease":["Huntington Disease"],"software":[""],"submitter_keywords":[""],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD074603"],"tissue":["Brain","Cerebrospinal Fluid"],"sample_protocol":["We employed a transgenic (TG) minipig model of Huntington's disease that contains two alleles of the wild-type (WT) porcine huntingtin and one copy of a human transgene encoding the N-terminal fragment of a huntingtin protein with the first 548 amino acids and 124 glutamines (mHTT). The brain samples were dissected from the TG minipigs and their WT siblings, deeply anaesthetized and perfused. Before cerebrospinal fluid (CSF) samples collection, the animals were starved, anaesthetized, and a lumbar puncture was performed. Tissue samples were homogenized and proteins were extracted using: i) radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 5 mM EDTA, 0.5% v/v Igepal CA-630, 1% w/v sodium deoxycholate, 1% v/v Triton X-100 and 0.1% w/v sodium dodecyl sulfate (SDS) in 50 mM Tris HCl pH 7.4); ii) SDS buffer (2% w/v SDS in 50 mM Tris HCl pH 7.4); and iii) octyl β-D-glucopyranoside (β-OG) buffer (10 mM Tris HCl, 0.15 M NaCl, 1 mM EDTA and 1.25% w/v β-OG). The albumin from chicken egg white (OVAL) and the alcohol dehydrogenase from yeast (ADH1) were spiked into RIPA, SDS, and β-OG quadruplicates. The samples were subjected to: i) on-filter digestion (RIPA and SDS samples); ii) in-solution digestion (β-OG samples); and iii) in-gel digestion (RIPA samples). Proteins were reduced, alkylated, and digested at 37 °C by lysyl endopeptidase (LysC) and trypsin for 2 h and overnight, respectively. Independent RIPA and β-OG samples were separated using a high-pH fractionation. The RIPA digests were injected into a Dionex Ultimate 300 (Thermo Fisher Scientific Inc.). The peptides were separated on a Kinetex C18 Biphenyl column, 5 μm, 100 Å 150 × 4.6 mm (Phenomenex) by a 30-min gradient from 2% to 40% (v/v) acetonitrile in 20 mM ammonium formate pH 10. The β-OG in-solution digest was injected using a 100 µl gastight microsyringe (SGE) onto the C18 microcolumn manually packed with Aeris Peptide XB, 3.6 µm, 40 mm x 250 µm (Phenomenex). A gradient was then formed by 20 mM ammonium formate (pH 10) in 2% to 40% (v/v) acetonitrile. Samples of WT and mHTT animals sacrificed at the age of 18, 36, 48 or 60 months were processed. Cerebral cortex samples were homogenized with a lysis buffer (13 mM Tris, 0.15 M NaCl, 1 mM EDTA in PBS and 1% w/v β-OG), the lysis buffer was exchanged for a wash buffer of 8 M urea and 5 mM EDTA in 50 mM Ambic. These samples were supplemented with sodium deoxycholate to a final concentration of 8 % (w/v). The nucleus caudatus samples were lyzed with the SDS buffer (2% w/v SDS in 50 mM Tris HCl pH 7.4) and supernatants were loaded onto the Microcon 30K using modified FASP protocol (https://doi.org/10.1111/bpa.13265). The CSF samples were supplemented with the OVAL and the ADH1 standard proteins and transferred into a wash buffer of 8 M urea and 5 mM EDTA in 50 mM Ambic. All samples were reduced, alkylated and digested. Peptide mixtures were desalted and analyzed by data-dependent acquisition (DDA) on an Eksigent 425 nanoLC (Sciex) online coupled to a TripleTOF5600+ mass spectrometer (Sciex). The peptide mixtures were separated in a trap-elute mode with a linear gradient from 5 to 35% (v/v) acetonitrile in 0.1% (v/v) FA over 120 min. Peptides were separated on an in-house fused-silica column, 250 mm × 75 μm manually packed with C18 beads (ProntoSIL, 3 μm, 120 Å; Bischoff Analysentechnik GmbH). The MS1 spectra were collected in a mass to charge range from 400 to 1,250 for 300 ms, with the 20 most intense ions selected for fragmentation and a dynamic exclusion of 13 s. For the high-pH RP-HPLC fractionated peptides, MS1 spectra were collected for 200 ms, and the 25 most intense ions were selected for fragmentation. The MS2 fragment ion spectra were collected in a mass to charge range from 170 to 1,500 for 150 ms. The RIPA and SDS on-filter peptide digests obtained from the pooled cerebral cortex samples were analyzed by data-independent acquisition (DIA) on the Eksigent 425 nanoLC online coupled to the TripleTOF5600+ mass spectrometer as described above. Data were collected with the sequential window acquisition of all theoretical mass spectra (SWATH-MS) method, 30 overlapping variable windows calculated with the SWATH Variable window calculator (Sciex) were monitored. The MS1 and MS2 spectra for the pooled cerebral cortex were collected in mass to charge ranges corresponding to DDA-MS, and the accumulation time was set to 150 ms and 99 ms for MS1 and MS2. The peptide digests obtained from the 48-month-old WT and mHTT animals were analyzed using the same instrumentation setup with modifications. For the cerebral cortices, 35 overlapping variable windows were monitored, and the accumulation time was set to 99 ms and 96 ms for MS1 and MS2. For the CSF samples, 30 overlapping variable windows were monitored with the accumulation time set to 96 ms for both MS1 and MS2. The nucleus caudatus samples obtained from the 60-month-old WT and TG animals were analyzed using the set-up applied to the cerebral cortices."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["The raw DDA files were processed and searched by the Pulsar algorithm implemented in Spectronaut 19.5.241126.62635 software (Biognosys) against a database derived from the porcine UniProtKB reference proteome (UP000008227; release 2024_06) (Uniprot 2024). The full non-redundant database with protein sequences reduced to one per gene contained 22,833 porcine proteins with the chicken OVAL (P01012), the yeast ADE1 (P00330), the human N-terminal fragment of huntingtin with the first 548 amino acids (P42858), universal contaminants (381) from (https://doi.org/10.1021/acs.jproteome.2c00145), and iRT peptides appended. The library was generated from Pulsar with default BGS Factory Settings. A specific cleavage rule was created for the combination of Lys-C and Trypsin cleavage after all lysines and after the arginine unless a proline proceeds, with two missed cleavages allowed. The carbamidomethylation of cysteine was set as a fixed modification, and protein N-terminal acetylation and methionine oxidation were set as variable modifications. A false discovery rate (FDR) cut-off 1% was applied at levels of peptide-spectrum matches, peptides, and proteins. Porcine proteins in the spectral library were annotated using a gene ontology annotation file (35497.S_scrofa, updated 22nd December 2024) from UniProtKB (https://ftp.ebi.ac.uk/pub/databases/GO/goa/proteomes/). Parameters of compliance were tested using the DIALib-QC software tool to systematically evaluate the quality of the spectral library.  The raw DIA data were processed in Spectronaut 19.5.241126.62635 with three different analysis workflows: i) a direct DIA - library-free DIA, ii) a library DIA - library-based DIA, and iii) a hybrid DIA - direct DIA enriched for extra spectra from the search archive of DDA data used for the spectral library generation. All DIA analysis workflows were performed using default BGS Factory Settings with the following modifications. A specific cleavage rule was created for the combination of Lys-C and Trypsin - cleavage after all lysines and after the arginine unless a proline proceeds. For the library DIA, the calibration using Biognosys's iRT kit was selected. For the hybrid DIA, the Hybrid Library option (DDA + DIA) was selected in DIA Analysis Workflow in Experiment Settings. Data were normalized in Spectronaut using cross-run local normalization. No filter type and automatic row selection were applied. Only RIPA and SDS samples with a qualitatively different composition caused by the different protein extraction strategy were analyzed without normalization. Differential protein abundance analyses were performed in Spectronaut using an unpaired t-test. Statistically significant changes in protein abundance were considered with a fold-change ≥ 1.5 and Q-value ≤ 0.05.  Protein group and peptide level quantification data for all three quantification strategies, as well as spectral library data, were exported from Spectronaut, and further data analysis and visualisation were performed in R 4.5.1. The tidyverse set of packages was used for data filtering and summarisation. The ggplot2 and cowplot packages were used for graph production, the ggvd and ggVennDiagram ggplot2 extensions were used to plot Venn diagrams and the ggbeeswarm package was used to produce column scatter plots. The porcine HTT and the N-terminal fragment of the human HTT were digested in silico using MS-Digest. Multiple sequence alignment was performed using Clustal Omega 1.2.4."],"omics_type":["Proteomics"],"labhead":["Petr Vodicka"],"instrument_platform":[""],"labhead_affiliation":["Laboratory of Applied Proteome Analyses, Institute of Animal Physiology and Genetics of the Czech Academy of Sciences, Libechov, Czech Republic"],"submission_type":["PARTIAL"],"species":["Sus Scrofa Domesticus (domestic Pig)"],"publication":["Not available"],"submitter_mail":["jakub.cervenka@lf1.cuni.cz"],"submitter_affiliation":["Laboratory of Proteomics, Institute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University, Prague, Czech Republic"],"submitter_country":["Czechia"],"additional_accession":[]},"is_claimable":false,"name":"A porcine spectral assay library to quantify brain proteome by DIA-MS","description":"The peptide-centric analysis requires spectral assay libraries that allow for the extraction of quantitative data in mass spectrometry-based methods, including data-independent acquisition (DIA), parallel reaction monitoring (PRM), or selected reaction monitoring (SRM). Such spectral libraries have been generated for humans (pan-human, T-cells, HLA subtypes) and other organisms such as mouse, E. coli, zebrafish, S. aureus, M. tuberculosis, etc. Here, we focused on the pig (Sus scrofa), a biomedical model capable of recapitulating the complexity of human pathology. We analyzed brain tissues and cerebrospinal fluids from a transgenic minipig model of Huntington's disease subjected to multiple extraction and fractionation steps. We generated a porcine spectral library for 8,321 proteins and demonstrated that this library provides a valuable resource to confidently and reproducibly quantify porcine proteins at the proteome-wide level. Via targeted assays, it enables the quantification of selected proteins with a key role not only in neuroscience.","dates":{"publication":"2026-05-18","submission":"2026-02-18"},"accession":"PXD074603","cross_references":{"TAXONOMY":["NEWT:108018","NEWT:377960","NEWT:1131","NEWT:2042546","NEWT:295546","NEWT:258594","NEWT:1126","NEWT:1129","NEWT:309807","NEWT:89184","NEWT:309800","NEWT:1323429","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:10034","NEWT:661410","NEWT:224326","NEWT:376619","NEWT:3765","NEWT:3767","NEWT:433727","NEWT:135622","NEWT:672127","NEWT:656061","NEWT:37965","NEWT:656064","NEWT:3760","NEWT:36630","NEWT:381198","NEWT:418106","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:44250","NEWT:317447","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:12637","NEWT:2164133","NEWT:2767846","NEWT:314344","NEWT:108061","NEWT:224308","NEWT:49928","NEWT:160621","NEWT:110368","NEWT:1916073","NEWT:1310161","NEWT:1230383","NEWT:77133","NEWT:1310165","NEWT:114727","NEWT:5516","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:1310173","NEWT:161934","NEWT:1148","NEWT:5508","NEWT:419481","NEWT:5507","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:1143","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:709964","NEWT:3712","NEWT:3711","NEWT:3714","NEWT:3715","NEWT:2065263","NEWT:1549675","NEWT:177437","NEWT:9014","NEWT:10891","NEWT:226126","NEWT:1616117","NEWT:34865","NEWT:28174","NEWT:550537","NEWT:284812","NEWT:3701","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:1246791","NEWT:9005","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:1883426","NEWT:657321","NEWT:10886","NEWT:3708","NEWT:536231","NEWT:219813","NEWT:215451","NEWT:460519","NEWT:272844","NEWT:1348799","NEWT:55784","NEWT:485","NEWT:2499525","NEWT:142786","NEWT:2853422","NEWT:487","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:216778","NEWT:376686","NEWT:237610","EFO:0001352","NEWT:219809","NEWT:219808","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:141469","NEWT:987059","NEWT:491","NEWT:460523","NEWT:85694","NEWT:85692","NEWT:3750","NEWT:291146","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:278161","NEWT:415978","NEWT:410289","NEWT:191701","NEWT:9054","NEWT:472","NEWT:29459","NEWT:28128","NEWT:470","NEWT:41122","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:29491","NEWT:911022","NEWT:446","NEWT:153481","NEWT:445","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:3726","NEWT:30455","NEWT:28141","NEWT:9031","NEWT:3728","NEWT:108931","NEWT:219799","NEWT:219798","NEWT:548","NEWT:150475","NEWT:256737","NEWT:51350","NEWT:8694","NEWT:8692","NEWT:542","NEWT:394503","NEWT:13076","NEWT:219785","NEWT:451515","NEWT:8673","NEWT:550","NEWT:10804","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:1225786","NEWT:771870","NEWT:51338","NEWT:9995","NEWT:28112","NEWT:49012","NEWT:28110","NEWT:10832","NEWT:507601","NEWT:520","NEWT:64657","NEWT:186441","NEWT:2585209","NEWT:643680","NEWT:214092","NEWT:40697","NEWT:13095","NEWT:162425","NEWT:242124","NEWT:9986","NEWT:8654","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:80833","NEWT:479096","NEWT:28108","NEWT:28104","NEWT:1114957","NEWT:568703","NEWT:568708","NEWT:52641","NEWT:353152","NEWT:353153","NEWT:227377","NEWT:1774284","NEWT:27689","NEWT:207769","NEWT:40674","NCBITaxon:1763","NEWT:443906","NEWT:13037","NEWT:519","NEWT:7394","NEWT:6063","NEWT:404330","NEWT:1328388","NEWT:300641","NEWT:39655","NEWT:38323","NEWT:206411","NEWT:80869","NEWT:214053","NEWT:80863","NEWT:665890","NEWT:52639","NEWT:52638","NEWT:8697","NEWT:861109","NEWT:8695","NEWT:1196866","NEWT:1045775","NEWT:1123869","NCBITaxon:1335626","NEWT:7370","NEWT:83906","NEWT:99488","NEWT:1134506","NEWT:255470","NEWT:27697","NEWT:86185","NEWT:9935","NEWT:287889","NEWT:1028307","NEWT:52603","NEWT:92867","NEWT:92866","NEWT:1344414","NEWT:202950","NEWT:741158","NEWT:1293091","NEWT:202956","NEWT:315750","NEWT:632957","NEWT:9925","NCBITaxon:9606","NEWT:1436183","NEWT:4232","NEWT:9922","NEWT:416348","NEWT:11298","NEWT:4236","NEWT:398031","NEWT:196627","NEWT:523840","NEWT:1076878","NEWT:200308","NEWT:200302","NEWT:9913","NEWT:9915","NEWT:105841","NEWT:2666255","NEWT:999810","NEWT:28995","NEWT:1392998","NEWT:1150621","NEWT:1085791","NEWT:745531","NEWT:226900","NEWT:231490","NEWT:590651","NEWT:449239","NEWT:244704","NEWT:9909","NEWT:9902","NEWT:430615","NEWT:563041","NEWT:36599","NEWT:41949","NEWT:326423","NEWT:326424","NEWT:13013","NEWT:749906","NEWT:418985","NEWT:749907","NEWT:431947","NEWT:142809","NEWT:1095183","NEWT:302409","NEWT:9979","NEWT:130821","NEWT:2320211","NEWT:27606","NEWT:290610","NEWT:1519788","NEWT:59202","NEWT:9975","NEWT:8643","NEWT:1801631","NEWT:1159899","NEWT:502780","NEWT:860688","NEWT:35231","NEWT:1774208","NEWT:8644","NEWT:1125630","NEWT:202914","NEWT:222523","NEWT:27602","NEWT:502779","NEWT:297246","NEWT:483139","NEWT:101491","NEWT:8639","NEWT:8637","NEWT:11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