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Samples were thawed on ice, 200 µl 100 mM ammonium bicarbonate, 200 µl trifluoroethanol and 10 µl 200 mM dithiothreitol added and mixed. The tissue was homogenized with ceramic beads using a Precellys 24 (Peqlab) 3 times for 15 seconds at 5000 rpm and incubated for 1 hour at 60°C. 200 µl 200 mM iodoacedamide were added before a 90 min incubation at room temperature.  An additional 60 min incubation at room temperature followed after addition of 10 µl 200 mM dithiothreitol. 350 µl of this sample were removed and mixed with 437 µl 200 mM ammonium bicarbonate and 874 µl water {Wisniewski, 2009 #2020}{Scheltema, 2014 #2016}. Protein concentrations were determined by DC assay (BioRad) and 100 µg protein was digested by trypsin (trypsin gold, Promega) at 37 °C overnight. Digested peptides were purified using SepPak columns (Waters, Milford, United States) and quantified with a nanodrop 2000 (peqlab, VWR, Radnor, Pennsylvania, United States).  Peptides were analyzed using a nanoLC (Ultimate 300, Thermo Fisher Scientific, Waltham, MA, US) coupled to a modified ESI-Orbitrap MS/MS (QExactive Plus, Thermo Fisher Scientific, Waltham, MA, US) equipped with an enrichment and analytical column (AcclaimTM PepMapTM 100 C18: 0.3 mm diameter, 5 mm length, 5 µm particle size; AcclaimTM PepMapTM 100 C18: 0.075 mm diameter, 250 mm length, 5 µm particle size). In this setup, the mass spectrometer is equipped with a Spectroglyph source (Spectroglyph, LLC, WA, US) including an ion funnel instead of an S-lense {Dilillo, 2019 #2045}. 1 µg of protein of each sample was loaded onto the enrichment column before switching in-line with the analytical column at a flow rate of 300 µL/min. The gradient length of the analytical column was adjusted to 67 (WT, DHET, DKO, AKO, BKO, Fig. 1A) or 187 (DHET, DKO, Fig. 1B) min and 4% to 50% of an 80% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA) solution. Two cohort experiments including three or five replicates of each genotype (WT, DHET, DKO, AKO, BKO) were analyzed in random order. The mass spectrometer was operated in data-dependent top 10 acquisition mode using a resolution of 70,000 (AGC target of 3e6 and 64 ms maximum IT) in full MS mode. For the dd-MS2, a resolution of 17,500, AGC target of 2e5 and a maximum IT of 100 ms was used."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["MaxQuant software (2.4.11.0) was used for database search against the UniProt Mus musculus (strain C57BL/6J) reviewed database {Cox, 2008 #2021}{Tyanova, 2016 #2013}. For protein identification in MaxQuant, the enzyme specificity was set to trypsin with a maximum number of two missed cleavages, as well as standard instrument settings for orbitrap-based data. For the variable modifcations, methionine oxidation and N-terminal acetylation were set. Fixed modifcations were set for the carbamidomethylation of cysteine. Label-free quantification was set to LFQ with fast LFQ enabled. Only unique peptides were used for quantification. The false discovery rate (FDR) for PSMs and proteins was set to 5%, whereas the FDR for site decoy fractions was used at the default of 1%. The decoy mode for FDR calculation was set to reverse. All other settings were set to default. The statistical analysis of the MaxQuant database search was performed in Perseus {Tyanova, 2016 #1988} software version 1.6.10.43 or 1.6.13.0. Proteins identified by MaxQuant as “Only identified by site”, “Reverse”, or “Potential contamination” were removed. For visualization of differential gene expression data were log2(x) transformed. Proteins with less than 2 identified peptides were filtered out. Additionally, only proteins with a minimal number of 3 valid values in each group were considered."],"omics_type":["Proteomics"],"labhead":["Gabriele Fischer von Mollard"],"instrument_platform":[""],"submission_type":["PARTIAL"],"labhead_affiliation":["Biochemistry III, Department of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany"],"species":["Mus Musculus (mouse)"],"publication":["41859820 Gottschalk J, Kotschnew K, Hahn J, Patschkowski T, von Fischer von Mollard G. Reduction in Synaptic Vesicle Protein Abundance but Increased Amounts of Nsg2 and Lpcat1 in Cerebral Cortices Without the Endosomal SNARE Proteins Vti1a and Vti1b. Proteomics. 2026:e70117 10.1002/pmic.70117"],"submitter_mail":["juvoss@cebitec.uni-bielefeld.de"],"submitter_affiliation":["Bielefeld University, Center for Biotechnology"],"submitter_country":["Germany"],"pubmed_abstract":["Absence of the endosomal SNAREs vti1a and vti1b results in perinatal death and severe neuronal phenotypes in mice, while lack of one of these proteins results in minor phenotypes. Proteomic differences were investigated to obtain a deeper insight into processes in which vti1a and vti1b are involved. Here we applied a bottom-up shotgun proteomic approach to investigate the differences in wild-type, double heterozygous (DHET), vti1a<sup>-/-</sup> vti1b<sup>-/-</sup> double knockout (DKO), vti1a<sup>-/-</sup> knockout and vti1b<sup>-/-</sup> knockout cerebral cortices. Single deletions did not affect protein levels significantly. A total of 1725 proteins were detected of which 69 were less abundant and 191 proteins were more abundant in DKO cortices. Many less abundant proteins belonged to cellular components and reactome pathways synapse, synaptic vesicle cycle, vesicle mediated transport, L1CAM interaction, and cholesterol biosynthesis in pathway enrichment analysis. More abundant proteins were enriched in cellular components and Kyoto Encyclopaedia of Genes and Genomes (KEGG)-pathways such as spliceosome, ribosome, carbon metabolism, and ribonucleoprotein complex. Immunoblotting validated reduced expression levels of the tested synaptic vesicle proteins as well as increased amounts of lysophosphatidylcholine acyltransferase 1 (Lpcat1) and neuron-specific gene 2 (Nsg2), which is involved in postsynaptic AMPA-receptor recycling. These data indicate that the synapse and cell adhesion were strongly affected in DKO brains. STATEMENT OF SIGNIFICANCE OF THE STUDY: Distinct populations of neurons and glia cells are generated and organize into layers during brain development. Neurons develop an elaborate morphology to transmit information via axons and synapses to dendrites in receiving neurons. These neurites form via several specialized pathways of vesicle secretion and endocytosis. Fusion between these membranes requires members of the SNARE protein family. Double knockouts of the endosomal SNAREs vti1a and vti1b (DKO) result in perinatal lethality in mice with massive defects in the brain. In this study we compared the proteome of DKO brain cortices with double heterozygous controls to obtain insights into the molecular alterations and affected pathways. DKO brains contained lower amounts of synaptic proteins and proteins involved in cell adhesion, membrane trafficking and cholesterol biosynthesis. Several proteins of spliceosomes, ribosomes and carbon metabolism were more abundant in DKO brains, which may be a consequence of the reduced amounts of synaptic proteins or a shift in cell populations. Lysophosphatidylcholine acyltransferase 1 (Lpcat1) and neuron-specific gene 2 (Nsg2), which is involved in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) recycling, were confirmed to be more abundant by Western blotting. These data point to defects in trafficking especially in the synapse and in cell adhesion, which is required for neurite outgrowth."],"pubmed_title":["Reduction in Synaptic Vesicle Protein Abundance but Increased Amounts of Nsg2 and Lpcat1 in Cerebral Cortices Without the Endosomal SNARE Proteins Vti1a and Vti1b."],"pubmed_authors":["Gottschalk Julia J, Kotschnew Katharina K, Hahn Julia J, Patschkowski Thomas T, von Fischer von Mollard Gabriele G"],"additional_accession":[]},"is_claimable":false,"name":"Reduction in synaptic vesicle proteins but increase in proteins involved in basic cell function in cerebral cortices without the endosomal SNARE proteins Vti1a and Vti1b","description":"Absence of the endosomal SNAREs vti1a and vti1b results in perinatal death and severe neuronal phenotypes in mice while lack of one of these proteins results in minor phenotypes. Proteomic differences were investigated to obtain a deeper insight into processes in which Vti1a and Vti1b are involved. Here we applied a bottom-up shotgun proteomic approach to investigate the differences in wild-type, double heterozygous (DHET),vti1a vt1ib double knockout (DKO), vti1a knockout and vti1b knockout cerebral cortices. Single deletions did not affect protein levels. A total of 1725 proteins were detected of which 69 were down and 191 proteins were upregulated in DKO cortices. Many less abundant proteins belonged to the gene ontology terms and Reactome pathways synapse, synaptic vesicle cycle, vesicle mediated transport and L1CAM interaction in pathway enrichment analysis. More abundant proteins were enriched in Reactome- and KEGG-pathways such as spliceosome, ribosome, carbon metabolism and gene expression. Immunoblotting validated reduced expression levels of the tested synaptic vesicle proteins as well as increased amounts of lysophosphatidylcholine acyltransferase 1 (Lpcat1) and neuron-specific gene 2 (Nsg2 ), which is involved in postsynaptic AMPA-receptor recycling. These data indicate that the synapse and cell adhesion were strongly affected in DKO brains.","dates":{"publication":"2026-03-31","submission":"2025-08-01"},"accession":"PXD066845","cross_references":{"TAXONOMY":["NEWT:108018","NEWT:377960","NEWT:1131","NEWT:190486","NEWT:2042546","NEWT:148942","NEWT:295546","NEWT:258594","NEWT:1126","NEWT:1129","NEWT:89187","NEWT:309807","NEWT:89184","NEWT:309800","NEWT:1323429","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:44270","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:10034","NEWT:661410","NEWT:488003","NEWT:224326","NEWT:376619","NEWT:3765","NEWT:2433","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:11308","NEWT:12637","NEWT:2164133","NEWT:1180","NEWT:2767846","NEWT:1343074","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:1410384","NEWT:1310173","NEWT:161934","NEWT:1148","NEWT:5508","NEWT:135666","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:65121","NEWT:3716","NEWT:3715","NEWT:2065263","NEWT:1549675","NEWT:177437","NEWT:9014","NEWT:10891","NEWT:118221","NEWT:34869","NEWT:226126","NEWT:1616117","NEWT:34865","NEWT:550537","NEWT:284812","NEWT:3701","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:1246791","NEWT:34875","NEWT:9005","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:1883426","NEWT:657321","NEWT:1423687","NEWT:657322","NEWT:10886","NEWT:3708","NEWT:536231","NEWT:219813","NEWT:1301262","NEWT:2305479","NEWT:215451","NEWT:460519","NEWT:272843","NEWT:272844","NEWT:1348799","NEWT:55784","NEWT:485","NEWT:2499525","NEWT:467144","NEWT:142786","NEWT:28196","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:272831","NEWT:987059","NEWT:491","NEWT:422203","NEWT:460523","NEWT:2423","NEWT:85694","NEWT:85692","NEWT:911045","NEWT:3750","NEWT:291146","NEWT:233271","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:278161","NEWT:415978","NEWT:410289","NEWT:191701","NEWT:9054","NEWT:472","NEWT:29459","NEWT:476","NEWT:28128","NEWT:470","NEWT:41122","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:63363","NEWT:3735","NEWT:29491","NEWT:911022","NEWT:446","NEWT:153481","NEWT:714485","NEWT:445","NEWT:2003758","NEWT:29486","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:3726","NEWT:30455","NEWT:28141","NEWT:9030","NEWT:9031","NEWT:3728","NEWT:108931","NEWT:219799","NEWT:1325292","NEWT:219798","NEWT:548","NEWT:150475","NEWT:546","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:40686","NEWT:1225786","NEWT:771870","NEWT:8665","NEWT:51338","NEWT:9995","NEWT:28112","NEWT:49012","NEWT:28110","NEWT:10832","NEWT:311339","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:536","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:51315","NEWT:52641","NEWT:353152","NEWT:116597","NEWT:353153","NEWT:116598","NEWT:227377","NEWT:1774284","NEWT:27689","NEWT:207769","NEWT:40674","NEWT:51329","NCBITaxon:1763","NEWT:443906","NEWT:13037","NEWT:519","NEWT:941638","NEWT:511","NEWT:7394","NEWT:6063","NEWT:404330","NEWT:1328388","NEWT:13064","NEWT:529120","NEWT:300641","NEWT:39655","NEWT:206411","NEWT:80869","NEWT:214053","NEWT:80863","NEWT:665890","NEWT:52639","NEWT:52638","NEWT:8697","NEWT:861109","NEWT:8695","NEWT:1196866","NEWT:6039","NEWT:1045775","NEWT:1123869","NCBITaxon:1335626","NEWT:7370","NEWT:280406","NEWT:83906","NEWT:99488","NEWT:99487","NEWT:1134506","NEWT:255470","NEWT:762903","NEWT:27697","NEWT:86185","NEWT:9935","NEWT:266940","NEWT:287889","NEWT:9938","NEWT:1028307","NEWT:52603","NEWT:92867","NEWT:92866","NEWT:1344414","NEWT:202950","NEWT:741158","NEWT:1293091","NEWT:202956","NEWT:483179","NEWT:315750","NEWT:99490","NEWT:632957","NEWT:265619","NEWT:11294","NEWT:754466","NEWT:9925","NCBITaxon:9606","NEWT:1436183","NEWT:1479235","NEWT:4232","NEWT:9922","NEWT:416348","NEWT:105812","NEWT:11298","NEWT:4233","NEWT:4236","NEWT:398031","NEWT:196627","NEWT:336685","NEWT:523840","NEWT:48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