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Alsherbiny"],"technology_type":["Mass Spectrometry","Bottom-up proteomics"],"software":[""],"submitter_keywords":["High-throughput","Liquid handling robotics","Tissue engineering","Induced pluripotent stem cells","Proteomics","Drug screening","Vascular smooth muscle cells"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD070184"],"tissue":["Cell Culture"],"sample_protocol":["Cells were harvested in RIPA buffer supplemented with protease inhibitor (cOmplete protease inhibitor; Roche) following aspiration of media and subsequent washes with PBS. Lysate was stored at -80C. Cells were then lysed by thawing and repeated trituration through a 26.5 G needle, prior to centrifugation (20,000 x g for 30 min at 4°C) and harvesting of the supernatant fraction. Protein levels were determined using a Bicinchoninic Acid (BCA) assay in accordance with the manufacturer's guidelines (Pierce Rapid Gold BCA Protein Assay Kit #A53226, Thermo Scientific, USA). Protein samples (100 µg in 100 µl of conditioned media) were prepared using the EasyPep Mini MS Sample Prep Kit, following the instructions provided by the manufacturer (EasyPep™ MS Sample Prep Kits, Thermo Fisher Scientific, Inc, USA, Kit #A45733). This process included a pre-digestion treatment with PNGaseF, a deglycosylating enzyme, before overnight trypsin digestion. The resulting clean peptide samples were then dried by vacuum centrifuge and subsequently reconstituted in 100 µL of 0.1% formic acid in water, in preparation for LC-MS analysis.LCMS data was acquired according to a previous study with some modifications in the liquid chromatography (Muenzner, Trébulle et al. 2024). Briefly, a micro liquid chromatography system, Waters M-Class, coupled to SCIEXTM 6600 Triple-TOF® mass spectrometer operating in positive electron spray ion mode (ESI +), was used to analyse the tryptic peptides within 33 minutes of acquisition. 4 µg of the peptide digest was injected onto nanoEase M/Z HSS T3 1.8μm 300μmx150mm (Waters, 186009249) column coupled to a Zorbax 300SB-C18, (5um, 5x0.3mm) guard column (Agilent, USA). The column temperature was maintained at 40 °C, and 98% water (2% acetonitrile) and acetonitrile containing 0.1% Formic acid were used as mobile phase A and B, respectively, at 5 µL/min flow rate with initial loading and later column cleaning and equilibration at 7µL/min. Briefly, the gradient started at a 7 µL/min flow rate at 2-10% B for 1.66 min and inclined to 10-25% B at 1.67-21.67 min at 5 µL/min flow rate, then 40-95% B at 23.33-24.67 min and kept for 2 minutes, then equilibrated for 9 min with 2% B at 7 µL/min flow rate. The TripleTOF® 6600 system was equipped with a DuoSpray source and 25 μm internal diameter electrode and controlled by Analyst 1.8.1 software and Analyst Device Driver for LC control. Ion source and MS parameters were set as follows: GS1 25; GS2 15; curtain gas 20; ion source temperature 150; ionSpray voltage floating of 5500; lower m/z limit 350; upper m/z limit 1250; window overlap (Da) 1.0; collision energy spread was set at 5. The Sequential windowed acquisition of all theoretical fragment ion spectra (SWATH™) acquisition method involved an MS1 scan ranging from m/z 350 to m/z 1,250 with an accumulation time of 50 ms, followed by 40 MS2 scans, each with a 35 ms accumulation time The MS2 scans utilized a variable precursor isolation width covering the m/z range of 400 to 1,250 (Supplementary Table 4). MS2 spectra were collected in the range of m/z 100 to 2000 in high-resolution mode, with ~1.5 s total cycle time. PepCalMix calibrant (SCIEX, P/N 5045759, 10fmol/µL solution that was prepared by 1:100 dilution of the stock solution in dilution buffer consisting of 5% acetic acid and 2% acetonitrile) was injected every 12 samples for mass calibration along with the pooled quality control sample to evaluate the whole batch. Six pooled QC samples were injected to generate DIA-only library using gas phase fractionation workflow (Pino, Just et al. 2020) with the following m/z mass ranges: 400–500, 500–600, 600–700, 700–800, 800–900, 900–1000, and 1000–1250. The precursor isolation window was set to m/z 5, with collision energy spread of 5 eV except for 700-990 and 990-1000 was 8 and 10 eV, respectively. The cycle time was 2.14 s, consisting of high- and low-energy scans with 40 ms MS2 accumulation time, and data were acquired in ‘high-resolution’ mode."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["Data were processed using Spectronaut v19.5 where DirectDIA+ analysis using BGS Factory workflow was implemented using canonical human FASTA database imported from Uniprot (25th September 2024; 20,420 entries). Briefly, Pulsar search was done using the following parameters trypsin/P and LysC/P cleavage rules, specific digest type with 7-52 peptide length and max 2 missed cleavages. Carbamidomethyl (C) was set as a fixed modification and Acetyl (protein N-term), and oxidation (M) were set as variable modification with max variable modification set to 5 and identification FDR of 1% for PSM, peptide and protein group were applied for directDIA+ workflow with Automatic LFQ protein quantification method using the areas at MS2 level and automatic normalisation strategy. Automatic protein inference workflow using IDPicker algorithm was used for protein inference. The raw and processed data have been deposited to the ProteomeXchange Consortium via the PRoteomics IDEntifications (PRIDE) repository (Perez-Riverol, Csordas et al. 2019)"],"omics_type":["Proteomics"],"labhead":["Professor Robert (Bob) M Graham"],"instrument_platform":[""],"labhead_affiliation":["1. Victor Chang Cardiac Research Institute, Darlinghurst, NSW, 2010 2. St Vincent’s Hospital, Darlinghurst, NSW, 2010 3. University of New South Wales Sydney, Kensington, NSW, 2052"],"submission_type":["PARTIAL"],"species":["Homo Sapiens (human)"],"submitter_mail":["muhammad.alsherbiny@pharma.cu.edu.eg"],"publication":["42774489 Bax M, Ramalingam J, Romanov V, Alsherbiny MA, Junday K, Thorpe J, Hurley E, Lim M, Tarr I, Hesselson S, Giannoulatou E, Liu R, Hill A, Iismaa S, Graham RM. Robotic handling preserves iPSC-derived vascular smooth muscle cell differentiation. Mater Today Bio. 2026 40:103638 10.1016/j.mtbio.2026.103638"],"submitter_affiliation":["1) Freedman Foundation Metabolomics Facility, Innovation Centre, Victor Chang Cardiac Research Institute UNSW\n2) Pharmacognosy Department, Faculty of Pharmacy, Cairo University"],"submitter_country":["Australia"],"pubmed_abstract":["To tackle the global cardiovascular disease epidemic, vascular cell culture requires scalable, efficient, and reproducible methods for differentiating cells. However, no studies have validated whether automation, essential for scale-up, preserves differentiation quality of cells. Here, we developed a differentiation protocol for induced pluripotent stem cell (iPSC)-derived vascular smooth muscle cells (iVSMCs) that is applicable to both manual, and automated robotic culture. iPSC lines generated from male and female donors across various ages were used to optimize the generation of iVSMCs. iVSMCs had consistent morphology and protein expression, which were largely similar to those of primary VSMCs, including expression of myosin heavy chain-11 (MYH11), α-smooth muscle actin (α-SMA), transgelin (TAGLN) and calponin (CNN1). Functionally, these iVSMCs responded to the vasoconstrictor carbachol. Coupling this protocol with an automated Hamilton liquid-handling robotics system allowed the generation of iVSMCs in large quantities, that were morphologically similar to manually differentiated iVSMCs. Comparative proteomic analysis confirmed that protein expression was substantially the same between automated and manual differentiation methods. Automation markedly reduced manual labor and facilitated increased production without sacrificing cell quality. This study demonstrates the feasibility of automating iVSMC differentiation, marking a significant step towards scalable VSMC manufacture for three-dimensional applications, and for the ever-growing demands of organoid and tissue engineering applications."],"pubmed_title":["Robotic handling preserves iPSC-derived vascular smooth muscle cell differentiation."],"pubmed_authors":["Bax Monique M, Ramalingam Jeya J, Romanov Valentin V, Alsherbiny Muhammad A MA, Junday Keerat K, Thorpe Jordan J, Hurley Emily E, Lim Melanie M, Tarr Ingrid I, Hesselson Stephanie S, Giannoulatou Eleni E, Liu Renjing R, Hill Adam A, Iismaa Siiri S, Graham Robert M RM"],"additional_accession":[]},"is_claimable":false,"name":"Robotic Handling Preserves Induced Pluripotent Stem Cell Derived Vascular Smooth Muscle Cells Differentiation using a Weekend-free, Automatable, Low-variability, Low-cost (WALL) Protocol","description":"As tissue engineering and high-throughput disease modeling develop further, necessitating large quantities of viable cells, automation will be essential. Robotic liquid handling systems are increasingly employed for generating large quantities of induced pluripotent stem cell (iPSC)-derived cells, which requires the critical development of scalable protocols. No studies have yet evaluated how using robotic liquid handling may alter the differentiation of cells compared to traditional manual handling. Here, we report a scalable differentiation protocol, which robustly generates iPSC-derived VSMCs (iVSMCs) from a cohort of female and male donors of varying age (N=5 lines). We demonstrate for the first time that these cells, differentiated through an automated robotic pipeline, had no significant differences in phenotype, as assessed morphologically and by proteomic analysis, compared to manual differentiation. The ability to automate this protocol facilitates the differentiation of iVSMC in large quantities, generating consistent, functionally relevant cells, setting a precedence for the application of automation in generating iPSC-derived cells.","dates":{"publication":"2026-09-28","submission":"2025-11-01"},"accession":"PXD070184","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:35795","NEWT:2042546","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:57577","NEWT:247480","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:77173","NEWT:112509","NEWT:475174","NEWT:111205","NEWT:1599","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:12215","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:1596","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NEWT:1748418","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:1907535","NEWT:135622","NEWT:4615","NEWT:67352","NEWT:672127","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:3760","NEWT:1584","NEWT:2086595","NEWT:36630","NEWT:1585","NEWT:383379","NEWT:381198","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:3357","NEWT:111225","NEWT:7719","NEWT:29073","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:29064","NEWT:108061","NEWT:60711","NEWT:2020713","NEWT:224308","NEWT:273791","NEWT:3347","NEWT:49928","NEWT:3349","NEWT:160621","NEWT:2502409","NEWT:212790","NEWT:110368","NEWT:55350","NEWT:1916073","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:1912919","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:370038","NEWT:498211","NEWT:345076","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:33554","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:200795","NEWT:37919","NEWT:2579791","NEWT:171719","NEWT:44685","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:260499","NEWT:410661","NEWT:329854","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:2649329","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:270643","NEWT:3715","NEWT:1538","NEWT:2065263","NEWT:199225","NEWT:177437","NEWT:717966","NEWT:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:28174","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:980415","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:66440","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:34878","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:2714896","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:1608994","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:56636","NEWT:2709072","NEWT:2499525","NEWT:1122981","NEWT:2853422","NEWT:1122984","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:45972","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:294607","NEWT:1502","NEWT:128017","NEWT:2836","NEWT:376686","NEWT:237610","NEWT:95486","EFO:0001352","NEWT:1385720","NEWT:119562","NEWT:1883446","NEWT:31345","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:1945864","NEWT:317047","NEWT:1509","NEWT:491","NEWT:460523","NEWT:45954","NEWT:235443","NEWT:1579","NEWT:133894","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:1922344","NEWT:108458","NEWT:5936","NEWT:85692","NEWT:321967","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:279010","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:415978","NEWT:1255228","NEWT:410289","NEWT:191701","NEWT:373153","NEWT:1640278","NEWT:9054","NEWT:472","NEWT:940825","NEWT:29459","NEWT:529507","NEWT:1071661","NEWT:1380096","NEWT:28128","NEWT:470","NEWT:41122","NEWT:5911","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:11723","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166314","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:3726","NEWT:906914","NEWT:81231006","NEWT:30455","NEWT:28141","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:3728","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:548","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT:2126348","NEWT:5180","NEWT:256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