Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/RawData.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_15_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_17_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_1_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_10_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_6_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_4_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_7_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_12_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_9_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_14_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_12_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_4_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_18_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Pool_GPF_500-600.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_17_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_2_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_13_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_15_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Pool_GPF_400-500.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_9_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_2_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_7_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_5_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_10_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Pool_GPF_700-800.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_8_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_13_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_3_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_18_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_16_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Pool_GPF_900-1000.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_5_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_11_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_1_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Pool_GPF_600-700.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_6_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Pool_GPF_800-900.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_3_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_16_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_11_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_8_1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD075611/Sample_14_2.rawprimaryOK200harald.hundsberger@imc.ac.atAdrian LendvaiMass SpectrometryData-independent acquisitionNot availablehttps://www.ebi.ac.uk/pride/archive/projects/PXD075611Bone Matrix2.1. Bone Graft Preparation Cortical and cancellous allograft samples were provided by the Cells + Tissuebank Austria gemeinnützige GmbH. Processing native bone into graft material follows the Allotec® purification procedure (compliant with the internal regulatory procedure). This includes mechanical cleaning (cutting, sawing, centrifugation), chemical cleaning (ultrasonic bath with water-for-injection (WFI), treatment with diethyl ether, ethanol, and hydrogen peroxide), followed by lyophilization and demineralization as well as heat treatment and sterilization (gamma irradiation). The grafts were categorized into three experimental groups reflecting increased processing: (1) demineralized bone granules (DBG), (2) wet heat-treated DBG, referred to as non-sterile Putty-heat-treated (PHT), and (3) gamma irradiated PHT, referred to as Putty-gamma-irradiated (PGI). Heat treatment, rehydration, and putty formulation were performed according to the validated national and international manufacturing protocols of the tissue bank and are therefore not disclosed in detail (European Directorate for the Quality of Medicines & HealthCare; Gewebebankenverordnung) [22, 23]. These procedures are essential for creating paste-like and moldable consistencies. Irradiation was performed by gamma irradiation according to the manufacturer’s validated sterilization protocol. Each group was prepared from both cortical and cancellous allograft products, resulting in a total of six conditions. Material from multiple donors was pooled to minimize donor-specific effects. Measurements represent technical replicates of pooled material. 2.2. Protein Extraction and Quantification Proteins were extracted from 50 mg of the bone grafts using 8 M urea (Carl Roth GmbH + Co. KG; 3941.3) in 20 mM ammonium bicarbonate (ABC) buffer (pH 8.0) (Merck KGaA; 09830-500G) at room temperature for 24 hours under constant agitation. Following extraction, lysates were isolated by centrifugation at 8,000 × g for 1 minute, followed by 12,000 × g for 1 minute. The supernatants were diluted to 1 M urea in 20 mM ABC buffer, and the total protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Cell Signaling Technology Europe; 7780S) employing a bovine serum albumin (BSA) standard curve ranging from 0 to 0.5 µg/µL in six dilutions. 2.3. Protein Digest Equal amounts of protein (6 µg per sample) were enzymatically digested using the iST Sample Preparation Kit, 96x (PreOmics GmbH; P.O.00027) according to the manufacturer’s protocol. The peptides were eluted twice with 90 µL elution buffer, which differed from the standard protocol. Accordingly, 180 µL of the eluted peptides were transferred into respective High-Performance Liquid Chromatography (HPLC) vials, followed by vacuum drying at 45 °C for approximately 4 hours (Eppendorf; Concentrator plus; Type 5305). The samples were stored at -80 °C. 2.4. High-Performance Liquid Chromatography with Mass Spectrometry (HPLC-MS) Samples were analyzed using an Ultimate 3000 RSLCnano System coupled to an Orbitrap Eclipse Tribrid Mass Spectrometer (both Thermo Fisher Scientific; Vienna; Austria). The dried peptides were resuspended in 12 µL of LC-MS grade water (Fisher Scientific; W/0112/17), 2 % acetonitrile (ACN) (Fisher Scientific; A/0638/17), and 0.1% formic acid (Carl Roth GmbH + Co. KG; 1EHK.1) prior to HPLC-MS analysis. 2 µL were injected onto a PepMap RSLC EASY-Spray column (C18, 2 µm, 100 Å, 75 µm x 50 cm; Thermo Fisher Scientific; ES903). Separation occurred at 300 nL/min with a linear gradient ranging from 2-35 % mobile phase B (2 % H2O, 98 % ACN, 0.1 % FA) within 60 minutes, resulting in a total method time of 80 minutes. The mass spectrometer was operated in data-independent-acquisition (DIA) mode with the FAIMS Pro System in positive ionization mode at CV-45. MS1 scans were acquired in a scan range of 350-1400 m/z with a resolution of 120000 @200 m/z. For DIA scans, the precursor mass range was set to 400-1000 m/z with a 14 m/z isolation window and 1 m/z window overlap, resulting in 43 independent scans. HCD fragmentation occurred at 30 % NCE and fragments were analyzed in the Orbitrap at a resolution of 30000 @200 m/z. To deepen the analysis, a pool of all samples was created and used for gas phase fractionation (GPF) [10.1038/s41467-020-15346-1]. Sample pool was analyzed six times consecutively with smaller precursor mass ranges of 100 m/z (400-500, 500-600, 600-700, 700-800, 800-900, 900-1000 m/z) and isolation windows of 4 m/z with 2 m/z window overlap. DIA-NN (version 18.1.1) [10.1038/s41592-019-0638-x] was used for protein identification and quantification. The GPF samples were first searched against the human protein database (Uniprot; version 10.2021; 20386 entries), and a spectral library was created using the identified peptides. The main samples were searched using the spectral library together with the human FASTA database to maximize the number of identified proteinsPride2.5. Proteomic Data Analysis HPLC–MS protein data analyzed in R (Posit Software; version 2024.09.0+375). Protein intensities, treated as missing if not detected, were log₂-transformed after adding a pseudocount (0.1× 1st percentile of all non-zero intensities). Proteins were retained if they were detected in at least one processing step x bone type subset fulfilling 4 of 6 valid sample HPLC-MS measurements per condition. No global detection or variance filtering was applied. RDA assessed proteome structure across bone types and steps, imputing missing data with Perseus-style Gaussian imputation. Differential abundance was analyzed per step with Limma, on log₂ intensities without imputation. Only proteins with at least two observed values per group were included in the statistical testing, using FDR <0.05 and log₂FC > 1. The results were visualized with volcano plots. Protein functions were annotated with STRING Gene Ontology (GO) (Biological Process) analyses (version 12.0) [24]. The protein panels were visualized on heat maps and projected onto the initial RDA space. All scripts in the Supplementary Materials.ProteomicsHarald HundsbergerIMC University of Applied Sciences KremsPARTIALHomo Sapiens (human)adrian.lendvai@hotmail.com42121943 Lendvai A, Weitzenböck HP, Klein C, Wiesner C, Seeboeck R, Entler B, Neuditschko B, Herzog F, Matzner M, Pichler M, De Luna A, Nehrer S, Hundsberger H. Distinct Extracellular Matrix Protein Signatures of Cortical and Cancellous Bone Allografts Following Processing for Clinical Use. Cells. 2026 15(9):842 10.3390/cells15090842IMC University of Applied Sciences KremsAustriaDemineralized bone matrices (DBMs) are widely used in bone replacement therapy. Bone tissue of either cancellous or cortical origin is decellularized, demineralized, and sterilized during processing, while retaining portions of native organic extracellular matrix (ECM) proteins that regulate cell-matrix interactions during bone repair. The ECM largely accounts for the distinct functions of cortical and cancellous bone. Differences in three-dimensional architecture and matrix density between cancellous and cortical bone may therefore affect ECM proteome signatures and the resulting cellular microenvironment. In this study, ECM proteins were extracted from processed cancellous and cortical allografts at multiple processing steps and analyzed by quantitative mass spectrometry. We identified distinct extractable proteome signatures associated with bone metabolic functions. Cancellous grafts were relatively enriched in proteins associated with inflammatory, coagulative, and immune-related processes, whereas cortical grafts showed higher abundance of structural and matrix-organization-associated proteins. More extensively processed product formats showed fewer significant protein differences between the cortical and cancellous bone type. Within the limitations of pooled donor material and absent functional validation, these findings provide a proteomic framework for future characterization and evaluation of DBM-based allograft products.Distinct Extracellular Matrix Protein Signatures of Cortical and Cancellous Bone Allografts Following Processing for Clinical Use.Lendvai Adrian A, Weitzenböck Hans Peter HP, Klein Christian C, Wiesner Christoph C, Seeboeck Rita R, Entler Barbara B, Neuditschko Benjamin B, Herzog Franz F, Matzner Michael M, Pichler Monika M, De Luna Andrea A, Nehrer Stefan S, Hundsberger Harald HfalseDistinct Extracellular Matrix Protein Signatures of Cortical and Cancellous Bone Allografts Following Processing for Clinical UseDemineralized bone matrices (DBMs) are widely used in bone replacement therapy. Bone tissue of either cancellous or cortical origin is decellularized, demineralized, and sterilized during processing, while retaining portions of native organic extracellular matrix (ECM) proteins that regulate cell-matrix interactions during bone repair. The ECM largely accounts for the distinct functions of cortical and cancellous bone. Differences in three-dimensional architecture and matrix density between cancellous and cortical bone may therefore affect ECM proteomic signatures and the resulting cellular microenvironment. In this study, ECM proteins were extracted from processed cancellous and cortical allografts at multiple processing steps and analyzed by quantitative mass spectrometry. We identified distinct extractable proteome signatures associated with bone metabolic functions. Cancellous grafts were enriched in proteins linked to inflammatory, coagulative, and immune-related processes, essential in early-phase bone healing and immune cell recruitment. Cortical grafts showed higher abundance of structural and matrix-organizing proteins linked to ECM organization and bone maturation, which are hallmarks of late-phase bone repair. Progressive processing reduces the number of differentially abundant proteins in both bone types. These findings are relevant for the selection, application, and quality assessment of DBM-based allograft products and their potential influence on the cellular microenvironment during bone regeneration.2026-06-032026-03-13PXD075611NEWT:1773NEWT:3555NEWT:38783NEWT:1182590NEWT:2NEWT:10090NEWT:935293NEWT:749200NEWT:35554NEWT:4120NEWT:5693NEWT:347515NEWT:1216979NEWT:307972NEWT:92867NEWT:990346NEWT:544496NEWT:5334NEWT:145953NEWT:257309NEWT:284812NEWT:115104NCBITaxon:1313NEWT:43330NEWT:67825NEWT:44544NEWT:13076NEWT:373995NEWT:544404NEWT:3702NEWT:8839NEWT:4232NEWT:990119NEWT:1736309NEWT:4113NEWT:7227NEWT:11298NEWT:7469NEWT:885318NEWT:171101NEWT:4081NEWT:876138NEWT:554NEWT:5691NEWT:408170NEWT:260710NEWT:106592NEWT:237561NEWT:9913NEWT:10036NEWT:4100NEWT:7574NEWT:1351NEWT:1076NEWT:6763NEWT:7215NEWT:803NEWT:8030NEWT:380394NEWT:272563NEWT:507601NEWT:1639NEWT:188229NEWT:4909NCBITaxon:79857NEWT:746360NEWT:6239NEWT:1589NEWT:135588NEWT:135622NEWT:216257NEWT:6915NEWT:9986NEWT:101510NEWT:95486NEWT:3880NEWT:58002NEWT:9103NEWT:4577NEWT:5664NEWT:2157NEWT:146479NEWT:1000589NEWT:145943NEWT:85962NEWT:160488NEWT:317447NEWT:3635NEWT:7955NCBITaxon:2NEWT:235443NEWT:985076NEWT:7959NEWT:2261NEWT:3197NEWT:9615NEWT:884019NEWT:4565NEWT:1264690NEWT:169963NCBITaxon:38727NEWT:36329NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:626528NEWT:139927NEWT:4558NEWT:9606NEWT:367830NEWT:243230NEWT:931281NEWT:373153NEWT:7029NEWT:1283300NEWT:334747NEWT:470NCBITaxon:79824NCBITaxon:4563NEWT:3218NEWT:5759NEWT:9838NCBITaxon:9615NEWT:1736231NEWT:1193501NEWT:6287NEWT:2242NEWT:6326NEWT:9796NEWT:2762NEWT:5476NEWT:562NEWT:260707NEWT:287NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1836NEWT:1735272NEWT:29760NEWT:260705NEWT:80863NEWT:1148NEWT:4932NEWT:70448NEWT:9825NEWT:3603NEWT:698936NEWT:2759NEWT:39946NEWT:11676NEWT:9823NEWT:100226NCBITaxon:6073NEWT:4530NEWT:4896NEWT:6279NEWT:7370NEWT:573NEWT:6282NEWT:7091NEWT:113450642121943