Journal of Proteome Research最新文献

{"title":"Optimized Hemoglobin Depletion Improves Clinical Proteomics from Dried Blood Spots","authors":"Hannah Ging,&nbsp;Rosemary E. Maher,&nbsp;Elin Davies,&nbsp;Philip J. Brownridge,&nbsp;Anirudh Rao,&nbsp;Alan D. Salama,&nbsp;Louise Oni,&nbsp;Claire E. Eyers,&nbsp;Andrew J. Chetwynd","doi":"10.1021/acs.jproteome.6c00534","DOIUrl":"https://doi.org/10.1021/acs.jproteome.6c00534","url":null,"abstract":"<p>Equitable access to large sample cohorts for robust,high-throughputproteomics for biomarker discovery is a major barrier to widescaleclinical implementation. Dried blood spots (DBS) offer a minimallyinvasive alternative to venous blood draws, enabling at-home microsampling(&lt;50 μL) for centralized analysis, thus enhancing researchparticipation. This approach is particularly relevant for under-representedgroups, including children, the elderly, minority backgrounds andthose with long-term health conditions such as chronic kidney disease(CKD), where disease fluctuations may occur outside the clinic, andvein preservation is critical. Proteomic analysis has demonstratedgreat utility in monitoring disease progression, and for biomarker/therapeutictarget discovery. However, liquid chromatography–tandem massspectrometry (LC–MS/MS) of whole blood is hindered by the widedynamic range and the relatively high-abundance of proteins such ashemoglobin, compromising biomarker discovery. Here, we establish anoptimized workflow for protein extraction and hemoglobin depletionfrom microsamples obtained using DBS, enabling sensitive and high-throughputproteomic analysis. We demonstrate that hemoglobin depletion increasesprotein identifications by ∼50%, mitigating ion suppressionand dynamic range effects, enabling the identification of putativebiomarkers from patients with stage 5 CKD on dialysis. We also evaluateda commercial cell-free DBS device which yielded a sample more representativeof plasma compared to traditional DBS and enabled greater depletionof hemoglobin compared to traditional DBS with hemoglobin depletionmethods. Our findings offer a scalable approach for biomarker discovery,facilitating remote, longitudinal clinical studies.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"25 11","pages":"6151–6165"},"PeriodicalIF":3.8,"publicationDate":"2026-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149330217","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mining of Small Open Reading Frame-Encoded Peptides from Chlamydomonas nivalis without Its Reference Genome","authors":"Sheng Yao,&nbsp;Zhao Peng,&nbsp;Gai Liu,&nbsp;Kaiyao Huang,&nbsp;Cuihong Wan","doi":"10.1021/acs.jproteome.6c00665","DOIUrl":"https://doi.org/10.1021/acs.jproteome.6c00665","url":null,"abstract":"<p>Small open reading frame (sORF)-encoded peptides (SEPs)participatein diverse biological processes, yet their identification in nonmodelorganisms is limited by the lack of high-quality reference genomes.Here, we developed an integrative proteogenomic pipeline to discoverSEPs in <em>Chlamydomonas nivalis</em> by combiningsORFs predicted from its transcriptome with those derived from thegenomes of the related species, <em>Chloromonas eustigma</em> and <em>Volvox carteri</em>. Using this strategy,we identified 978 endogenous SEPs supported by transcript evidenceand 1 999 candidate SEPs supported by cross-species proteogenomicevidence. SEPs predicted from transcriptomic and related-species genomicdata showed broadly similar physicochemical and evolutionary features.Importantly, incorporation of related-species genomic data substantiallyincreased identification coverage, with over 95% of SEPs identifiedthrough these databases absent from the transcriptome-derived dataset. Functional analysis identified several cold-responsive candidates,including an Lhca6-homologous SEP and several intrinsically disorderedregion-rich SEPs. Heterologous expression of selected candidates in <em>C. reinhardtii</em> further showed that two SEPs enhancedcold tolerance. These results demonstrate the value of integratingtranscriptomic and related-species genomic resources for SEP discoveryand provide functional evidence for SEP involvement in cold adaptation.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"25 11","pages":"6392–6405"},"PeriodicalIF":3.8,"publicationDate":"2026-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149330225","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Integrative Single-Cell Proteomics and Network Analysis Identifies DHX9 as a Key Regulator of Oocyte Maturation and a Therapeutic Target for Oocyte Developmental Arrest","authors":"Guang-Yi Cao,&nbsp;Si-Pei Liu,&nbsp;Ao-Lei Guo,&nbsp;Jing-Xin Li,&nbsp;Ru-Yu Jiang,&nbsp;Shan-Shan Wang","doi":"10.1021/acs.jproteome.6c00419","DOIUrl":"https://doi.org/10.1021/acs.jproteome.6c00419","url":null,"abstract":"<p>Oocyte developmental arrest at the germinal vesicle (GV)or metaphaseI (MI) stage is a major cause of infertility, yet its molecular mechanismsremain poorly understood. Here, we performed ultradeep single-cellproteomics on 8 human oocytes (4 GV-stage and 4 MI-stage) and 12 mouseoocytes (3 GV, 3 MI, 3 MII, and 3 FGO) to investigate the proteomiclandscape of developmental arrest. We identified an average of 5,574proteins per human oocyte (range: 4,350–6,157) and 3,477 permouse oocyte (range: 3,315–3,634), revealing aberrant activationof mRNA splicing pathways in arrested oocytes. Integrative networkanalysis identified the RNA helicase DHX9 as a central hub withina conserved coexpression module. Mechanistically, elevated DHX9 expressionmaintained transcriptional hyperactivity, preventing the transitionto transcriptional quiescence required for maturation. Critically,pharmacological inhibition of DHX9 with ATX968 restored transcriptionalsilencing and significantly rescued meiotic maturation, achieving95% GVBD and 80% PBE at 1 μM ATX968, comparable to wild-typecontrols (98%/82%) and significantly exceeding vehicle-treated arrestedoocytes (8% GVBD; <em>p</em> &lt; 0.001). In summary, ourstudy demonstrates that DHX9-driven dysregulation of RNA splicingis a key mechanism underlying oocyte developmental arrest. These findingshighlight DHX9 as a promising therapeutic target and provide a potentialstrategy for improving oocyte competence in clinical infertility treatment.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"25 11","pages":"6056–6069"},"PeriodicalIF":3.8,"publicationDate":"2026-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149330345","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Integrated Proteomic and Ubiquitinomic Reveal Ubiquitination Remodeling in Pelvic Organ Prolapse","authors":"Wei Liu,&nbsp;Yafei Gao,&nbsp;Yike Gao,&nbsp;Yiwei Zhang,&nbsp;Yaqian Li,&nbsp;Yuxin Dai,&nbsp;Jian Sun,&nbsp;Juntao Yang","doi":"10.1021/acs.jproteome.6c00091","DOIUrl":"https://doi.org/10.1021/acs.jproteome.6c00091","url":null,"abstract":"<p>Pelvicorgan prolapse (POP) is characterized by localized vaginal-walltissue failure, but post-translational regulation remains incompletelyunderstood. We integrated quantitative proteomic and ubiquitinomicprofiling of vaginal tissues from 10 women with POP and 8 controls.Differentially expressed proteins were enriched in ECM-associated,cytoskeletal, and adherens-junction pathways, whereas the ubiquitinomerevealed reproducible site-specific changes. Integrated analysis highlightedβ-catenin (CTNNB1) and collagen type III alpha 1 chain (COL3A1),including the previously reported CTNNB1 K233 site and two newly identifiedubiquitination sites, CTNNB1 K672 and COL3A1 K674. RepresentativeMS/MS spectra supported assignment of these sites. Together, thesedata define POP-associated ubiquitination remodeling and nominateadherens-junction and ECM-linked proteins for future functional validation.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"25 11","pages":"5750–5762"},"PeriodicalIF":3.8,"publicationDate":"2026-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149330290","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative Proteomics Reveals CBX4 Promotes Cervical Cancer Cell Proliferation by Regulating PFKFB4","authors":"Rui Su,&nbsp;Aiping Sun,&nbsp;Tiesuo Zhao,&nbsp;Hui Wang","doi":"10.1021/acs.jproteome.6c00611","DOIUrl":"https://doi.org/10.1021/acs.jproteome.6c00611","url":null,"abstract":"<p>CBX4is an E3 SUMO ligase involved in the initiation and progressionof multiple malignancies. However, the molecular mechanism underlyingthe functional significance of CBX4 in cervical cancer (CC) cellsremains unclear. Here, we used CRISPR/Cas9 genome editing to generateCBX4-knockout cell lines and found that CBX4 loss markedly suppressedHeLa cell proliferation. A label-free quantitative proteomics approachbased on data-independent acquisition (DIA) was employed to systematicallyprofile CBX4-regulated proteins in HeLa cells. In total, 263 proteinsshowed significant changes upon CBX4 knockout, among which 171 proteinswere downregulated, and 92 were upregulated. Bioinformatics analysisrevealed that these differentially expressed proteins (DEPs) are associatedwith diverse cellular pathways, especially metabolic pathways. Furthermore,our results demonstrated that CBX4 promotes CC cell proliferationby regulating the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase4 (PFKFB4). Mechanistically, PFKFB4 is a direct target of the transcriptionalactivator SP1, and CBX4 interacts with SP1 and increases its SUMOylation.Therefore, CBX4 may exert its effects in CC cells through enhancingSP1-mediated <em>PFKFB4</em> transcription. In conclusion,the current study establishes a proteome-wide CBX4 regulatory networkand provides novel insights into the molecular pathogenesis of CC.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"25 11","pages":"6289–6300"},"PeriodicalIF":3.8,"publicationDate":"2026-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149330214","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Proximity Labeling: An Empowering Tool for Deciphering Molecular Mechanisms","authors":"Hao Liang,&nbsp;Shaoxiang Chen,&nbsp;Kai Gan,&nbsp;Renjie Chai,&nbsp;Zhenghong Bi","doi":"10.1021/acs.jproteome.6c00523","DOIUrl":"https://doi.org/10.1021/acs.jproteome.6c00523","url":null,"abstract":"<p>Proximitylabeling (PL) has become a widely used approach for studyingprotein–protein interactions (PPIs) and addressing limitationsof conventional techniques in capturing transient, weak interactions,and physiological protein networks within living systems. Over thepast decade, core PL enzymes, notably APEX2, BirA* (BioID), and TurboID,have been iteratively engineered, with reported improvements in temporalresolution, targeting specificity, and biocompatibility. This reviewsummarizes how PL technologies have developed and describes the enzymaticproperties, catalytic mechanisms, and reported performance trade-offsof the commonly used tools. We then survey applications of PL in neuroscience,cardiology, oncology, and infectious disease, where it has been usedto map compartment-specific proteomes and spatiotemporally regulatednetworks, including neural activity-driven PPIs, tumor microenvironmentcross-talk, and host–pathogen interfaces. Many of the resultingcandidates await orthogonal validation. We also describe recentlyreported conditional PL systems with optical, calcium- or oxygen-dependentcontrol. Finally, we discuss open questions and possible directionsfor PL development, including enzyme-directed evolution, multisignalresponsive regulation, cross-omics integration, and translationalapplications. This review is intended as a practical reference forresearchers to support the selection and application of PL platformsin specific experimental contexts from mechanistic studies to theidentification of candidate therapeutic targets.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"25 11","pages":"5648–5666"},"PeriodicalIF":3.8,"publicationDate":"2026-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149330279","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protein Aggregation Capture for Top-Down Proteomics","authors":"Isabella G. Feltenstein,&nbsp;Bryon S. Drown","doi":"10.1021/acs.jproteome.6c00583","DOIUrl":"https://doi.org/10.1021/acs.jproteome.6c00583","url":null,"abstract":"<p>Proteins are dynamically regulated by a myriad of post-translationalmodifications (PTMs) that control their stability, conformation, activity,subcellular localization, and local interactions. Capturing the precisecomposition of these various modification states, or proteoforms,is a principal objective of top-down proteomics (TDP). By ionizingintact proteoforms and combining measurements of precursor ion andfragment ion masses, the position, stoichiometry, and combinationof PTMs can be determined. Despite the highly valuable measurementsthat TDP can provide, it is typically less sensitive than correspondingpeptide-level analysis, with many reports utilizing input materialin the microgram to milligram range. Contributing to this lack ofsensitivity is the risk of sample loss due to nonspecific bindingto surfaces during sample preparation. The most widely employed samplepreparation approaches for TDP either require high sample input (e.g.,precipitation and ultrafiltration) or fail to effectively remove surfactants(e.g., solid-phase extraction). These limitations have hindered advancementof targeted TDP applications involving immunoprecipitation and otherenrichment strategies. Bead-assisted protein aggregation, also referredto as single-pot, solid-phase-enhanced sample preparation (SP3), hasemerged as a popular sample preparation strategy for bottom-up proteomicworkflows, but has only been used in TDP with high concentrationsof formic acid (40%) or a secondary ion exchange chromatography cleanupstep. We envisioned a magnetic bead-based protein cleanup approachthat proceeds directly to MS analysis with a judicious choice of beadsurface chemistry and elution conditions. Here we report a samplepreparation method using hydroxyl-functionalized magnetic beads thatproceeds directly to MS analysis. Applied to SDS-containing HeLa lysate,hydroxyl beads with 0.5% TFA recovery identified as many proteinsas methanol-chloroform-water precipitation (231 vs 228), spanningthe same ranges of mass, isoelectric point, hydrophobicity, and cellularcompartment, with comparable precursor intensity at a modest costin precursor signal-to-noise.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"25 11","pages":"6245–6255"},"PeriodicalIF":3.8,"publicationDate":"2026-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149330409","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Stage-Specific Proteomic Signatures of Matrix Vesicles Support Functional Transitions during Biomineralization","authors":"Juçara Gastaldi Cominal,&nbsp;Larwsk Hayann,&nbsp;Maryanne Trafani de Melo,&nbsp;Iara Souza Lima,&nbsp;Angie Lissette Atoche Puelles,&nbsp;Lucas Fabricio Bahia Nogueira,&nbsp;Saida Mebarek,&nbsp;Ana Paula Ramos,&nbsp;José Luis Millán,&nbsp;Massimo Bottini,&nbsp;Pietro Ciancaglini","doi":"10.1021/acs.jproteome.6c00508","DOIUrl":"https://doi.org/10.1021/acs.jproteome.6c00508","url":null,"abstract":"<p>Matrix vesicles (MVs)are extracellular vesicles releasedby mineralizing-competentcells, including chondrocytes and osteoblasts. Although MVs are classicallyassociated with biomineralization, whether distinct MV populationsare produced throughout osteoblast differentiation remains poorlyunderstood. Here, we investigated the biophysical, biochemical, andproteomic profiles of MVs isolated throughout osteogenic differentiationto evaluate the hypothesis of functional transitions during biomineralizationprogression. MVs were isolated from osteoblast cultures at three timepoints, representing the onset, establishment, and propagation stagesof biomineralization. Biophysical and biochemical characterizationrevealed temporal differences in MV properties. Stage-specific proteomicsignatures were identified among MV groups. Early stage MVs (7–14days) exhibited enrichment of proteins associated with the extracellularmatrix (ECM) organization, intracellular trafficking, cytoskeletaldynamics, and adhesion, suggesting a role in preparing the mineralizingmicroenvironment. In contrast, later-stage MVs (14–21 days)showed enrichment of proteins related to ECM interactions, calciumresponse, proteoglycan binding, metabolism, and regulatory signalingpathways, consistent with enhanced mineral propagation. Collectively,our findings demonstrate that distinct MVs are produced throughoutosteoblast differentiation and support a functional transition modelduring biomineralization, in which MV cargo and properties are dynamicallycoordinated with the temporal demands of ECM mineralization.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"25 11","pages":"6380–6391"},"PeriodicalIF":3.8,"publicationDate":"2026-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149330412","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Targeted Analysis of >1500 Plasma Proteoforms via Individual Ion Mass Spectrometry","authors":"Aniel Sanchez,&nbsp;Indira Pla,&nbsp;Katrina N. Peterson,&nbsp;Vincent White,&nbsp;Troy D Fisher,&nbsp;Michael A. R. Hollas,&nbsp;Taojunfeng Su,&nbsp;Nhat Hoang Van Le,&nbsp;Claire R. Harrington,&nbsp;Paolo Cravedi,&nbsp;David N. Assis,&nbsp;Paola Barrios,&nbsp;Therese Elaine Banea,&nbsp;Daniela P. Ladner,&nbsp;Eleonora Forte,&nbsp;Matthew Lucky,&nbsp;John T. Wilkins,&nbsp;Douglas E Vaughan,&nbsp;Michael A. Caldwell,&nbsp;John P. McGee,&nbsp;Neil L. Kelleher","doi":"10.1021/acs.jproteome.6c00449","DOIUrl":"https://doi.org/10.1021/acs.jproteome.6c00449","url":null,"abstract":"<p>Plasma proteomics has emerged as a powerful approachfor interrogatinghuman biology, identifying disease-associated proteomic changes, andadvancing precision diagnostics. In recent years, affinity-based plasmaproteomics platforms with predefined protein panels, such as Olinkand SomaScan, have emerged to complement bottom-up mass spectrometry(BUP). This study leverages a novel mass spectrometry platform tocapture targeted proteoform information lost by mainline antibody-,aptamer-, and BUP-driven workflows. The Plasma Proteoform Assay (PPA)uses Individual Ion Mass Spectrometry (I²MS) to resolvemixtures of intact proteins presented by direct injection. Two proteoformpanels, <strong>PPA 526</strong> and <strong>PPA 1514,</strong> were definedfrom human plasma samples obtained from 81 individuals. The panelsquantify 526 proteoforms derived from 59 genes and 1,514 proteoformsfrom 155 genes, respectively. Across both panels, the proportion oftargeted proteoforms with CVs below 20% ranged from 59% to 80%. PPAwas benchmarked in studies including 9 pooled plasma samples derivedfrom subjects with hepatic cirrhosis (N = 30 individuals) and a cohortof resilient agers carrying a SERPINE1 (PAI-1) mutation (N = 27 individuals).PPA signatures distinguished SERPINE1 mutation carriers from affectedindividuals and provided sufficient resolution to discriminate amongcirrhosis disease stages. In summary, we present <strong>PPA 526</strong> and <strong>PPA 1514</strong>, the first scalable plasma proteoformpanels capable of tracking hundreds to thousands of targets in a fewminutes per sample.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"25 11","pages":"5985–6002"},"PeriodicalIF":3.8,"publicationDate":"2026-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149330187","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of the SPEED Sample Preparation Protocol for Metabolomics","authors":"Shuhd BinEshaq,&nbsp;Serine Boucherabine,&nbsp;Omar Bashir,&nbsp;Nesrin Mohamed,&nbsp;Abiola Senok,&nbsp;Mohammed Uddin,&nbsp;Alexander D. Giddey","doi":"10.1021/acs.jproteome.6c00450","DOIUrl":"https://doi.org/10.1021/acs.jproteome.6c00450","url":null,"abstract":"<p>Multiomic (paired proteomic and metabolomic) analysesallow researchersto better decipher the underlying biology being studied. While samplepreparation techniques for multiomic analyses exist, they are oftenautomation-unfriendly or secondary to preferred techniques for proteomicanalyses. The recently published, popular Sample Preparation by EasyExtraction and Digestion (SPEED) protocol offers a single sample preparationtechnique for near-universal sample preparation across sample typesand is used routinely in many proteomics laboratories; however, thisprotocol has not previously been investigated as to its suitabilityfor metabolomic analyses. We tested the compatibility of two variationsof the SPEED protocol: trifluoroacetic acid (TFA) lysis followed bydrying and methanol extraction and TFA lysis and neutralization followedwith SP4 cleanup. We compared these protocols with standard metabolomicssample preparation protocols of direct extraction with methanol orACN and bead-beating in ammonium acetate lysis. The results show thatTFA lysis with drying and methanol extraction yielded quantificationresults that were comparable to or better than those of the otherprotocols tested. We additionally demonstrated the utility of thistechnique by performing multiomic analysis of iPSCs before and afterdifferentiation into neurons and identified coordinated dysregulationof metabolites and proteins in key neuronal differentiation pathways.We also observed that ACN extraction performed similarly to the goldstandard methanol extraction. Consequently, we conclude that laboratoriesthat already routinely use the SPEED protocol for dedicated proteomicscan obtain functional metabolomics samples for little to no additionaleffort or cost with regard to sample preparation.</p>","PeriodicalId":48,"journal":{"name":"Journal of Proteome Research","volume":"25 11","pages":"6497–6506"},"PeriodicalIF":3.8,"publicationDate":"2026-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149330188","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}