{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T15:00:39Z","timestamp":1785942039788,"version":"3.56.0"},"reference-count":25,"publisher":"American Chemical Society (ACS)","issue":"8","license":[{"start":{"date-parts":[[2026,7,10]],"date-time":"2026-07-10T00:00:00Z","timestamp":1783641600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,8,5]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Single-cell (SC) metabolomics holds great potential in the development of novel diagnostic tools and mechanistic insights into cell biology. Using high-resolution mass spectrometry (HRMS), the masses of a single cell\u2019s constituents can be determined with an accuracy high enough to derive their respective elemental compositions. Using a molecule\u2019s mass and its MS fragmentation pattern, in many cases a molecular structure can be assigned or looked up in databases. Due to the small measurement cell volume of an Orbitrap HRMS instrument, samples need to be scanned multiple times, which necessitates across-scan clustering per sample, and across-sample alignment of m\/z values. However, existing HRMS data processing software is not designed to process SC HRMS data, as it typically requires liquid chromatography retention times or reference spectra for m\/z clustering and alignment. Herein, a novel, robust SC HRMS m\/z clustering and alignment algorithm is presented and compared with two commercially available and industry standard algorithms used by Sciex MarkerView and Thermo FreeStyle. Furthermore, output is compared with clustering results from DBSCAN and MaldiQuant binning. Our algorithm, Global Clustering unTargeted Analysis (GCTA), enforces a strict maximum on the cluster size, thereby reducing the chance of peak aggregation. Furthermore, by design, GCTA enables noise filtering based on intensity and number of peaks. Across-scan clustering and across-sample alignment were contrasted for accuracy in finding peaks identified by commercial software output and peaks with known m\/z values corresponding to standards and HMDB and LipidMaps database hits. Comparisons are made based on data recorded for quality control samples containing standard mixes as well as SC HRMS data recorded for two different cell lines. This work shows that the presented algorithm is comparable in accuracy with respect to MarkerView and FreeStyle, reliably identifies compounds, is less prone to peak splitting than MaldiQuant binning while providing similar levels of error in clustering peaks, and successfully filters noise. Furthermore, it is shown to be competitive with DBSCAN, MaldiQuant binning and MarkerView when compared to theoretical m\/z values based on database hits. GCTA encompasses both m\/z clustering and reference-free alignment, which makes it pivotal to further development of untargeted SC HRMS metabolomics.<\/jats:p>","DOI":"10.1021\/jasms.6c00150","type":"journal-article","created":{"date-parts":[[2026,7,10]],"date-time":"2026-07-10T17:34:33Z","timestamp":1783704873000},"page":"1942-1951","source":"Crossref","is-referenced-by-count":0,"title":["An\n                    <i>m<\/i>\n                    \/\n                    <i>z<\/i>\n                    - and Intensity-Based\nHRMS Clustering Algorithm Targeted toward Single-Cell Metabolomics"],"prefix":"10.1021","volume":"37","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-3392-5603","authenticated-orcid":true,"given":"Dirk","family":"Wevers","sequence":"first","affiliation":[{"name":"Leiden Academic Centre for Drug Research , , Wassenaarseweg 76 , ,","place":["AL Leiden, The Netherlands, 2333"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-9173-3128","authenticated-orcid":true,"given":"Laura","family":"Koldenhof","sequence":"additional","affiliation":[{"name":"Leiden Academic Centre for Drug Research , , Wassenaarseweg 76 , ,","place":["AL Leiden, The Netherlands, 2333"]},{"name":"Universiteit Utrecht , Heidelberglaan 8 , ,","place":["CS Utrecht, The Netherlands, 3584"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6922-9423","authenticated-orcid":true,"given":"Laura","family":"Castaneda","sequence":"additional","affiliation":[{"name":"Leiden Academic Centre for Drug Research , , Wassenaarseweg 76 , ,","place":["AL Leiden, The Netherlands, 2333"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anouschka","family":"van der Zeeuw","sequence":"additional","affiliation":[{"name":"Hogeschool Leiden , Zernikedreef 11 , ,","place":["CK Leiden, The Netherlands, 2333"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hellen","family":"Fass","sequence":"additional","affiliation":[{"name":"Leiden Academic Centre for Drug Research , , Wassenaarseweg 76 , ,","place":["AL Leiden, The Netherlands, 2333"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7871-2073","authenticated-orcid":true,"given":"Thomas","family":"Hankemeier","sequence":"additional","affiliation":[{"name":"Leiden Academic Centre for Drug Research , , Wassenaarseweg 76 , ,","place":["AL Leiden, The Netherlands, 2333"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Charlie","family":"Clark","sequence":"additional","affiliation":[{"name":"Leiden Academic Centre for Drug Research , , Wassenaarseweg 76 , ,","place":["AL Leiden, The Netherlands, 2333"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2157-4399","authenticated-orcid":true,"given":"Ahmed","family":"Ali","sequence":"additional","affiliation":[{"name":"Leiden Academic Centre for Drug Research , , Wassenaarseweg 76 , ,","place":["AL Leiden, The Netherlands, 2333"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,7,10]]},"reference":[{"issue":"7631","key":"2026080510111922400_cit1","doi-asserted-by":"publisher","first-page":"153","DOI":"10.1038\/540153a","article-title":"Metabolomics:\nSmall Molecules, Single Cells","volume":"540","author":"Fessenden","year":"2016","journal-title":"Nature"},{"issue":"30","key":"2026080510111922400_cit2","doi-asserted-by":"publisher","first-page":"10567","DOI":"10.1021\/acs.analchem.2c02279","article-title":"Single-Cell\nMass Spectrometry Enables Insight into Heterogeneity\nin Infectious Disease","volume":"94","author":"Nguyen","year":"2022","journal-title":"Anal. Chem."},{"issue":"5","key":"2026080510111922400_cit3","doi-asserted-by":"publisher","first-page":"3667","DOI":"10.1021\/acs.analchem.8b05739","article-title":"Metabolic Discrimination\nof Breast Cancer Subtypes\nat the Single-Cell Level by Multiple Microextraction Coupled with\nMass Spectrometry","volume":"91","author":"Wang","year":"2019","journal-title":"Anal. Chem."},{"key":"2026080510111922400_cit4","doi-asserted-by":"publisher","first-page":"339621","DOI":"10.1016\/j.aca.2022.339621","article-title":"Single Cell\nMass Spectrometry Analysis of Drug-Resistant\nCancer Cells: Metabolomics Studies of Synergetic Effect of Combinational\nTreatment","volume":"1201","author":"Chen","year":"2022","journal-title":"Anal. Chim. Acta"},{"issue":"24","key":"2026080510111922400_cit5","doi-asserted-by":"publisher","first-page":"2000","DOI":"10.1002\/elps.202300105","article-title":"Opportunities and Challenges for Sample Preparation and Enrichment\nin Mass Spectrometry for Single-Cell Metabolomics","volume":"44","author":"Wevers","year":"2023","journal-title":"Electrophoresis"},{"issue":"1","key":"2026080510111922400_cit6","doi-asserted-by":"publisher","first-page":"67","DOI":"10.1002\/mas.21704","article-title":"Mass Spectrometry-Based Strategies\nfor Single-Cell Metabolomics","volume":"42","author":"Hu","year":"2023","journal-title":"Mass Spectrom.\nRev."},{"key":"2026080510111922400_cit7","doi-asserted-by":"publisher","first-page":"124","DOI":"10.1016\/j.aca.2020.11.020","article-title":"Single Cell Metabolomics\nUsing Mass Spectrometry: Techniques and\nData Analysis","volume":"1143","author":"Liu","year":"2021","journal-title":"Anal. Chim. Acta"},{"issue":"1","key":"2026080510111922400_cit8","doi-asserted-by":"publisher","first-page":"9110","DOI":"10.1038\/s41467-024-52213-9","article-title":"METASPACE-ML:\nContext-Specific Metabolite Annotation\nfor Imaging Mass Spectrometry Using Machine Learning","volume":"15","author":"Wadie","year":"2024","journal-title":"Nat. Commun."},{"issue":"1","key":"2026080510111922400_cit9","doi-asserted-by":"publisher","first-page":"165","DOI":"10.1002\/jms.3512","article-title":"GridMass:\nA Fast Two-Dimensional Feature Detection Method for LC\/MS","volume":"50","author":"Trevin\u0303o","year":"2015","journal-title":"J. Mass Spectrom."},{"issue":"1","key":"2026080510111922400_cit10","doi-asserted-by":"publisher","first-page":"256","DOI":"10.1186\/s12859-019-2848-8","article-title":"ROIMCR: A Powerful Analysis Strategy\nfor LC-MS Metabolomic Datasets","volume":"20","author":"Gorrochategui","year":"2019","journal-title":"BMC Bioinf."},{"issue":"8","key":"2026080510111922400_cit11","doi-asserted-by":"publisher","first-page":"64","DOI":"10.1007\/s11306-022-01923-6","article-title":"Spectral Binning as\nan Approach to Post-Acquisition\nProcessing of High Resolution FIE-MS Metabolome Fingerprinting Data","volume":"18","author":"Finch","year":"2022","journal-title":"Metabolomics"},{"key":"2026080510111922400_cit12","unstructured":"Ester, M.; Kriegel, H.-P.; Xu, X.\n          A Density-Based\nAlgorithm for\nDiscovering Clusters in Large Spatial Databases with Noise; AAAI Press kdd 1996, 96(34), 226\u2013231."},{"issue":"3","key":"2026080510111922400_cit13","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1145\/3068335","article-title":"DBSCAN Revisited,\nRevisited: Why and How You Should (Still) Use DBSCAN","volume":"42","author":"Schubert","year":"2017","journal-title":"ACM Trans. Database Syst."},{"issue":"2","key":"2026080510111922400_cit14","doi-asserted-by":"publisher","first-page":"49","DOI":"10.1145\/304181.304187","article-title":"OPTICS: Ordering Points\nto Identify the Clustering Structure","volume":"28","author":"Ankerst","year":"1999","journal-title":"SIGMOD\nRec"},{"key":"2026080510111922400_cit15","doi-asserted-by":"publisher","first-page":"14","DOI":"10.1016\/j.aca.2013.03.048","article-title":"Warping\nMethods for Spectroscopic and Chromatographic Signal Alignment: A\nTutorial","volume":"781","author":"Bloemberg","year":"2013","journal-title":"Anal. Chim. Acta"},{"issue":"24","key":"2026080510111922400_cit16","doi-asserted-by":"publisher","first-page":"16138","DOI":"10.1021\/acs.analchem.0c03833","article-title":"A General Approach\nto Mass Alignment in Mass Spectrometry Imaging","volume":"92","author":"Eriksson","year":"2020","journal-title":"Anal. 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