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As most DIA data sets are acquired with wide isolation windows, highly complex MS\/MS spectra are generated, which negatively impacts obtaining peptide information through classical protein database searches. Therefore, the analysis of DIA data mainly relies on the evidence of the existence of peptides from prebuilt spectral libraries. Consequently, one major weakness of this method is that it does not account for peptides that are not included in the spectral library, precluding the use of DIA for discovery studies. Here, we present a strategy termed Precursor ion And Small Slice-DIA (PASS-DIA) in which MS\/MS spectra are acquired with small isolation windows (slices) and MS\/MS spectra are interpreted with accurately determined precursor ion masses. This method enables the direct application of conventional spectrum-centric analysis pipelines for peptide identification and precursor ion-based quantitation. The performance of PASS-DIA was observed to be superior to both data-dependent acquisition (DDA) and conventional DIA experiments with 69 and 48% additional protein identifications, respectively. Application of PASS-DIA for the analysis of post-translationally modified peptides again highlighted its superior performance in characterizing phosphopeptides (77% more), N-terminal acetylated peptides (56% more), and N-glycopeptides (83% more) as compared to DDA alone. Finally, the use of PASS-DIA to characterize a rare proteome of human fallopian tube organoids enabled 34% additional protein identifications than DDA alone and revealed biologically relevant pathways including low abundance proteins. Overall, PASS-DIA is a novel DIA approach for use as a discovery tool that outperforms both conventional DDA and DIA experiments to provide additional protein information. We believe that the PASS-DIA method is an important strategy for discovery-type studies when deeper proteome characterization is required.<\/jats:p>","DOI":"10.1021\/acs.analchem.0c02513","type":"journal-article","created":{"date-parts":[[2020,10,20]],"date-time":"2020-10-20T16:43:53Z","timestamp":1603212233000},"page":"14466-14475","source":"Crossref","is-referenced-by-count":26,"title":["PASS-DIA: A Data-Independent Acquisition Approach\nfor Discovery Studies"],"prefix":"10.1021","volume":"92","author":[{"given":"Dong-Gi","family":"Mun","sequence":"first","affiliation":[{"name":"Mayo Clinic , , , ,","place":["Rochester, Minnesota, United States, 55905"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Santosh","family":"Renuse","sequence":"additional","affiliation":[{"name":"Mayo Clinic , , , ,","place":["Rochester, Minnesota, United States, 55905"]},{"name":"Mayo Clinic , , , ,","place":["Rochester, Minnesota, United States, 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India, 560029"]},{"name":"Institute of Bioinformatics , International Technology Park , , ,","place":["Bangalore, Karnataka, India, 560066"]},{"name":"Manipal Academy of Higher Education (MAHE) , ,","place":["Manipal, Karnataka, India, 576104"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Savita","family":"Udainiya","sequence":"additional","affiliation":[{"name":"Mayo Clinic , , , ,","place":["Rochester, Minnesota, United States, 55905"]},{"name":"Mayo Clinic , , , ,","place":["Rochester, Minnesota, United States, 55905"]},{"name":"National Institute of Mental Health and Neurosciences (NIMHANS) , , Hosur Road , ,","place":["Bangalore, India, 560029"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hokeun","family":"Kim","sequence":"additional","affiliation":[{"name":"Korea University , , ,","place":["Seoul, Republic of Korea, 136-701"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8759-6799","authenticated-orcid":true,"given":"Sung-Kyu Robin","family":"Park","sequence":"additional","affiliation":[{"name":"The Scripps Research Institute , , 10550 North Torrey Pines Road , , ,","place":["La Jolla, California, United States, 92037"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hui","family":"Zhao","sequence":"additional","affiliation":[{"name":"Mayo Clinic , , , ,","place":["Rochester, Minnesota, United States, 55905"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raja Sekhar","family":"Nirujogi","sequence":"additional","affiliation":[{"name":"University of Dundee , , ,","place":["Dundee, United Kingdom, DD1 5EH"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chan Hyun","family":"Na","sequence":"additional","affiliation":[{"name":"Johns Hopkins University School of Medicine , , , ,","place":["Baltimore, Maryland, United States, 21205"]},{"name":"Johns Hopkins University School of Medicine , , , ,","place":["Baltimore, Maryland, United States, 21205"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nagarajan","family":"Kannan","sequence":"additional","affiliation":[{"name":"Mayo Clinic , , , ,","place":["Rochester, Minnesota, United States, 55905"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5267-1672","authenticated-orcid":true,"suffix":"III","given":"John R.","family":"Yates","sequence":"additional","affiliation":[{"name":"The Scripps Research Institute , , 10550 North Torrey Pines Road , , ,","place":["La Jolla, California, United States, 92037"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5042-0084","authenticated-orcid":true,"given":"Sang-Won","family":"Lee","sequence":"additional","affiliation":[{"name":"Korea University , , ,","place":["Seoul, Republic of Korea, 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