{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T22:31:59Z","timestamp":1784327519948,"version":"3.55.0"},"reference-count":52,"publisher":"Wiley","issue":"S1","license":[{"start":{"date-parts":[[2023,4,24]],"date-time":"2023-04-24T00:00:00Z","timestamp":1682294400000},"content-version":"am","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"},{"start":{"date-parts":[[2023,4,24]],"date-time":"2023-04-24T00:00:00Z","timestamp":1682294400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["CHE 2003379"],"award-info":[{"award-number":["CHE 2003379"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Rapid Comm Mass Spectrometry"],"published-print":{"date-parts":[[2023,9]]},"abstract":"<jats:sec>\n                    <jats:title>Rationale<\/jats:title>\n                    <jats:p>Purification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI).<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      A protein standard, His\u2010Ubq, and two recombinant proteins, His\u2010SHAN and His\u2010CS, expressed in\n                      <jats:styled-content style=\"fixed-case\">\n                        <jats:italic>Escherichia coli<\/jats:italic>\n                      <\/jats:styled-content>\n                      were immobilized on two immobilized metal affinity systems, Cu\u2013nitriloacetic acid (Cu\u2010NTA) and Ni\u2010NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96\u2010well plate form factor, or analyzed directly from immobilized metal affinity\u2010coated microscope slides by DESI\u2010MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      Small proteins (His\u2010Ubq) and medium proteins (His\u2010SAHN) could readily be detected from 96\u2010well plates by direct infusion ESI, or from microscope slides by DESI\u2010MS after purification on surface from clarified\n                      <jats:italic>E. coli<\/jats:italic>\n                      cell lysate. Protein oxidation was observed for immobilized proteins on both Cu\u2010NTA and Ni\u2010NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His\u2010SAHN and the methylation product of His\u2010CS (theobromine to caffeine) were detected.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>The immobilization, purification, release and detection of His\u2010tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI\u2010MS or ambient DESI\u2010MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/rcm.9516","type":"journal-article","created":{"date-parts":[[2023,4,4]],"date-time":"2023-04-04T02:55:55Z","timestamp":1680576955000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Analysis of histidine\u2010tagged recombinant proteins from nickel and copper coated surfaces by direct electrospray ionization and desorption electrospray ionization mass spectrometry"],"prefix":"10.1002","volume":"37","author":[{"given":"Roshan","family":"Javanshad","sequence":"first","affiliation":[{"name":"Department of Chemistry Western Michigan University  Kalamazoo MI USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christopher James","family":"Taylor","sequence":"additional","affiliation":[{"name":"Department of Chemistry Western Michigan University  Kalamazoo MI USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Niusha","family":"Delavari","sequence":"additional","affiliation":[{"name":"Department of Chemistry Western Michigan University  Kalamazoo MI USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Todd J.","family":"Barkman","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences Western Michigan University  Kalamazoo MI USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Frederick","family":"Stull","sequence":"additional","affiliation":[{"name":"Department of Chemistry Western Michigan University  Kalamazoo MI USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6912-7446","authenticated-orcid":false,"given":"Andre R.","family":"Venter","sequence":"additional","affiliation":[{"name":"Department of Chemistry Western Michigan University  Kalamazoo MI USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2023,4,24]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1042\/BA20010099"},{"key":"e_1_2_7_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbiotec.2011.10.009"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00253\u2010016\u20108045\u2010z"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1080\/23312025.2019.1665406"},{"key":"e_1_2_7_6_1","doi-asserted-by":"publisher","DOI":"10.2174\/1389203721666200606220109"},{"key":"e_1_2_7_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biotechadv.2020.107653"},{"key":"e_1_2_7_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0076\u20106879(09)63025\u20101"},{"key":"e_1_2_7_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0076\u20106879(09)63022\u20106"},{"key":"e_1_2_7_10_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0076\u20106879(09)63027\u20105"},{"key":"e_1_2_7_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/258598a0"},{"key":"e_1_2_7_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.f.202"},{"key":"e_1_2_7_13_1","doi-asserted-by":"publisher","DOI":"10.1385\/0\u201089603\u2010258\u20102:371"},{"key":"e_1_2_7_14_1","article-title":"Characterizing endogenous protein complexes with biological mass spectrometry","author":"Rogawski R","year":"2021","journal-title":"Chem Rev"},{"key":"e_1_2_7_15_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1124619"},{"key":"e_1_2_7_16_1","doi-asserted-by":"publisher","DOI":"10.1021\/cr068289b"},{"key":"e_1_2_7_17_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.0c02162"},{"key":"e_1_2_7_18_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00216\u2010020\u201002649\u2010x"},{"key":"e_1_2_7_19_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.9b05018"},{"key":"e_1_2_7_20_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41596\u2010019\u20100233\u20108"},{"key":"e_1_2_7_21_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.analchem.7b00398"},{"key":"e_1_2_7_22_1","doi-asserted-by":"publisher","DOI":"10.1038\/s42003\u2010018\u20100231\u20103"},{"key":"e_1_2_7_23_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41596\u2010019\u20100281\u20100"},{"key":"e_1_2_7_24_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.biochem.1c00138"},{"key":"e_1_2_7_25_1","doi-asserted-by":"publisher","DOI":"10.1007\/s13361\u2010015\u20101221\u2010z"},{"key":"e_1_2_7_26_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac049848m"},{"key":"e_1_2_7_27_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1044\u20100305(97)00284\u20105"},{"key":"e_1_2_7_28_1","unstructured":"Scientific\u2122 T. 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