title: Online Hydrophobic Interaction Chromatography-Mass Spectrometry for Top-Down Proteomics.
abstract: Recent progress in top-down proteomics has led to a demand for mass spectrometry (MS)-compatible chromatography techniques to separate intact proteins using volatile mobile phases. Conventional hydrophobic interaction chromatography (HIC) provides high-resolution separation of proteins under nondenaturing conditions but requires high concentrations of nonvolatile salts. Herein, we introduce a series of more-hydrophobic HIC materials that can retain proteins using MS-compatible concentrations of ammonium acetate. The new HIC materials appear to function as a hybrid form of conventional HIC and reverse phase chromatography. The function of the salt seems to be preserving protein structure rather than promoting retention. Online HIC-MS is feasible for both qualitative and quantitative analysis. This is demonstrated with standard proteins and a complex cell lysate. The mass spectra of proteins from the online HIC-MS exhibit low charge-state distributions, consistent with those commonly observed in native MS. Furthermore, HIC-MS can chromatographically separate proteoforms differing by minor modifications. Hence, this new HIC-MS combination is promising for top-down proteomics.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin, United States.
authors.forename: Bifan
authors.initials: B
authors.lastname: Chen
authors.affiliation: Department of Cell and Regenerative Biology, University of Wisconsin-Madison , Madison, Wisconsin, United States.
authors.forename: Ying
authors.initials: Y
authors.lastname: Peng
authors.affiliation: Department of Cell and Regenerative Biology, University of Wisconsin-Madison , Madison, Wisconsin, United States.
authors.forename: Santosh G
authors.initials: SG
authors.lastname: Valeja
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin, United States.
authors.forename: Lichen
authors.initials: L
authors.lastname: Xiu
authors.affiliation: Department of Cell and Regenerative Biology, University of Wisconsin-Madison , Madison, Wisconsin, United States.|PolyLC, Inc., 9151 Rumsey Rd., Suite 180, Columbia, Maryland, United States.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Alpert
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin, United States.|Department of Cell and Regenerative Biology, University of Wisconsin-Madison , Madison, Wisconsin, United States.|Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison , Madison, Wisconsin, United States.
authors.forename: Ying
authors.initials: Y
authors.lastname: Ge
mesh_terms: D000818:Animals; D007611:Aprotinin / Q000032:analysis; D002417:Cattle; D002645:Chickens; D002845:Chromatography; D002919:Chymotrypsinogen / Q000032:analysis; D006736:Horses; D057927:Hydrophobic and Hydrophilic Interactions; D020407:Internet; D007782:Lactoglobulins / Q000032:analysis; D013058:Mass Spectrometry; D009113:Muramidase / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012259:Ribonuclease, Pancreatic / Q000032:analysis / Q000378:metabolism; D014362:Trypsinogen / Q000032:analysis
chemical_list: D007782:Lactoglobulins; D014362:Trypsinogen; D002919:Chymotrypsinogen; D007611:Aprotinin; D012259:Ribonuclease, Pancreatic; D009113:Muramidase