title: Identification and Characterization of Human Proteoforms by Top-Down LC-21 Tesla FT-ICR Mass Spectrometry.
abstract: Successful high-throughput characterization of intact proteins from complex biological samples by mass spectrometry requires instrumentation capable of high mass resolving power, mass accuracy, sensitivity, and spectral acquisition rate. These limitations often necessitate the performance of hundreds of LC-MS/MS experiments to obtain reasonable coverage of the targeted proteome, which is still typically limited to molecular weights below 30 kDa. The National High Magnetic Field Laboratory (NHMFL) recently installed a 21 T FT-ICR mass spectrometer, which is part of the NHMFL FT-ICR User Facility and available to all qualified users. Here we demonstrate top-down LC-21 T FT-ICR MS/MS of intact proteins derived from human colorectal cancer cell lysate. We identified a combined total of 684 unique protein entries observed as 3238 unique proteoforms at a 1% false discovery rate, based on rapid, data-dependent acquisition of collision-induced and electron-transfer dissociation tandem mass spectra from just 40 LC-MS/MS experiments. Our identifications included 372 proteoforms with molecular weights over 30 kDa detected at isotopic resolution, which substantially extends the accessible mass range for high-throughput top-down LC-MS/MS.
authors.affiliation: Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory , Tallahassee, Florida 32310, United States.
authors.forename: Lissa C
authors.initials: LC
authors.lastname: Anderson
authors.affiliation: Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory , Tallahassee, Florida 32310, United States.|Proteomics Center of Excellence, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Caroline J
authors.initials: CJ
authors.lastname: DeHart
authors.affiliation: Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory , Tallahassee, Florida 32310, United States.
authors.forename: Nathan K
authors.initials: NK
authors.lastname: Kaiser
authors.affiliation: Proteomics Center of Excellence, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Fellers
authors.affiliation: Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory , Tallahassee, Florida 32310, United States.
authors.forename: Donald F
authors.initials: DF
authors.lastname: Smith
authors.affiliation: Proteomics Center of Excellence, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Joseph B
authors.initials: JB
authors.lastname: Greer
authors.affiliation: Proteomics Center of Excellence, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Richard D
authors.initials: RD
authors.lastname: LeDuc
authors.affiliation: Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory , Tallahassee, Florida 32310, United States.
authors.forename: Greg T
authors.initials: GT
authors.lastname: Blakney
authors.affiliation: Proteomics Center of Excellence, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Paul M
authors.identifier: 0000-0003-2887-4765
authors.initials: PM
authors.lastname: Thomas
authors.affiliation: Proteomics Center of Excellence, Northwestern University , Evanston, Illinois 60208, United States.|Departments of Chemistry and Molecular Biosciences and the Division of Hematology-Oncology, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Neil L
authors.identifier: 0000-0002-8815-3372
authors.initials: NL
authors.lastname: Kelleher
authors.affiliation: Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory , Tallahassee, Florida 32310, United States.|Department of Chemistry and Biochemistry, Florida State University , Tallahassee, Florida 32304, United States.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Hendrickson
mesh_terms: D000595:Amino Acid Sequence; D015179:Colorectal Neoplasms / Q000737:chemistry* / Q000473:pathology; D045424:Complex Mixtures / Q000737:chemistry; D017357:Cyclotrons / Q000295:instrumentation; D005583:Fourier Analysis; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D009363:Neoplasm Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*
chemical_list: D045424:Complex Mixtures; D009363:Neoplasm Proteins; D020543:Proteome
keywords: 21 tesla; FT-ICR; FTMS; top-down proteomics