title: Nano-LC FTICR tandem mass spectrometry for top-down proteomics: routine baseline unit mass resolution of whole cell lysate proteins up to 72 kDa.
abstract: Current high-throughput top-down proteomic platforms provide routine identification of proteins less than 25 kDa with 4-D separations. This short communication reports the application of technological developments over the past few years that improve protein identification and characterization for masses greater than 25 kDa. Advances in separation science have allowed increased numbers of proteins to be identified, especially by nanoliquid chromatography (nLC) prior to mass spectrometry (MS) analysis. Further, a goal of high-throughput top-down proteomics is to extend the mass range for routine nLC MS analysis up to 80 kDa because gene sequence analysis predicts that ~70% of the human proteome is transcribed to be less than 80 kDa. Normally, large proteins greater than 50 kDa are identified and characterized by top-down proteomics through fraction collection and direct infusion at relatively low throughput. Further, other MS-based techniques provide top-down protein characterization, however at low resolution for intact mass measurement. Here, we present analysis of standard (up to 78 kDa) and whole cell lysate proteins by Fourier transform ion cyclotron resonance mass spectrometry (nLC electrospray ionization (ESI) FTICR MS). The separation platform reduced the complexity of the protein matrix so that, at 14.5 T, proteins from whole cell lysate up to 72 kDa are baseline mass resolved on a nano-LC chromatographic time scale. Further, the results document routine identification of proteins at improved throughput based on accurate mass measurement (less than 10 ppm mass error) of precursor and fragment ions for proteins up to 50 kDa.
authors.affiliation: Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-4005, USA.
authors.forename: Jeremiah D
authors.initials: JD
authors.lastname: Tipton
authors.forename: John C
authors.initials: JC
authors.lastname: Tran
authors.forename: Adam D
authors.initials: AD
authors.lastname: Catherman
authors.forename: Dorothy R
authors.initials: DR
authors.lastname: Ahlf
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Durbin
authors.forename: Ji Eun
authors.initials: JE
authors.lastname: Lee
authors.forename: John F
authors.initials: JF
authors.lastname: Kellie
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Hendrickson
authors.forename: Alan G
authors.initials: AG
authors.lastname: Marshall
mesh_terms: D002851:Chromatography, High Pressure Liquid; D005583:Fourier Analysis; D006367:HeLa Cells; D006801:Humans; D007525:Isoelectric Focusing; D008970:Molecular Weight; D036103:Nanotechnology; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D011506:Proteins