title: Size-sorting combined with improved nanocapillary liquid chromatography-mass spectrometry for identification of intact proteins up to 80 kDa.
abstract: Despite the availability of ultra-high-resolution mass spectrometers, methods for separation and detection of intact proteins for proteome-scale analyses are still in a developmental phase. Here we report robust protocols for online LC-MS to drive high-throughput top-down proteomics in a fashion similar to that of bottom-up proteomics. Comparative work on protein standards showed that a polymeric stationary phase led to superior sensitivity over a silica-based medium in reversed-phase nanocapillary LC, with detection of proteins >50 kDa routinely accomplished in the linear ion trap of a hybrid Fourier transform mass spectrometer. Protein identification was enabled by nozzle-skimmer dissociation and detection of fragment ions with <10 ppm mass accuracy for highly specific database searching using tailored software. This overall approach led to identification of proteins up to 80 kDa, with 10-60 proteins identified in single LC-MS runs of samples from yeast and human cell lines prefractionated by their molecular mass using a gel-based sieving system.
authors.affiliation: Department of Chemistry, 600 South Mathews Avenue, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Adaikkalam
authors.initials: A
authors.lastname: Vellaichamy
authors.forename: John C
authors.initials: JC
authors.lastname: Tran
authors.forename: Adam D
authors.initials: AD
authors.lastname: Catherman
authors.forename: Ji Eun
authors.initials: JE
authors.lastname: Lee
authors.forename: John F
authors.initials: JF
authors.lastname: Kellie
authors.forename: Steve M M
authors.initials: SM
authors.lastname: Sweet
authors.forename: Leonid
authors.initials: L
authors.lastname: Zamdborg
authors.forename: Paul M
authors.initials: PM
authors.lastname: Thomas
authors.forename: Dorothy R
authors.initials: DR
authors.lastname: Ahlf
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Durbin
authors.forename: Gary A
authors.initials: GA
authors.lastname: Valaskovic
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D036103:Nanotechnology; D011108:Polymers / Q000737:chemistry; D016062:Porosity; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D020543:Proteome / Q000032:analysis / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000737:chemistry; D013997:Time Factors
chemical_list: D011108:Polymers; D011506:Proteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins