title: An integrated top-down and bottom-up strategy for characterization of protein isoforms and modifications.
abstract: Bottom-up and top-down strategies are two commonly used methods for mass spectrometry (MS) based protein identification; each method has its own advantages and disadvantages. In this chapter, we describe an integrated top-down and bottom-up approach facilitated by concurrent liquid chromatography-mass spectrometry (LC-MS) analysis and fraction collection for comprehensive high-throughput intact protein profiling. The approach employs a high resolution reversed phase (RP) LC separation coupled with LC eluent fraction collection and concurrent on-line MS with a high field (12 T) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer. Protein elusion profiles and tentative modified protein identification are made using detected intact protein mass in conjunction with bottom-up protein identifications from the enzymatic digestion and analysis of corresponding LC fractions. Specific proteins of biological interest are incorporated into a target ion list for subsequent off-line gas-phase fragmentation that uses an aliquot of the original collected LC fraction, an aliquot of which was also used for bottom-up analysis.
authors.affiliation: Pacific Northwest National Laboratory, Richland, WA, USA.
authors.forename: Si
authors.initials: S
authors.lastname: Wu
authors.forename: Nikola
authors.initials: N
authors.lastname: Tolić
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Tian
authors.forename: Errol W
authors.initials: EW
authors.lastname: Robinson
authors.forename: Ljiljana
authors.initials: L
authors.lastname: Paša-Tolić
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D024402:Carbonic Anhydrase II / Q000737:chemistry / Q000378:metabolism; D005591:Chemical Fractionation; D002853:Chromatography, Liquid; D056148:Chromatography, Reverse-Phase; D005583:Fourier Analysis; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009862:Online Systems; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D012015:Reference Standards; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013223:Statistics as Topic
chemical_list: D020033:Protein Isoforms; D046988:Proteasome Endopeptidase Complex; D024402:Carbonic Anhydrase II