title: Protein fragmentation via liquid chromatography-quadrupole time-of-flight mass spectrometry: the use of limited sequence information in structural characterization.
abstract: Fragmentation and "top-down" sequencing of intact proteins by mass spectrometry (MS) is most commonly performed by infusion of protein solutions into Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. However, the high cost of this instrumentation, coupled with the need to infuse "clean" solutions (lacking standard biological buffers), limits broad application of this technique. The current study describes an alternative approach to top-down sequencing using in-source fragmentation on quadrupole time-of-flight (Q-Tof) instrumentation coupled with reversed-phase liquid chromatography (LC). Application of this technique to purified recombinant samples yielded protein fragments during routine LC-MS analysis. The presence of multiple N- and C-terminal fragments allowed localization of structural modifications without proteolytic digestion. The method was extended to complex samples by using LC conditions that provided high-resolution protein separation. Utility of the method was illustrated by real-time monitoring of protein modifications occurring in reconstituted apoptosomes. These experiments illustrate that intact protein mass and limited sequence information can be obtained simultaneously on an LC timescale. This approach will allow a wide variety of laboratories to routinely apply top-down sequencing to problems in structural characterization, protein purification, and biomarker identification.
authors.affiliation: Department of Structural Chemistry, Abbott Laboratories, 200 Abbott Park Road, Abbott Park, IL 60064, USA. robert.w.johnson@abbott.com
authors.forename: Robert W
authors.initials: RW
authors.lastname: Johnson
authors.forename: Tanveer F
authors.initials: TF
authors.lastname: Ahmed
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Miesbauer
authors.forename: Rohinton
authors.initials: R
authors.lastname: Edalji
authors.forename: Richard
authors.initials: R
authors.lastname: Smith
authors.forename: John
authors.initials: J
authors.lastname: Harlan
authors.forename: Sarah
authors.initials: S
authors.lastname: Dorwin
authors.forename: Karl
authors.initials: K
authors.lastname: Walter
authors.forename: Tom
authors.initials: T
authors.lastname: Holzman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002645:Chickens; D002853:Chromatography, Liquid; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins