title: Parallel detection of intrinsic fluorescence from peptides and proteins for quantification during mass spectrometric analysis.
abstract: Direct mass spectrometric quantification of peptides and proteins is compromised by the wide variabilities in ionization efficiency which are hallmarks of both the MALDI and ESI ionization techniques. We describe here the implementation of a fluorescence detection system for measurement of the UV-excited intrinsic fluorescence (UV-IF) from peptides and proteins just prior to their exit and electrospray ionization from an ESI capillary. The fluorescence signal provides a quantifiable measure of the amount of protein or peptide present, while direct or tandem mass spectrometric analysis (MS/MS) on the ESI-generated ions provides information on identity. We fabricated an inexpensive, modular fluorescence excitation and detection device utilizing an ultraviolet light-emitting diode for excitation in a ∼300 nL fluorescence detection cell integrated into the fused-silica separation column. The fluorescence signal is linear over 3 orders of magnitude with on-column limits of detection in the low femtomole range. Chromatographically separated intact proteins analyzed using UV-IF prior to top-down mass spectrometry demonstrated sensitive detection of proteins as large as 77 kDa.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Russell
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Hilger
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Ladror
authors.forename: Mark A
authors.initials: MA
authors.lastname: Tervo
authors.forename: Mark
authors.initials: M
authors.lastname: Scalf
authors.forename: Michael R
authors.initials: MR
authors.lastname: Shortreed
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
authors.forename: Lloyd M
authors.initials: LM
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D012822:Silicon Dioxide / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D018511:Systems Integration; D014357:Trypsin / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D010455:Peptides; D011506:Proteins; D012997:Solvents; D012822:Silicon Dioxide; D014357:Trypsin