Parallel Detection of Intrinsic Fluorescence from Peptides and Proteins for Quantification during Mass Spectrometric Analysis

Direct mass spectrometric quantification of peptides and protein is compromised by the wide variabilities in ionization efficiency which are hallmarks of both the MALDI and ESI ionization techniques. We describes 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 of protein or peptide present, while direct or tandem mass spectrometric analysis (MS/MS).on the ESI-generated ions provides informatikit:on.iideritity. We fabricated an inexpensive, modular fluorescence excitation and detection device utilizing an ultraviolet light-emitting diode for excitation in a similar to 300 nL fluorescence detection cell integrated 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.