Effect of Fluorescent Labels on Peptide and Amino Acid Sample Dimensionality in Two Dimensional nLC × μFFE Separations

Matthew Geiger, Michael Bowser

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

Multidimensional separations present a unique opportunity for generating the high peak capacities necessary for the analysis of complex biological mixtures. We have coupled nano liquid chromatography with micro free flow electrophoresis (nLC × μFFE) to produce high peak capacity separations of peptide and amino acid mixtures. Currently, μFFE largely relies on laser-induced fluorescence (LIF) detection. We have demonstrated that the choice of fluorescent label significantly affects the fractional coverage and peak capacity of nLC × μFFE separations of peptides and amino acids. Of the labeling reagents assessed, Chromeo P503 performed the best for nLC × μFFE separations of peptides. A nLC × μFFE analysis of a Chromeo P503-labeled BSA tryptic digest produced a 2D separation that made effective use of the available separation space (48%), generating a corrected peak capacity of 521 in a 5 min separation window (104 peaks/min). nLC × μFFE separations of NBD-F-labeled peptides produced similar fractional coverage and peak capacity, but this reagent was able to react with multiple reaction sites, producing an unnecessarily complex analyte mixture. NBD-F performed the best for nLC × μFFE separations of amino acids. NBD-F-labeled amino acids produced a 2D separation that covered 36% of the available separation space, generating a corrected peak capacity of 95 in a 75 s separation window (76 peaks/min). Chromeo P503 and Alexa Fluor 488-labeled amino acids were not effectively separated in the μFFE dimension, giving 2D separations with poor fractional coverage and peak capacity.

Original languageEnglish (US)
Pages (from-to)2177-2187
Number of pages11
JournalAnalytical chemistry
Volume88
Issue number4
DOIs
StatePublished - Feb 16 2016

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