Pharmaceutical Sciences Faculty Publications

Comparison of Fluorescence, Laser-Induced Fluorescence, and Ultraviolet Absorbance Detection for Measuring HPLC Fractionated Protein/Peptide Mixtures

Document Type

Article

Publication Date

2-14-2011

Journal Title

Analytical Chemistry

Volume

83

Issue

6

First Page

2394

Last Page

2396

DOI

10.1021/ac1032462

Abstract

Proteomics is the study of all proteins in a biological sample. High-pressure liquid chromatography coupled online with mass spectrometry (HPLC/MS) is currently the method of choice for proteomic analysis. Proteins are extracted, separated at the protein or peptide level (after enzymatic digestion), and fractions are analyzed by HPLC/MS. Detection during off-line fractionation is generally conducted using UV−vis, which is not sensitive enough to distinguish fractions having the largest concentration of proteins/peptides and should not be combined prior to HPLC/MS. To overcome this deficiency, we utilize fluorescence or UV-laser induced fluorescence (UV-LIF) detection for measuring proteins/peptides during the off-line fractionation. Fluorescence detection allows low-abundance proteins/peptides that contain aromatic amino acids to be measured. In this study, peptide/protein samples fractionated using ion-exchange chromatography were detected using UV absorbance, fluorescence, and UV-LIF. The results indicated that fluorescence and UV-LIF were able to detect the lower abundance proteins/peptides to give a more representative chromatogram, allowing the analyst to decide which fractions should be combined prior to HPLC/tandem mass spectrometry (MS/MS) analysis.

Keywords

Fractionation, peptides, proteins, fluorescence, detection, absorption

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