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

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• 作者：King C. Chan ; Timothy D. Veenstra ; Haleem J. Issaq
• 刊名：Analytical Chemistry
• 出版年：2011
• 出版时间：March 15, 2011
• 年：2011
• 卷：83
• 期：6
• 页码：2394-2396
• 全文大小：663K
• 年卷期：v.83,no.6(March 15, 2011)
• ISSN：1520-6882

文摘

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鈭抳is, 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.