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

  1. Author:
    Chan, K. C.
    Veenstra, T. D.
    Issaq, H. J.

  2. Author Address

    [Chan, King C.; Veenstra, Timothy D.; Issaq, Haleem J.] NCI, Lab Prote & Analyt Technol, Adv Technol Program, SAIC Frederick Inc, Frederick, MD 21702 USA.;Issaq, HJ, NCI, Lab Prote & Analyt Technol, Adv Technol Program, SAIC Frederick Inc, POB B, Frederick, MD 21702 USA.;issaqh@mail.nih.gov
    1. Year: 2011
    2. Date: Mar
  2. Journal: Analytical Chemistry
    1. 83
    2. 6
    3. Pages: 2394-2396
  4. Type of Article: Article
  5. ISSN: 0003-2700
  1. 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.

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External Sources

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  2. DOI: 10.1021/ac1032462
  3. View record in Web of Science®
  4. WOS: 000288182900073

Library Notes

  1. Fiscal Year: FY2010-2011
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