Identification of proteins in a human pleural exudate using two-dimensional preparative liquid-phase electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry

Carol Lynn Nilsson, Maja Puchades, Ann Westman, Ka Blennow, Pia Davidsson

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Pleural effusion may occur in patients suffering from physical trauma or systemic disorders such as infection, inflammation, or cancer. In order to investigate proteins in a pleural exudate from a patient with severe pneumonia, we used a strategy that combined preparative two-dimensional liquid-phase electrophoresis (2-D LPE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Western blotting. Preparative 2-D LPE is based on the same principles as analytical 2-D gel electrophoresis, except that the proteins remain in liquid phase during the entire procedure. In the first dimension, liquid-phase isoelectric focusing allows for the enrichment of proteins in liquid fractions. In the Rotofor cell, large volumes (up to 55 mL) and protein amounts (up to 1-2 g) can be loaded. Several low abundance proteins, cystatin C., haptoglobin, transthyretin, ß2-microglobulin, and transferrin, were detected after liquid-phase isoelectric focusing, through Western blotting analysis, in a pleural exudate (by definition,>25 g/L total protein). Direct MALDI-TOF-MS analysis of proteins in a Rotofor fraction is demonstrated as well. MALDI-TOF-MS analysis of a tryptic digest of a continuous elution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fraction confirmed the presence of cystatin C. By applying 2-D LPE, MALDI-TOF-MS, and Western blotting to the analysis of this pleural exudate, we were able to confirm the identity of proteins of potential diagnostic value. Our findings serve to illustrate the usefulness of this combination of methods in the analysis of pathological fluids.

Original languageEnglish (US)
Title of host publicationFrom Genome to Proteome: Advances in the Practice & Application of Proteomics
PublisherWiley Blackwell
Pages280-285
Number of pages6
ISBN (Electronic)9783527613489
ISBN (Print)9783527301546
DOIs
StatePublished - Dec 26 2007
Externally publishedYes

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Keywords

  • Biological fluid
  • ionization time-of-flight mass spectrometry
  • Matrix-assisted laser desorption
  • Peptide mass mapping
  • Protein identification
  • Two-dimensional liquid-phase electrophoresis

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Nilsson, C. L., Puchades, M., Westman, A., Blennow, K., & Davidsson, P. (2007). Identification of proteins in a human pleural exudate using two-dimensional preparative liquid-phase electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry. In From Genome to Proteome: Advances in the Practice & Application of Proteomics (pp. 280-285). Wiley Blackwell. https://doi.org/10.1002/9783527613489.ch36