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, Kaj Blennow, Pia Davidsson

Research output: Contribution to journalArticle

45 Citations (Scopus)

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-TOFMS) 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, β2-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)
Pages (from-to)860-865
Number of pages6
JournalElectrophoresis
Volume20
Issue number4-5
DOIs
StatePublished - 1999
Externally publishedYes

Fingerprint

Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Exudates and Transudates
Electrophoresis
Ionization
Mass spectrometry
Desorption
Lasers
Liquids
Proteins
Cystatin C
Western Blotting
Isoelectric Focusing
Prealbumin
Haptoglobins
Electrophoresis, Gel, Two-Dimensional
Pleural Effusion
Transferrin
Protein C
Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis

Keywords

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

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Identification of proteins in a human pleural exudate using two- dimensional preparative liquid-phase electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry. / Nilsson, Carol Lynn; Puchades, Maja; Westman, Ann; Blennow, Kaj; Davidsson, Pia.

In: Electrophoresis, Vol. 20, No. 4-5, 1999, p. 860-865.

Research output: Contribution to journalArticle

Nilsson, Carol Lynn ; Puchades, Maja ; Westman, Ann ; Blennow, Kaj ; Davidsson, Pia. / Identification of proteins in a human pleural exudate using two- dimensional preparative liquid-phase electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry. In: Electrophoresis. 1999 ; Vol. 20, No. 4-5. pp. 860-865.
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