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

Pleural effusion may occur in patients suffering from physical trauma or sy stemic disorders such as infection, inflammation, or cancer. In order to in vestigate proteins in a pleural exudate from a patient with severe pneumoni a, we used a strategy that combined preparative two-dimensional liquid-phas e electrophoresis (2-D LPE), matrix-assisted laser desorption/ionization ti me-of-flight mass spectrometry (MALDI-TOF-MS) and Western blotting. Prepara tive 2-D LPE is based on the same principles as analytical 2-D gel electrop horesis, 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, l arge volumes (up to 55 mL) and protein amounts (up to 1-2 g) can be loaded. Several low abundance proteins, cystatin C, haptoglobin, transthyretin, be ta(2)-microglobulin, and transferrin, were detected after liquid-phase isoe lectric focusing, through Western blotting analysis, in a pleural exudate ( by definition, > 25 g/L total protein). Direct MALDI-TOF-MS analysis of pro teins in a Rotofor fraction is demonstrated as well. MALDI-TOF-MS analysis of a tryptic digest of a continuous elution sodium dodecyl sulfate-polyacry lamide gel electrophoresis (SDS-PAGE) fraction confirmed the presence of cy statin C. By applying 2-D LPE, MALDI-TOF-MS, and Western blotting to the an alysis of this pleural exudate, we were able to confirm the identity of pro teins of potential diagnostic value. Our findings serve to illustrate the u sefulness of this combination of methods in the analysis of pathological fl uids.