Aberrant Processing Forms of Lung Surfactant Proteins SP–B and SP–C Revealed by High-Resolution Mass Spectrometry

The mutation (g.1286T>C) of the pulmonary surfactant-associated protein C gene (SFTPC) leads to the I73T substitution in the precursor protein (pro–SP–C) and results in interstitial lung disease with the histological pattern of non-specific interstitial pneumonia and pulmonary alveolar proteinosis. Central for the disease is the abnormal processing of the SP–C pro–protein to mature SP–C; however little is known about the nature of intermediates and processing products. We report here the application of high resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry to the characterization of processing intermediates of hydrophobic pulmonary surfactant proteins SP–B and SP–C in intra-alveolar surfactant material of a patient with I73T mutation. SP–C and SP–B processing forms were separated from broncho-alveolar lavage fluid using chloroform/methanol extraction and sodium dodecyl sulfate polyacrylamide gel electrophoreis, detected by Western blot and identified by electrospray- and matrix-assisted laser desorption/ionization-FT-ICR mass spectrometry. The mass spectrometric and immuno-analytical results show the intra-alveolar accumulation of an aberrant C-terminal SP–C processing products in which the mature SP–C protein part is missing, and aberrant processing intermediates of SP–B.