Comprehensive characterization and proteoform analysis of the hydrophobic surfactant proteins B and C in calf pulmonary surfactant

J Pharm Biomed Anal. 2019 Sep 10:174:625-632. doi: 10.1016/j.jpba.2019.06.027. Epub 2019 Jun 21.

Abstract

Calf pulmonary surfactant (CPS), which contains about 98% lipids and 2% hydrophobic surfactant proteins B (SP-B) and C (SP-C), has been used as a surfactant preparation for the clinical replacement therapy of respiratory distress syndrome (RDS). Characterization of SP-B and SP-C in CPS is informative for quality control and the evaluation of their biological activities. However, analysis of SP-B and SP-C is impeded by the high content of lipids in CPS. Here, we describe an integrated method by combining size exclusion chromatography (SEC)-based delipidation, SDS-PAGE separation, in-gel digestion and mass spectrometric analysis for comprehensive characterization and proteoform analysis of the extremely hydrophobic SP-B and SP-C in CPS. This study has shown that 30 proteoforms of SP-C with different truncations and modifications were identified and SP-B was found to be existed as a dimer form in the CPS.

Keywords: Calf pulmonary surfactant; Mass spectrometry; Protein palmitoylation; SP-B; SP-C.

MeSH terms

  • Animals
  • Bronchoalveolar Lavage Fluid
  • Cattle
  • Chloroform / chemistry
  • Chromatography, Liquid / methods*
  • Hydrophobic and Hydrophilic Interactions
  • Lipids / chemistry
  • Lipoproteins / analysis*
  • Lipoylation
  • Methanol / chemistry
  • Protein Isoforms
  • Protein Multimerization
  • Pulmonary Surfactants / analysis*
  • Quality Control
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Surface-Active Agents
  • Tandem Mass Spectrometry / methods*

Substances

  • Lipids
  • Lipoproteins
  • Protein Isoforms
  • Pulmonary Surfactants
  • Surface-Active Agents
  • Chloroform
  • Methanol