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Efficient delipidation of a recombinant lung surfactant lipopeptide analogue by liquid-gel chromatography.

ABSTRACT: Pulmonary surfactant preparations extracted from natural sources have been used to treat millions of newborn babies with respiratory distress syndrome (RDS) and can possibly also be used to treat other lung diseases. Due to costly production and limited supply of animal-derived surfactants, synthetic alternatives are attractive. The water insolubility and aggregation-prone nature of the proteins present in animal-derived surfactant preparations have complicated development of artificial surfactant. A non-aggregating analog of lung surfactant protein C, SP-C33Leu is used in synthetic surfactant and we recently described an efficient method to produce rSP-C33Leu in bacteria. Here rSP-C33Leu obtained by salt precipitation of bacterial extracts was purified by two-step liquid gel chromatography and analyzed using mass spectrometry and RP-HPLC, showing that it is void of modifications and adducts. Premature New Zealand White rabbit fetuses instilled with 200mg/kg of 2% of rSP-C33Leu in phospholipids and ventilated with a positive end expiratory pressure showed increased tidal volumes and lung gas volumes compared to animals treated with phospholipids only. This shows that rSP-C33Leu can be purified from bacterial lipids and that rSP-C33Leu surfactant is active against experimental RDS.

SUBMITTER: Basabe-Burgos O

PROVIDER: S-EPMC6892477 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Project description:Targeted glycoproteomics represents an attractive approach for conducting peripheral blood based cancer biomarker discovery due to the well-known altered pattern of protein glycosylation in cancer and the reduced complexity of the resultant glycoproteome. Here we report its application to a set of pooled nonsmall cell lung cancer (NSCLC) case sera (9 adenocarcinoma and 6 squamous cell carcinoma pools from 54 patients) and matched controls pools, including 8 clinical control pools with computed tomography detected nodules but being nonmalignant as determined by biopsy from 54 patients, and 8 matched healthy control pools from 106 cancer-free subjects. The goal of the study is to discover biomarkers that may enable improved early detection and diagnosis of lung cancer. Immunoaffinity subtraction was used to first deplete the topmost abundant serum proteins; the remaining serum proteins were then subjected to hydrazide chemistry based glycoprotein capture and enrichment. Hydrazide resin in situ trypsin digestion was used to release nonglycosylated peptides. Formerly N-linked glycosylated peptides were released by peptide-N-glycosidase F (PNGase F) treatment and were subsequently analyzed by liquid chromatography (LC)-tandem mass spectrometry (MS/MS). A MATLAB based in-house tool was developed to facilitate retention time alignment across different LC-MS/MS runs, determination of precursor ion m/z values and elution profiles, and the integration of mass chromatograms based on determined parameters for identified peptides. A total of 38 glycopeptides from 22 different proteins were significantly differentially abundant across the case/control pools (P < 0.01, Student's t test) and their abundances led to a near complete separation of case and control pools based on hierarchical clustering. The differential abundances of three of these candidate proteins were verified by commercially available ELISAs applied in the pools. Strong positive correlations between glycopeptide mass chromatograms and ELISA-measured protein abundance was observed for all of the selected glycoproteins.

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Efficient delipidation of a recombinant lung surfactant lipopeptide analogue by liquid-gel chromatography.

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