Methods
Establishment of LC-MS methods for the analysis of palmitoylated surfactant proteins[S]

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The surfactant proteins (SPs), SP-B and SP-C, are important components of pulmonary surfactant involved in the reduction of alveolar surface tension. Quantification of SP-B and SP-C in surfactant drugs is informative for their quality control and the evaluation of their biological activity. Western blot analysis enabled the quantification of SP-B, but not SP-C, in surfactant drugs. Here, we report a new procedure involving chemical treatments and LC-MS to analyze SP-C peptides. The procedure enabled qualitative analysis of SP-C from different species with discrimination of the palmitoylation status and the artificial modifications that occur during handling and/or storage. In addition, the method can be used to estimate the total amount of SP-C in pulmonary surfactant drugs. The strategy described here might serve as a prototype to establish analytical methods for peptides that are extremely hydrophobic and behave like lipids. The new method provides an easy measurement of SP-C from various biological samples, which will help the characterization of various experimental animal models and the quality control of surfactant drugs, as well as diagnostics of human samples.

pulmonary surfactant
mass spectrometry
palmitoylation
proteomics
lung
drug therapy
surfactant drug
liquid chromatography

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This work was supported by JSPS KAKENHI Grants 24229003 (T.S.), 25116707 (Y.K.), 23790324 (H.S.), 24790805 (T.H.), and the grants for National Center for Global Health and Medicine 24-001 and 25-201 (T.S.). Materials and fees for this research were provided, in part, by Mitsubishi Tanabe Pharma Corporation. The Department of Lipidomics, Graduate School of Medicine, University of Tokyo is financially supported by Shimadzu Corporation and Ono Pharmaceutical Company, LTD

    Abbreviations:

    bSP-C

    bovine surfactant protein C

    DPPC

    dipalmitoylphosphatidylcholine

    mSP-C

    murine surfactant protein C

    SP

    surfactant protein

    SRM

    selected reaction monitoring

    TFA

    trifluoroacetic acid

    UPLC

    ultra-performance LC

[S]

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of two figures and one table.

1

Present address of T. Harayama: Department of Biochemistry, Sciences II, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland.