Lecture
An Integrative MS Workflow Reveals New Insights into Lipid Transfer Proteins
- at -
- ICM Saal 4b
- Type: Lecture
Lecture description
Native mass spectrometry (MS) is a powerful technique for studying protein–lipid interactions. However, mass analysis alone is often insufficient to obtain comprehensive structural information. To overcome this limitation, we recently established a multistage native MS workflow that enables deep structural characterization of protein-bound lipids[1], together with a native top-down MS strategy for identifying and localizing post-translational modifications (PTMs)[2]. Here, we demonstrate that this integrative native MS workflow, combined with lipidomics and proteomics, yields new insights into the endogenous ligands and regulation of human lipid transfer proteins (LTPs). LTPs mediate the intracellular trafficking of lipids and establish the distinct lipid profiles of cellular organelles. Yet, due to the difficulty of monitoring protein-mediated lipid transport at a molecular level, the lipid cargo and membrane-targeting mechanism of many LTPs remain elusive.
Here we study the human phosphatidylcholine (PC) transfer proteins STARD2, STARD7 and STARD10 to identify their native lipid cargoes and regulatory PTMs. Using affinity purification and native MS, we preserve the native protein conformation and protein–lipid interactions from the cell to the final analysis step. Analysis of the copurified endogenous lipids revealed that the three LTPs selectively bind PC lipids with distinct acyl chain combinations. This specificity correlates with their differential expression across different tissues and feeds back into the overall lipid metabolism. Furthermore, native top-down MS and proteomic profiling uncovered phosphorylation sites near membrane-interacting regions, which attenuate lipid transfer activity. Collectively, our results show that cellular lipid metabolism is tightly coupled to the expression levels and acyl chain selectivities of LTPs and their regulation through reversible PTMs.
[1] C. Kirschbaum, J. L. Bennett, C. V. Robinson, Anal. Chem. 2025, 97, 19331–19339. DOI: 10.1021/acs.analchem.5c03691
[2] J. L. Bennett, T. J. El-Baba, K. C. Zouboulis, C. Kirschbaum, H. Song, F. I. Butroid, J. L. P. Benesch, C. A. Lutomski, C. V. Robinson, Nat. Methods 2025, 22, 2127–2137. DOI: 10.1038/s41592-025-02846-5
Here we study the human phosphatidylcholine (PC) transfer proteins STARD2, STARD7 and STARD10 to identify their native lipid cargoes and regulatory PTMs. Using affinity purification and native MS, we preserve the native protein conformation and protein–lipid interactions from the cell to the final analysis step. Analysis of the copurified endogenous lipids revealed that the three LTPs selectively bind PC lipids with distinct acyl chain combinations. This specificity correlates with their differential expression across different tissues and feeds back into the overall lipid metabolism. Furthermore, native top-down MS and proteomic profiling uncovered phosphorylation sites near membrane-interacting regions, which attenuate lipid transfer activity. Collectively, our results show that cellular lipid metabolism is tightly coupled to the expression levels and acyl chain selectivities of LTPs and their regulation through reversible PTMs.
[1] C. Kirschbaum, J. L. Bennett, C. V. Robinson, Anal. Chem. 2025, 97, 19331–19339. DOI: 10.1021/acs.analchem.5c03691
[2] J. L. Bennett, T. J. El-Baba, K. C. Zouboulis, C. Kirschbaum, H. Song, F. I. Butroid, J. L. P. Benesch, C. A. Lutomski, C. V. Robinson, Nat. Methods 2025, 22, 2127–2137. DOI: 10.1038/s41592-025-02846-5
