Our project aims to elucidate the structural interactions within the LCN2-MMP9 protein complex involved in cancer metastasis. A critical step in this process is obtaining precise experimental data through cross-linking mass spectrometry (XLMS) to identify interaction interfaces and develop accurate structural models. Although our comprehensive XLMS analysis is ongoing, we have made significant progress in the experimental preparation for LCN2 analysis, yielding promising initial results.

Successful In-Gel Digestion of LCN2

To begin our exploration of the LCN2-MMP9 complex, we performed in-gel digestion of purified LCN2 following cross-linking reactions. This step is vital, as this way, we obtain mass spectra for non-cross-linked proteins. (We also used an LCN2 mutant, the model of which we plan to build in IMPROViSeD, to compare the results with the wild type.)

After running the cross-linked samples on SDS-PAGE, we successfully identified the LCN2 band based on its expected molecular weight. This band was carefully excised from the gel, and we proceeded with the established protocol for in-gel tryptic digestion. The LCN2 protein was digested into peptides overnight, ensuring thorough cleavage into smaller fragments amenable to mass spectrometry.

The digestion process was confirmed to be successful, as subsequent steps yielded well-resolved peptide fragments. This success sets a solid foundation for further analysis of LCN2 and its interaction with MMP9

Mass Spectrometry Analysis of LCN2 Peptides

Following the in-gel digestion, the resulting peptides were extracted and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). The high-resolution mass spectrometry data were meticulously analyzed, focusing on identifying the peptide sequences derived from LCN2 and assessing potential cross-linked regions that could provide insights into protein interactions.

Our mass spectrometry analysis yielded a robust set of LCN2 peptide spectra, confirming the successful identification of multiple peptide fragments corresponding to different regions of the LCN2 protein. This data provides a rich source of information for further interpretation and cross-linking analysis. We performed a nano-LC-MS/MS and the analysis was done using the Thermo xCalibur Qual Browser software.

In particular, we observed peptides that cover key regions of the LCN2 structure, which could serve as critical points of interaction in its complex with MMP9. These peptide spectra will be analyzed in more detail to identify any cross-linked pairs that indicate proximity between the two proteins.

LCN2 (wild type) : Base peaks

LCN2 (wild type) : TIC (Total Ion Chromatogram)

Next Steps: Cross-Linking Data Integration

While our mass spectrometry results have so far focused on LCN2 alone, this marks a crucial first step in mapping the interaction interfaces between LCN2 and MMP9. Moving forward, we will integrate the cross-linking data derived from our XLMS experiments with these peptide fragments, and feed into our Integrated Modelling Platform for Protein Complexes (IMPROViSeD) to refine the structural models of this critical cancer-related protein interaction. As we continue to build on these results, we anticipate that our experimental pipeline will reveal new insights into the mechanisms underlying tumor metastasis, with potential applications in therapeutic design.