Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

The overall goal of this procedure is to extract lipoproteins from bacterial cells for direct applications and to further prepare N-terminal lipopeptides for structural analysis by MALDI-TOF mass spectrometry. This method can help answer key questions in the immunology field, as the lipoproteins'N-terminal structure influences Toll-like receptor recognition and signaling, impacting the host's immune response. The unique advantage of this technique is the optional but intentional formation of sodium adducts, which promotes fragmentation towards the diagnostic dehydroalanyl ion, aiding in structural assignment of the lipoprotein's acylation state.

Grow bacteria in 15 milliliters of tryptic soy broth or similar rich media to late exponential phase. Harvest the cells by centrifugation before washing once with Tris-buffered saline EDTA or TBSE. resuspend the cells in 800 microliters of TBSE with one millimolar PMSF and 0.5 milligram per milliliter lysozyme.