Sample Preparation Strategies for Microbial Cell Surface Proteomics: Integrating Shaving and Shotgun Approaches

A range of mass spectrometry-based proteomic approaches is now employed to characterize the surface proteome-surfaceome-of pathogenic microorganisms, including fungi capable of causing infections in humans. Nevertheless, the preparation of surface-enriched samples remains technically challenging because of the persistent risk of intracellular protein contamination. Cell surface shaving represents a focused sample treatment approach in which proteolytic enzymes act on intact cells to release surface-exposed peptides, thereby enriching this fraction for high-resolution liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) analysis. Optimizing sample preparation for fungal surface proteomics is critical, as reducing sample complexity, enriching surface-exposed components, and ensuring LC-MS/MS compatibility must be achieved while avoiding matrix interferences, partial lysis, and biases toward only trypsin-accessible epitopes. These methodological challenges, as well as strategies for validating sample-treatment selectivity with appropriate controls, are discussed in this chapter.