Bacterial
surface proteins are of outmost importance as they play critical roles in the interaction between cells and their environment. In addition, they can be targets of either
vaccines or
antibodies. Proteomic analysis through "shaving" live cells with
proteases has become a successful approach for a fast and reliable identification of
surface proteins. However, this protocol has not been able to reach the goal of excluding cytoplasmic contamination, as cell lysis is an inherent process during culture and experimental manipulation. In this work, we carried out the optimization of the "shaving" strategy for the Gram-positive human pathogen Streptococcus pneumoniae, a bacterium highly susceptible to
autolysis, and set up the conditions for maximizing the identification of
surface proteins containing sorting or exporting signals, and for minimizing cytoplasmic contamination. We also demonstrate that cell lysis is an inherent process during culture and experimental manipulation, and that a low level of lysis is enough to contaminate a "surfome" preparation with
peptides derived from cytoplasmic
proteins. When the optimized conditions were applied to several clinical isolates, we found the majority of the
proteins described to induce protection against
pneumococcal infection. In addition, we found other
proteins whose protection capacity has not been yet tested. In addition, we show the utility of this approach for providing
antigens that can be used in serological tests for the diagnosis of
pneumococcal disease.