Selectivity of bacterial proteome fractionation based on differential solubility: A mass spectrometry evaluation

We investigate the selectivity achieved after differential solubilization of bacterial proteomes following two procedures, both based on successive extraction of proteins in solutions of increasing solubilizing power. Recently, these procedures have gained notable popularity and several commercial kits are now available. A total of 225 proteins in one case and 227 proteins in the other were identified by LC MSMS analysis; 146 of them were identified in both procedures. The proportions of proteins identified as present in only one fraction were 64 and 57%, respectively. The distribution of cytosolic, membrane, and ribosomal proteins among the successive extracts was analyzed in detail. The effect of (1) replacement of low-speed with high-speed centrifugation, (2) omission of detergents in urea solutions, (3) successive washes of pellets, and (4) reproducibility was evaluated. Proteins with positive grand averages of hydropathicity values and membrane proteins were found in all fractions. This study highlights the benefits and limitations of differential solubilization methods, focusing on practical aspects that may strongly influence their selectivity.