In an effort to optimize reverse-phase liquid chromatography (RPLC) for 
proteomics, we studied the impact of composition of the sample injection 
solution on protein on-column selection and retention. All the proteins studied 
were retained on-column when injections were made in 50% formic acid, 0.1% TFA 
or 8.3M urea. When formic acid was increased to 80%, the superoxide dismutase 
standard (MW 26,159) and 58 mouse microsomal proteins that possessed low-range 
molecular weights, high pIs or basic amino acid clusters were non-retained, 
resulting in retention selectivity during sample injection. Introducing to the 
80% formic acid injection solution an organic solvent such as acetonitrile or 
acetonitrile-DMSO induced further retention selectivity, and increasing levels 
of organic solvents reduced on-column retention. The proteome was split into the 
proteins that were retained on-column which eluted at higher retention times 
(RTs), vs the proteins that collected in the injection flow-through which 
normally eluted at lower RTs. This protein selectivity was confirmed after 
fraction collection, 1D-GE and nano-LC-MS/MS. The significance of this procedure 
is that it can be exploited for fast extraction of small basic proteins from the 
bulk of the proteome and for on-column enrichment of hydrophobic proteins.