Direct injection of complex protein mixtures, e.g. those derived from crude biological fluids, is often incompatible with conventional LC supports, because of column clogging and rapid deterioration of chromatographic performance. In this paper, we report the use of restricted access media to rapidly enrich and fractionate human breast milk. This resin, combining size exclusion and anion exchange functionalities, yielded a fraction enriched in soluble CD14 and showing specific sCD14-dependant activity. This fraction was split into five aliquots, which were individually characterized using multidimensional protein identification technology. Reproducibility of the results was addressed by analysing and comparing five datasets using different protein identification tools available within the Sequest software. Furthermore, a comparison of three major releases of the Ensembl human protein database was performed to examine the effect of database updates on our results. We report here the benefit of repeated analysis of aliquots of the same fraction: first to increase the confidence in peptide identification by repeated confirmation in several aliquots; and second to assess experimental reproducibility. We demonstrate furthermore the effect of database modifications on the results and the importance of constantly re-analysing data with new releases to keep them consistent and up to date with the latest protein identities and predictions available.