The combination of the two types of software does not increase significantly the total number of proteins identified, but it positively affects the average sequence coverage. The limited number of proteins identified in this study, despite a multi-step approach,ASB14780 highlights the difficulty of analysing the plasma proteome. The merging of all sets of data allowed the identification of a total of 279 unique protein groups with a 99% confidence. However, our aim was not to develop a protocol for the identification of the maximum number of proteins in plasma, but rather to evaluate which of the two methods, between HAPs depletion and LAPs enrichment, is more suitable as the first step for a plasma proteomic analysis. Despite the different number of identifications, all the fractionation approaches primarily led to the detection of proteins related to acute phase reaction,T-26c and complement and coagulation, including proteins which can be classified as high- and mid- abundance plasma proteins. To show the overlap among the fractionation methods, we reporta Venn diagram of the protein groups identified with 99% confidence and one peptide per protein. From this diagram it is clear that the three experimental protocols are complementary: only 69 protein groups are common to all the approaches, which represent only 37, 42, and 50% of all groups associated to ProteoPrep20, ProteoExtract+ProteoPrep20, and ProteoMiner respectively. By looking at the list of proteins identified and the Venn diagram, we conclude that the great majority of proteins, regardless the fact that they are identified with one or more methods, belong to the above mentioned categories. Therefore, all methods yielded similar performance in terms of concentration range of identified proteins. These data may suggest that the depletion and the enrichment methods we have compared exhibit a similar performance and lead to partially overlapping results. However, there are several other aspects to be taken into account. The aim of a plasma proteome analysis is to study proteins with a concentration under 100 ng/ml, so other steps will necessarily follow the first to improve sensitivity of the analysis. In this regard, the use of ProteoPrep20, as a first-line fractionation step, does not seem very practical.