Plasma proteins may often serve as indicators of disease and are a rich source for biomarker discovery. However, the intrinsic large dynamic range of plasma proteins makes the analysis very challenging because a large number of low abundance proteins are often masked by a few high abundance proteins. The use of prefractionation methods, such as depletion of higher abundance proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may ultimately prove to be informative biomarkers. But there are few studies on comprehensive investigation of the proteins both in the fractions depleted and remainder. In the present study, two different immunoaffinity fractionation columns for the top-6 or the top-12 proteins in plasma were investigated and both the proteins in column-bound and flow-through fractions were subsequently analyzed. A two-dimensional peptide separation strategy, utilizing chromatographic separation techniques, combined with tandem mass spectrometry (MS/MS) was employed for proteomic analysis of the four fractions. Using the established HUPO PPP criteria, a total of 2401 unique plasma proteins were identified. The Multiple Affinity Removal System yielded 921 and 725 unique proteins from the flow-through and bound fractions, respectively, whereas the Seppro MIXED 12 column yielded identification of 897 and 730 unique proteins from the flow-through and bound fractions, respectively. When more stringent criteria, based on searching against the reversed database, were implemented, 529 unique proteins were identified from the four fractions with the confidence in peptide identification increased from 73.6% to 99%. To determine whether the presence of nontarget proteins in the immunoaffinity-bound fraction could be attributed to their interaction with high abundance proteins, co-immunoprecipitation analysis with an antibody to human plasma albumin was performed, which resulted in an identification of 40 unique proteins from the coimmunoprecipitate with the more stringent criteria. This study illustrated that combining the column-bound and flow-through fractions from immunoaffinity separation affords more extensive profiling of the protein content of human plasma. The presence of nontarget proteins in the column-bound fractions may be induced by their binding to the higher abundance proteins targeted by the immunoaffinity column.