Utilizing spectral counting to quantitatively characterize tandem removal of abundant proteins (TRAP) in human plasma

Biomarker discovery efforts in serum and plasma are greatly hindered by the presence of high abundance proteins that prevent the detection and quantification of less abundant, yet biologically significant, proteins. The most common method for addressing this problem is to specifically remove the few abundant proteins through immunoaffinity depletion/subtraction. Herein, we improved upon this method by utilizing multiple depletion columns in series, so as to increase the efficiency of the abundant protein removal and augment the detection/identification of less abundant plasma proteins. Spectral counting was utilized to make quantitative comparisons between undepleted plasma, plasma depleted with a single depletion column, and plasma depleted using two or three depletion columns in tandem. In the undepleted plasma only 29 lower abundance protein groups were identified with the top-scoring protein from each group having a median spectral count of 3, while in the plasma processed using a single HSA depletion column 61 such protein groups were identified with a median spectral count of 8. In comparison, 76 lesser abundant protein groups were identified with a median spectral count of 11.5 in the two column setup (i.e., HSA followed by MARS Hu14). However, in the ultimate depleted plasma sample, which was created using three depletion columns in tandem, the number of less abundant protein groups identified increase to 81 and the median spectral count for the top-scoring proteins from each group increased to 15 counts per protein. Moreover, exogenous B-type natriuretic peptide-32, which was added to the plasma as a detection benchmark at 12 μg/mL, was only detected in the plasma sample depleted using three depletion columns in tandem. Collectively, these data demonstrate that this method, tandem removal of abundant proteins or TRAP, provides superior removal efficiency compared to traditional applications and improves the depth of proteome coverage in plasma.