High Abundance Proteins Depletion vs Low Abundance Proteins Enrichment: Comparison of Methods to Reduce the Plasma Proteome Complexity

Abstract

BackgroundTo date, the complexity of the plasma proteome exceeds the analytical capacity of conventional approaches to isolate lower abundance proteins that may prove to be informative biomarkers. Only complex multistep separation strategies have been able to detect a substantial number of low abundance proteins (<100 ng/ml). The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity columns or, alternatively, the enrichment of by the use of solid phase hexapeptides ligand libraries.Methodology/Principal FindingsHere we present a direct comparison of these two approaches. Following either approach, the plasma sample was further fractionated by SCX chromatography and analyzed by RP-LC-MS/MS with a Q-TOF mass spectrometer. The depletion of the 20 most abundant plasma proteins allowed the identification of about 25% more proteins than those detectable following low abundance proteins enrichment. The two datasets are partially overlapping and the identified proteins belong to the same order of magnitude in terms of plasma concentration.Conclusions/SignificanceOur results show that the two approaches give complementary results. However, the enrichment of low abundance proteins has the great advantage of obtaining much larger amount of material that can be used for further fractionations and analyses and emerges also as a cheaper and technically simpler approach. Collectively, these data indicate that the enrichment approach seems more suitable as the first stage of a complex multi-step fractionation protocol.

Fluorescence Anisotropy Reduction of Allosteric Aptamer for Sensitive and Specific Protein Signaling

“…It is known that more than 10 abundant proteins (about 90% of total protein in amount) are present in human serum. 29 In addition, our fluorescence emission spectroscopy and FA analysis suggest the diluted serum can produce a strong fluorescence background (see Figure S2 in the Supporting Information) and high FA response (r > 0.5).…”

High Abundance Proteins: Proteomer’s Thorns in the Flesh?

“…The development and utilization of latest mass spectrometers have increased the sensitivity of the protein identification, fostering the identification of proteins present in extremely small amounts (up to attomoles, in isolation) [3]. However, the identification of low-abundance proteins during the analysis of whole cell/tissue proteome is still not achievable without any prefractionation of the samples [4]. One of the major reasons of this limited resolution, during total proteome analysis, is the presence of high-abundance proteins which occupies a major portion of the cell/ tissue proteome.…”

Designing biomedical proteomics experiments: state-of-the-art and future perspectives

“…One of the most typical examples are biomedical projects where (human) serum/plasma samples are frequently used to deplete highly abundant proteins in order to decrease the dynamic range and thus to increase coverage of lower abundant proteins. Several different tools exist to achieve the depletion of most of the highly abundant proteins, including Multiple Affinity Removal System (MARS) and ProteoPrep [29].…”