Ideally shotgun proteomics would facilitate the identification of an entire proteome with 100% protein sequence coverage. In reality, the large dynamic range and complexity of cellular proteomes results in oversampling of abundant proteins, while peptides from low abundance proteins are undersampled or remain undetected. We tested the proteome equalization technology, ProteoMiner, in conjunction with Multidimensional Protein Identification Technology (MudPIT) to determine how the equalization of protein dynamic range could improve shotgun proteomics methods for the analysis of cellular proteomes. Our results suggest low abundance protein identifications were improved by two mechanisms: (1) depletion of high abundance proteins freed ion trap sampling space usually occupied by high abundance peptides and (2) enrichment of low abundance proteins increased the probability of sampling their corresponding more abundant peptides. Both mechanisms also contributed to dramatic increases in the quantity of peptides identified and the quality of MS/MS spectra acquired due to increases in precursor intensity of peptides from low abundance proteins. From our large data set of identified proteins, we categorized the dominant physicochemical factors which facilitate proteome equalization with a hexapeptide library. These results illustrate that equalization of the dynamic range of the cellular proteome is a promising methodology to improve low abundance protein identification confidence, reproducibility, and sequence coverage in shotgun proteomics experiments, opening a new avenue of research for improving proteome coverage.

Low-abundance protein detection in biological samples is one of the main challenges in proteomics investigations. One approach that makes the detection of rare species possible is the treatment of biological samples with solidphase combinatorial peptide ligand libraries. However, the use of combinations of ligands opens an uncertainty in that, since the diversity of the library is very large, aliquots of beads sampled from the library might not have fully comparable bead species each time. Reproducibility of experimental data with highly diverse libraries is therefore a main concern to address. This paper reports reproducibility data when aliquots of similar and different volumes of libraries are used at a certain sample to library ratio. Eluates from ligand libraries and other fractions are analyzed using various complementary methods such as two-dimensional gel electrophoresis, immunoassay and mass spectrometry.The collected data show a high level of consistency from sample to sample when processed with similar and variable bead volumes. Analytical determinations are all convergent with each other in considering the similarity of results. It is anticipated that this demonstration reinforces the possibility that differential proteomics studies, in particular for the discovery of protein targets of interest, can effectively be accomplished with combinatorial peptide libraries.

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Background: Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE).

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