Improvements in Proteomic Metrics of Low Abundance Proteins through Proteome Equalization Using ProteoMiner Prior to MudPIT

Abstract

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.

Directed shotgun proteomics guided by saturated RNA-seq identifies a complete expressed prokaryotic proteome

“…The enrichment for shorter proteins can be appreciated in the upper left panel of Figure 3B. Lowabundance proteins were targeted using ProteoMiner (Guerrier et al 2008;Fonslow et al 2011). These experiments (Fig.…”

Detailed Methodologies and Protocols

“…For very complex extracts like eukaryotic cell lysates, Fonslow et al (2011) found that the addition of 0.1% SDS allows increasing the number of captured proteins and hence the number of spots found by 2D gel analysis. These authors justified this approach based on the assumption that this detergent denatures proteins and further exposes high-affinity epitopes, disrupts protein interactions within protein complexes, and may minimize hydrophobic hexapeptide-protein interactions.…”

“…However, if the objective is limited to identification of captured proteins by means of the analysis of peptides, it is possible to hydrolyze the captured proteins directly on the beads (Fonslow et al, 2011;Meng et al, 2011). This approach is also recommended when the volume of beads is very small.…”

Proteomics of Endosomal Compartments from Plants Case Study: Isolation of Trans-Golgi Network Vesicles

“…Proteomic analysis can be carried out using a variety of approaches, depending on expertise and availability of equipment. When the protein content is low, a gel-free strategy of direct on-beads digestion can be employed and the resulting tryptic peptides can be analyzed by MudPIT nano-LC-MS/ MS [ 22 ]. The resultant MS/MS data can be investigated with the latest version of the Arabidopsis protein database using Mascot (Matrix Sciences).…”