Developing top down proteomics to maximize proteome and sequence coverage from cells and tissues

Abstract

Mass spectrometry based proteomics generally seeks to identify and characterize protein molecules with high accuracy and throughput. Recent speed and quality improvements to the independent steps of integrated platforms have removed many limitations to the robust implementation of top down proteomics (TDP) for proteins below 70 kDa. Improved intact protein separations coupled to high-performance instruments have increased the quality and number of protein and proteoform identifications. To date, TDP applications have shown >1000 protein identifications, expanding to an average of ∼3-4 more proteoforms for each protein detected. In the near future, increased fractionation power, new mass spectrometers and improvements in proteoform scoring will combine to accelerate the application and impact of TDP to this century's biomedical problems.

Figure 1

A schematic depiction of one work and data flow used for high-throughput top-down proteomics. Total protein content is quantified after cell purification or sub-cellular fractionation and loaded onto a GELFREE column (see Figure 2) for molecular weight-based separation. Protein fractions of increasing molecular weight are processed to remove SDS before injection onto reversed phase columns for online MS or MS/MS analysis. LC-MS/MS files are processed with ProSight and accompanying software for high-throughput protein identification and characterization.

Figure 2

Gel Eluted Liquid Fractionation Entrapment Electrophoresis (GELFrEE). The device consists of a resolving gel which can be cast to analogous to an SDS-PAGE gel depending on the separation desired. Fractions are manually removed from the collection chamber in a time-based manner. A portion of each fraction can be removed and optionally visualized on a traditional SDS-PAGE gel (at right) to assess separation performance. This mass-based separation allows instrument parameters to be selected for acquisition of optimal top down MS and MS/MS data sets.

Figure 3

An overview of some mass analyzers used for top down proteomics analysis. From custom instruments (top left) that were used to pioneer the technique to hybrid FT-ICR systems (top right), FTMS continues to be the workhorse mass spectrometers for top down proteomics. Recently however, several research groups have shown the capability of the Orbitrap line as a promising option for many labs (bottom).

Figure 4

A conceptual comparison of how proteomics measurements (in $/proteoform) might be reduced in the future from the same type of public/private sector construct used in the Human Genome Project. Cost per measurement can fall with investment and focused effort. Figures are approximate and projections are not based on advanced modeling.(Adapted from [8]).