Pharmaceutical proteomics: a journey from discovery and characterisation of targets to development of high-throughput assays
Posted: 15 December 2013 |
Proteomics has evolved during the last few years from a time-intensive, cost-intensive and hard-to-reproduce technique in basic research to a versatile and reliable tool in various areas of pharmaceutical research. The exploding progress in mass-spectrometry-compatible protein and peptide-separation methods led to the development of new approaches particularly suited for monitoring a multitude of specific targets in highly complex matrices in a highly sensitive, specific and parallel fashion. These new technologies have caused a paradigm shift in proteomics from mostly gel-based, hypothesis-generating studies towards fast, cost-effective and mostly LC-MS-based assays. Therefore, proteomics emerges for pharmaceutical researchers aiming to identify and verify proteinaceous biomarkers as proteomics technology comes of age.
This mini-review highlights some of the recent technical developments that facilitate the use of proteomics as a tool for important steps in target protein identification, characterisation, biomarker discovery, validation and monitoring and discuss advantages and disadvantages of individual techniques.
Proteomics: from past to present
The term ‘proteome’ was coined as the entirety of all expressed proteins encoded in a genome1. In contrast to the genome which remains rather unchanged during an organisms` life and is the same for every cell, the proteome is not only altered by cell-type but is also highly dynamic due to various intrinsic and extrinsic factors that may affect it even within a few seconds. Proteomics therefore aims at a quantitative description of all proteins of a cell in parallel and their regulations by different stimuli. The necessity to identify and quantify thousands of proteins in parallel, typically from rather tiny available amounts of sample, poses a great challenge to analytical methods.
Protein analytical techniques used in proteomics nowadays like gel-based protein separations in one or two dimensions and chromatographic techniques for separation of peptides have been known and widely used for decades. Yet, protein identification e.g. by Edman-sequencing was restricted to pure proteins in rather large amounts and was therefore very time-consuming and often not successful. While protein and peptide separation techniques were continuously improved in separation power, sensitivity and dynamic range, protein identification remained the bottleneck in protein analysis and prevented their analysis on a proteomic scale.
The evolution of proteomics from protein analysis really gained momentum with the availability of genomic databases from the genome era and the development of soft ionisation techniques enabling the analysis of peptides and proteins by (tandem-) mass spectrometry. Electrospray ionisation (ESI) particularly became a widespread technique because it facilitates the direct coupling of liquid chromatography to tandem mass spectrometry, thereby allowing reliable high-throughput protein identification also from high-complexity mixtures.
Development and continuous improvement of the core techniques of proteomics, most notably protein electrophoresis, peptide chromatography and tandem mass spectrometry, paved the way for proteomics as a versatile tool in all stages of pharmaceutical research.
Analysis of intact proteins: top-down analyses
The mass spectrometric measurement of intact proteins has the benefit of reflecting the overall modification state of the respective protein and recent developments in mass spectrometric instrumentation also facilitates top-down sequencing of proteins2. By these means, the proteins can not only be identified but modifications on the proteins such as acetylations or phosphorylations can be localised to individual amino acid residues. These analyses are typically carried out on high-resolution mass spectrometers with FT-ICR-, orbitrap- or QTOF-analysers that feature the necessary mass accuracy and resolution for the analysis of the highly-charged analyte ions. The major limitations of the top-down mass spectrometry-techniques still lie in the insufficiency in the analysis of high-complexity mixtures and the fragmentation of large proteins as MS/MS-spectrum complexity rises tremendously with protein size and full sequence coverage is typically not obtained above 20 kDa.