The separation sciences, the front end to proteomics: An historical perspective

Abstract

It is now over 25 years since the term proteomics (analysis of the entire protein complement of a cell, tissue, or organism under a specific, defined set of conditions) was originally coined. Since then, the field has advanced rapidly and there are now more than 135,500 publications addressing the field. With current instrumentation it is possible to detect over 10,000 protein forms in a single experiment. The separation of proteins and peptides has been a key component of many of these studies for both sample concentration and enrichment and to reduce the complexity of the samples under analysis, allowing deeper mining of the individual proteomes. In this review, the roles of chromatography, electrophoresis, and other allied techniques in the advancement of the field will be investigated. Key technologies will be presented, and examples given of their application showing how the field has now advanced to a stage where it is enhancing our understanding of the human biology underlying health and disease, and clinical translation, supporting precision/personalized medicine, is now feasible. Clearly the separation sciences have played a key role in many of these advances.

Proteomics, Personalized Medicine and Cancer

“…MS-based proteomics relies on success in three main areas: sample pretreatment and analysis and data analysis. Two-dimensional gel electrophoresis (2-DE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were the original mainstays for sample separation before MS analysis, with the ability to separate over 10,000 proteoforms [ 13 ], and indeed these systems are still in use [ 20 ]. In this example, proteome information of tumor tissues and normal tissues was obtained by SDS-PAGE for a comparative proteomic analysis of different stages of BC.…”

“…More recently chromatographic methods have been well recognized as methodologies worthy of consideration with particular advantages, especially in the areas of sample manipulation, recovery and automation. Multidimensional purification has been found to be particularly efficacious, giving high purification factors and reducing sample complexity prior to MS analysis, enabling deeper mining of the proteome [ 13 , 21 , 22 , 23 , 24 ]. As exemplars, Kaur et al have designed a simple fractionation workflow to extend the coverage of the plasma proteome [ 25 ].…”

“…This has been largely facilitated by the detailed analysis of the human genome and proteome [ 9 , 10 , 11 ], and as predicted, corresponding technological advances [ 12 ]. Proteomics is defined as the characteristic analysis of the whole protein complement of a cell, tissue or organism at a particular condition, including protein interactions, posttranslational modifications and localization [ 13 ]. Proteins are the molecules directly responsible for life, driving the structure, function and regulation of the body’s tissues and organs.…”

On the Applicability of Electrophoresis for Protein Quantification

“…Protein chemistry methods are currently used to control, optimize, and elaborate novel technologies in molecular biology, pharmacology, bioengineering, and food technology [1][2][3][4][5]. Such efficient, fast, illustrative, and reproducible methods as HPLC, HPLC-MS, and PCR coupled with Sanger sequencing are used for protein quantification [6][7][8][9].…”

On the Applicability of Electrophoresis for Protein Quantification

“…Protein chemistry methods are currently used to control, optimize, and elaborate novel technologies in molecular biology, pharmacology, bioengineering, and food technology [1][2][3][4][5]. Such efficient, fast, illustrative, and reproducible methods as high-performance liquid chromatography (HPLC) with different detectors and polymerase chain reaction (PCR) coupled with Sanger sequencing are used for protein quantification [6][7][8][9].…”