Different Stationary Phase Selectivities and Morphologies for Intact Protein Separations

Chromatographia, Sep 2016

The central dogma of biology proposed that one gene encodes for one protein. We now know that this does not reflect reality. The human body has approximately 20,000 protein-encoding genes; each of these genes can encode more than one protein. Proteins expressed from a single gene can vary in terms of their post-translational modifications, which often regulate their function within the body. Understanding the proteins within our bodies is a key step in understanding the cause, and perhaps the solution, to disease. This is one of the application areas of proteomics, which is defined as the study of all proteins expressed within an organism at a given point in time. The human proteome is incredibly complex. The complexity of biological samples requires a combination of technologies to achieve high resolution and high sensitivity analysis. Despite the significant advances in mass spectrometry, separation techniques are still essential in this field. Liquid chromatography is an indispensable tool by which low-abundant proteins in complex samples can be enriched and separated. However, advances in chromatography are not as readily adapted in proteomics compared to advances in mass spectrometry. Biologists in this field still favour reversed-phase chromatography with fully porous particles. The purpose of this review is to highlight alternative selectivities and stationary phase morphologies that show potential for application in top-down proteomics; the study of intact proteins.

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Different Stationary Phase Selectivities and Morphologies for Intact Protein Separations

Chromatographia Different Stationary Phase Selectivities and Morphologies for Intact Protein Separations A. Astefanei 0 1 2 I. Dapic 0 1 2 M. Camenzuli 0 1 2 0 Centre for Analytical Science in Amsterdam (CASA), Van't Hoff Institute for Molecular Sciences, University of Amsterdam , Science Park 904, 1098 XH Amsterdam , The Netherlands 1 M. Camenzuli 2 Published in the topical collection Young Investigators in Separation Science with editors D. Mangelings , G. Massolini, G. K. E. Scriba, R. M. Smith and A. M. Striegel The central dogma of biology proposed that one gene encodes for one protein. We now know that this does not reflect reality. The human body has approximately 20,000 protein-encoding genes; each of these genes can encode more than one protein. Proteins expressed from a single gene can vary in terms of their post-translational modifications, which often regulate their function within the body. Understanding the proteins within our bodies is a key step in understanding the cause, and perhaps the solution, to disease. This is one of the application areas of proteomics, which is defined as the study of all proteins expressed within an organism at a given point in time. The human proteome is incredibly complex. The complexity of biological samples requires a combination of technologies to achieve high resolution and high sensitivity analysis. Despite the significant advances in mass spectrometry, separation techniques are still essential in this field. Liquid chromatography is an indispensable tool by which lowabundant proteins in complex samples can be enriched and separated. However, advances in chromatography are not as readily adapted in proteomics compared to advances in mass spectrometry. Biologists in this field still favour reversed-phase chromatography with fully porous particles. LC-MS; Liquid chromatography; Top-down proteomics; Intact proteins - A. Astefanei and I. Dapic contributed equally to this work. Introduction Proteomics is often applied to clinical studies in the search of biomarkers [ 1 ]. These biomarkers are mostly proteins that are found in the tissue or plasma of patients suffering from a particular disease yet may be expressed in different amounts in healthy patients. While this sounds simple, the reality is that there are approximately 20,000 proteinencoding genes in the human body [ 2 ]. Many of these genes code for more than one protein isoform (proteoforms). These proteoforms arise from various post-translational modifications (PTMs) including phosphorylation, methylation and ubiquitination to name but a few, which can change the function of the protein in addition to modifying its structure. Since there are a number of amino acids that can act as sites for PTMs it follows that proteoforms can have varying degrees of PTMs in addition to multiple types of PTM. Consequently from any given proteinencoding gene, a large number of proteins can be produced. Proteins are expressed in varying abundance with some proteins such as albumin in blood, being much more abundant than other proteins present in biological material. It follows that these aspects significantly complicate the study of any proteome. Such samples require analytical techniques capable of providing high resolving power and sensitivity. Mass spectrometry (MS) is an obvious choice for proteomics research given its separation power and the ability to characterize protein structure through the interpretation of the fragmentation patterns in mass spectra. However, the analysis of intact proteins using MS faces a number of technical challenges. The large dynamic range in protein abundances within a sample can result in the suppression of the ionization of low abundant proteins, reducing their ability to be detected. Once ionized, intact proteins feature multiple charge states all corresponding to the same protein species, with multiple isotopes for each charged state. Different types of mass spectrometers present different levels of resolving power which may or may not be enough for the isotopic distributions of each protein multiple charge states to be resolved. Developments in Fourier Transform Ion Cyclotron Resonance (FTICR) MS has been an important step towards improving our ability to analysis proteins. In an MS imaging application using FTICR and secondary ion MS, resolving power in the order of 3,000,000 has been reported [ 3 ]. This compares to a resolving power of 2000– 10,000 using time-of-flight (TOF) in a similar setup. Even with the high resolving power of FTICR MS, hyphenation of MS with other separation techniques is necessary to reduce the sample complexity. Liquid chromatography (LC) is widely used for this purpose due to its high separation power and the ability to hyphenate it with MS, typically via electrospray ionization (ESI). However, using LC for protein separations faces its own technical challenges. Proteoforms often have similar physio-chemical properties making their separati (...truncated)


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A. Astefanei, I. Dapic, M. Camenzuli. Different Stationary Phase Selectivities and Morphologies for Intact Protein Separations, Chromatographia, 2016, pp. 665-687, Volume 80, Issue 5, DOI: 10.1007/s10337-016-3168-z