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
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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)