36 February / March 2019
Mobile Affinity Sorbent Chromatography Of Proteins
by Fred Regnier+,‡ , JinHee Kim+ , Meena Narsimhan+ , Kanon Goodrich+ +Novilytic Laboratories, West Lafayette, Indiana 47906, USA; ‡ . Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
A new three-phase partitioning system is described in which a nanoparticulate analyte sequestering transport phase (ASTP) causes analytes to elute in the column void volume followed by impurities. The function of the ASTP is to structure specifically sequester an analyte (An
Fluorescent sandwich methods were used for detection in the examples shown. ) with high
affinity and be of sufficient size to be excluded from a size exclusion chromatography (SEC) column. The linear velocity of non-analytes in contrast is retarded by diffusion into pores of the column packing during SEC. Analytes in the ASTP:An
complex are detected by an analyte specific method.
Introduction
Based on advances in mass spectrometry analysis of proteins and their peptide fragments it has been predicted that the human proteome of many organisms is composed of more than a million proteins, many of which are proteoforms of very similar structure [1]. Although gas phase analysis is very powerful and exciting, most of these proteins have never been ‘held in the hand’, so to speak. Total characterisation and analysis require protein purification. There is also the issue of cost and speed of analysis. Sample preparation can take hours with mass spectral methods [2]. There is a need for inexpensive methods that rapidly identify proteoforms in routine analyses. Millions of protein specific analyses are performed annually in clinical diagnostics [3]. With samples as complicated as blood, existing separation systems do not differentiate well between structural isoforms [1]. Improvements in protein separation methods are needed.
Liquid chromatography (LC) systems have been at the core of protein purification and structure analysis for more than 50 years. The underlying two-phase partitioning mechanism enabling protein resolution remains unchanged [4]. Although improvements in stationary phases, enhanced particle fabrication methods, and reductions in particle size have greatly advanced the resolution and speed of this separation mode, the peak capacity of most liquid chromatography columns is still a
hundred or less. With biological extracts of 105
probability individual peaks will contain many proteins.
The discussion below will focus on a new, inexpensive mobile affinity sorbent chromatography (MASC) approach that increases resolution and speed in routine analyses by incorporating a transport phase into conventional two-phase LC systems [5]. The major advantage of MASC is that it rapidly increases selectivity.
or more components there is a high
The Two-Phase Separation Mechanism
Proteins are separated in LC by repetitive differential partitioning between a stationary phase (PS
) and mobile phase (Pm achieved by Pm ) [6]. This is transport of mixtures through
long, narrow channel networks bearing a stationary phase (PS
or in stagnant Pm elute. Stagnant Pm ) embedded in a packed
particle bed. Substances spending time on a solid Ps
take longer to is located within porous, insoluble supporting matrices (Figure 1).
Figure 1. Schematic of a longitudinal channel segment in a two-phase separation system. For the sake of illustration relative dimensions in the schematic are not equivalent to those in LC systems. Support structures are matrix domains within particles that provide pore networks and surfaces to which bonded phases may be covalently linked. The concept of laminar flow between particles in a packed bed is well documented in hydrodynamic chromatography [7,8]. Bonded phases in protein SEC systems are hydrophilic and neutral, their function being to diminish non-specific binding. Stagnant mobile phase fills the pore matrices in all two-phase separation systems and serves as the Ps various forms of adsorption based separations.
in SEC. Proteins bind directly to the bonded phase in the
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