Neal F. Gordon

Very high speed separation of proteins with a 20-μm reversed-phase sorbent

Abstract A five-protein mixture was successfully separated in less than 15 s by reversed-phase gradient elution on a 20-μm polymeric flow-through type chromatographic packing material (POROS R/M). The same mixture could also be separated with far better resolution in less than 60 s on the same column by simply extending the gradient volume. Pressure drop across the column was less than 130 bar, despite a superficial linear velocity of nearly 9000 cm/h. Frontal chromatography at different flow-rates was used to demonstrate that the ability to separate at high speed was due to greatly enhanced mass transport within the particles due to the flow-through (perfusion chromatography) effect.

Characterization of large-pore polymeric supports for use in perfusion biochromatography

Perfusion chromatography is uniquely characterized by the flow of a portion of the column eluent directly through the resin in the packed bed. The benefits of this phenomenon and some of the properties of perfusive resins have been described before, and can be summarized as enhanced mass transport to interior binding sites. Here we extend the understanding of this phenomenon by comparing resins with different pore size distributions. Resins are chosen to give approximately the same specific pore volumes (as shown in the characterization section) but the varying contribution of large pores is used to control the amount of liquid flowing through the beads. POROS R1 has the largest contribution of throughpores, and therefore the greatest intraparticle flow. POROS R2 has a lower contribution of throughpores, and a higher surface area coming from a greater population of diffusive pores, but still shows significant mass transport enhancements relative to a purely diffusive control. Oligo R3 is dominated by a high population of diffusive pores, and is used comparatively as a non-perfusive resin. Although the pore size distribution can be engineered to control mass transport rates, the resulting surface area is not the only means by which binding capacity can be controlled. Surface coatings are employed to increase binding capacity without fundamentally altering the mass transport properties. Models are used to describe the amount of flow transecting the beads, and comparisons of coated resins to uncoated (polystyrene) resins leads to the conclusion that these coatings do not obstruct the throughpore structures. This is an important conclusion since the binding capacity of the coated product, in some cases, is shown to be over 10-fold higher than the precursor polystyrene scaffold (i.e., POROS R1 or POROS R2).

Quantitative analysis of cell signaling and drug action via mass spectrometry-based systems level phosphoproteomics

Protein phosphorylation is a primary form of information transfer in cell signaling pathways and plays a crucial role in regulating biological responses. Aberrant phosphorylation has been implicated in a number of diseases, and kinases and phosphatases, the cellular enzymes that control dynamic phosphorylation events, present attractive therapeutic targets. However, the innate complexity of signaling networks has presented many challenges to therapeutic target selection and successful drug development. Approaches in phosphoproteomics can contribute functional, systems‐level datasets across signaling networks that can provide insight into suitable drug targets, more broadly profile compound activities, and identify key biomarkers to assess clinical outcomes. Advances in MS‐based phosphoproteomics efforts now provide the ability to quantitate phosphorylation with throughput and sensitivity to sample a significant portion of the phosphoproteome in clinically relevant systems. This review will discuss recent work and examples of application data that demonstrate the utility of MS, with a particular focus on the use of quantitative phosphoproteomics and phosphotyrosine‐directed signaling analyses to provide robust measurement for functional biological interpretation of drug action on signaling and phenotypic outcomes.

Multidimensional chromatography coupled with mass spectrometry for target-based screening

SummaryThe synthesis of structural analogs and the process of drug discovery have evolved dramatically through recent advances in solid-phase synthesis reagents and automated screening systems. As molecular diversity strategies emerge, the need for automated target-based selection of lead candidates becomes equally important. Multidimensional automated chromatographic techniques coupled to electrospray ionization mass spectrometry facilitate the selection process and provide maximum characterization information in a single screening run. The capture of tightly bound affinity leads by target biomolecules, followed by subsequent release and high-resolution separation with sensitive detection, significantly reduces the time required to identify and characterize lead compounds. This automated multidimensional chromatographic approach coupled with mass spectrometry, Selectronics™, was used with several organic and natural libraries to demonstrate an automated target-based screening technique to select for high-affinity binders as potential lead compounds.

Automated proteolytic mapping of proteins

Abstract A new, rapid, automated method for peptide mapping has been developed that requires less than two hours to complete. This method first (i) reduces the protein with dithiothreitol (DTT) or s-mercaptoethanol at 50°C, (ii) then alkylates it with an alkylating agent selected from iodoacetamide, iodoacetic acid, or vinylpyridine, (iii) digests the protein completely with immobilized, TPCK treated trypsin, and (iv) finally analyzes the tryptic fragments by high resolution, reversed-phase liquid chromatography (RPLC) in less than two hours. Reduction and alkylation are achieved in the autosampler of the instrument where the sample, reagents, and reaction protocol are specified by the operator in the system computer. Proteins with up to seven disulfide bridges were quantitatively reduced and alkylated by the system. Immobilized enzyme columns coupled in tandem with an RPLC column were shown to generate protein digests and reproducibly separate the fragments for many cycles of analysis. Based on the fact that any one of several alkylating agents could be used in the mapping process, it was demonstrated that a campaign of experiments could be executed automatically in a search for the optimum alkylating agent. The mapping technique was applied to five different proteins.

Accelerated recombinant protein purification process development: Automated, robotics-based integration of chromatographic purification and analysis

Recovery of recombinant proteins from endogenous, host molecules can be an experimentally intensive and time-consuming task. Often the time to analyze material during development of recovery procedures is the rate-limiting step. Nowadays, modern techniques and equipment are being specifically engineered to make this effort much more efficient. We present a case study to illustrate how a new, automation tool, designed for easy, systematic methods development, can be used for very rapid process and analytical optimization. This tool uses robotics to integrate process development with rapid LC-based analysis requiring no user intervention. The methods and procedures described can be generalized to any recombinant protein recovery campaign.

Semi-targeted plasma proteomics discovery workflow utilizing two-stage protein depletion and off-line LC-MALDI MS/MS.

A quantitative proteomics workflow was implemented that provides extended plasma protein coverage by extensive protein depletion in combination with the sensitivity and breadth of analysis of two-dimensional LC-MS/MS shotgun analysis. Abundant proteins were depleted by a two-stage process using IgY and Supermix depletion columns in series. Samples are then extensively fractionated by two-dimensional chromatography with fractions directly deposited onto MALDI plates. Decoupling sample fractionation from mass spectrometry facilitates a targeted MS/MS precursor selection strategy that maximizes measurement of a consistent set of peptides across experiments. Multiplexed stable isotope labeling provides quantification relative to a common reference sample and ensures an identical set of peptides measured in the set of samples (set of eight) combined in a single experiment. The more extensive protein depletion provided by the addition of the Supermix column did not compromise overall reproducibility of the measurements or the ability to reliably detect changes in protein levels between samples. The implementation of this workflow is presented for a case study aimed at generating molecular signatures for prediction of first heart attack.

An overview of continuous protein purification processes

As the sphere of influence of recombinant technology moves away from the laboratory bench, towards product commercialization, development of manufacturing and large scale process technology is becoming a major challenge and determinant for commercial success. The challenge is particularly acute for protein purification process development where protein purification costs tend to dominate overall process economics. The primary objective for process scale purification is to minimize cost for a purified product which meets specifications. Continuous processes may be used to facilitate achievement of the overall objectives. This review critically examines the use of continuous processing for protein purification and recovery operations. The processes have been divided into three general areas: adsorptive and chromatographic, electrophoretic, and extractive. Consideration is given to the operational advantages and limitations of the reviewed processes.

Mathematical modelling of the continuous affinity-recycle extraction purification technique

Abstract Continuous affinity-recycle extraction (CARE), a continuous protein purification unit operation, has been designed to address design and optimization criteria relevant for process scale chromatographic separation of proteins. The development and application of a mathematical model describing purification in the CARE process are described. The model incorporates adsorption-desorption kinetics into material balance equations describing the operation of two well-mixed reactors operating with recycle. An accurate mathematical model of CARE has aided in its development as a new unit operation for protein purification, in the assessment of its performance tradeoffs, and in its optimization.

Quantitative measurement of epidermal growth factor receptor-mitogen-activated protein kinase signal transduction using a nine-plex, peptide-based immunoassay

Aberrant epidermal growth factor receptor (EGFR, ErbB1) signaling is implicated in cell transformation, motility, and invasion in a variety of cell types, and EGFR is the target of several anticancer drugs. However, the kinetics of EGFR signaling and the individual contributions of site-specific phosphorylation events remain largely unknown. A peptide-based, multiplex immunoassay approach was developed to simultaneously measure both total and phosphorylated protein in a single sample. The approach involves the proteolytic digestion of proteins prior to the isolation and quantitation of site-specific phosphorylation events within an individual protein. Quantitation of phosphorylated and total proteins, in picomolar to nanomolar concentrations, were interpolated from standard curves generated with synthetic peptides that correspond to the peptide targets used in the immunoassays. In this study, a bead-based, nine-plex immunoassay measuring total and phosphorylated protein was constructed to measure temporal, site-specific phosphorylation of key members of the EGFR pathway (ErbB1 receptor, MEK1, MEK2, ERK1, and ERK2) in A431 cells stimulated with epidermal growth factor. The effect of MEK inhibition on this pathway was determined using a known MEK kinase inhibitor, SL327. The results reported herein are the first quantitative measurements of site-specific phosphorylation events and total proteins in a single sample, at the same time representing a new paradigm for standardized protein and phosphorylation analysis using multiplexed, peptide-based, sandwich immunoassays.