Cancer proteomics: many technologies,one goal

A major goal of the National Cancer Institute is to alleviate patient pain, suffering and death associated with cancer by the year 2015. This goal does not insinuate a cure for cancer, but rather the development of diagnostics and therapeutics that will eventually decrease cancer morbidity and mortality. A part of meeting this goal is to leverage the enormous data-gathering capabilities of proteomic technologies to discover disease-specific biomarkers in serum, plasma, urine, tissues and other biologic samples. The rapid advance in available technologies that have been spurred by the -omics era, has enabled biologic samples to be surveyed for biomarkers in ways never before possible. However, it is not yet clear which specific technologies will be the most successful. Therefore, proteomic laboratories within the National Cancer Institute are taking a multipronged approach to identify disease-specific biomarkers. This review discusses some of these approaches in their context of meeting the National Cancer Institute’s 2015 goal.     

Superficially Porous Particle Based Hydroxypropyl‐β‐cyclodextrin Stationary Phase for High‐Efficiency Enantiomeric Separations

A superficially porous particle (SPP)‐based hydroxypropyl‐β‐cyclodextrin (HPBCD) chiral stationary phase (CSP) was produced and its chromatographic performance was compared to both 5 µm and 3 µm fully porous particle (FPP)‐based CSPs. The relative surface coverage of the HPBCD chiral selector on each particle was approximately equal, which resulted in equivalent enantiomeric selectivity (α) values on each phase when constant mobile phase conditions were used. Under such conditions, the SPP column resulted in greatly reduced analysis times and three times greater efficiencies compared to the FPP columns. When higher flow rates were used, efficiency gains per analysis times were five times greater for the SPP column compared to the FPP‐based columns. When the mobile phases were altered to give similar analysis times on each column, resolution values were doubled for the SPP column. Finally, the novel SPP based HPBCD column proved to be stable for 500 injections under high flow rate (4.5 mL/min) and high pressure (400 bar) conditions used for an ultrafast (~45 sec) enantiomeric separation. Chirality 27:788–794, 2015. © 2015 Wiley Periodicals, Inc.     

Surfactant structure effects in protein separations using nonionic microemulsions

In this article, the extraction of cytochrome c utilizing various nonionic surfactant microemulsions has been tested to determine the effect of surfactant structure on protein partitioning. Surfactants tested include a linear alcohol ethoxylate (Neodol 91-2.5), two alkyl phenol ethoxylates (lgepal CO-520, Trycol 6985), and a series of alkyl sorbitan esters that are either ethoxylated (Tweens) or un-ethoxylated (Spans). Initial attempts to extract hemoglobin into Neodol 91-2.5 Winsor II microemulsions (oil-continuous) appeared successful based on heme estimation. Careful analysis showed that the hemoglobin had dissociated prior to extraction and that only the heme was extracted with false positive results. In fact, Neodol 91-2.5 microemulsions were unable to extract a variety of proteins with differing biophysical properties. Among all the other nonionic surfactant microemulsions tested only those made using sorbitan esters extracted significant amounts of cytochrome c. The partition coefficients achieved in this study are more than an order of magnitude higher than that seen previously in the literature for comparable sorbitan systems. However, this partition coefficient is extremely sensitive to ionic strength. At an ionic strength as low as 0.001 M, the partition coefficient is reduced to that seen in previous studies. We have found that protein partitioning in sorbitan ester microemulsions is not a function of water content. In addition, extraction is not a function of either alkyl chain length, or polyethylene oxide molecular weight. Hence, the sorbitan group appears to have an important role in extraction, possibly through a weak electrostatic protein-surfactant interaction. (c) 1995 John Wiley & Sons, Inc.     

Steric hindrance influence on the enantiorecognition ability of tyrosine-derived chiral stationary phases

Four chiral stationary phases (CSPs) derived from N-(3,5-dinitrobenzoyl)tyrosine have been synthesized. They differ by the substituent nature (methyl, ethyl, isopropyl, tert-butyl) of the aliphatic amide function. The enantiorecognition ability of these CSPs was evaluated with 10 racemates. For the majority of them, the stereoselectivity increases with the steric hindrance of the substituent. The chiral selector enantiomeric separation on the resulting CSPs has evidenced a reversal of elution order only for CS 4 on CSP 4 (tert-butyl substituent), suggesting a change in its conformation.     

Optimizing the sample size and the retention parameters to achieve maximum production rates for enantiomers in chiral chromatography

The optimum experimental conditions (sample size and mobile phase composition) are calculated for maximum production rate of either one of two enantiomers contained in feeds of different compositions (1/1, 1/10, and 10/1). The products are obtained at 99% purity. The calculations use the equilibrium-dispersive model of chromatography and the equilibrium isotherms determined experimentally from the rear, diffuse boundary of overloaded elution profiles. The production rate measured experimentally under the optimum conditions calculated agree with 4% of the calculated values. There is an optimum value for the retention factor which is higher than predicted by a model assuming constant separation factor, because both separation factor and retention decrease with increasing organic solvent concentration in the mobile phase. (c) 1992 John Wiley & Sons, Inc.     

Solvent-impregnated resins as an in situ product recovery tool for phenol recovery from Pseudomonas putida S12TPL fermentations

The sustainable production of fine/bulk chemicals is often hampered by product toxicity and inhibition to the producing micro-organisms. Consequently, the product must be removed from the micro-organisms' environment. To achieve this, so-called solvent-impregnated resins (SIRs) as well as commercial resins have been added to a Pseudomonas putida S12TPL fermentation that produces phenol as a model compound from glucose. The SIRs contained an ionic liquid which extracts phenol effectively. It was observed that the addition of these particles resulted in an increased phenol production of more than a fourfold while the commercial resin (XAD-4) which is widely used in aromatic removal from aqueous phases, only gave a 2.5-fold increase in volumetric production.     

Evaluation of a ristocetin bonded stationary phase for subcritical fluid chromatography of enantiomers

A stationary phase derived from ristocetin was evaluated for chiral separation in subcritical fluid chromatography. Separation of various enantiomers having different structures and pK(a) values were investigated using carbon dioxide and polar modifiers. The influence of modifiers, additives, temperature, and mobile phase flow rate on separations is presented. It is concluded that this stationary phase can be used for SFC despite its structural similarity with protein-derived stationary phases that can only be used in HPLC. The separation mechanisms could not be elucidated or predicted using these initial experiments. The separations of warfarin and, especially, efavirenz demonstrate the potential of this type of stationary phase for rapid SFC chiral separations.     

Additive concentration effects on enantioselective separations in supercritical fluid chromatography

Polar additive concentration effects in supercritical fluid chromatography were studied on chiral stationary phases having either a macrocyclic glycopeptide or a derivatized polysaccharide as the chiral selector. Two basic additives, isopropylamine and triethylamine, were incorporated into the methanol modifier at various concentrations and the effects on retention, selectivity, and resolution were monitored. Many of the analytes failed to elute from the macrocyclic glycopeptide stationary phase in the absence of an additive and the most noticeable effect of increasing additive concentration was a significant decrease in retention. On the derivatized polysaccharide stationary phase the additives had little effect on retention, but they did foster significant improvements in peak shape and resolution.