Fred E. Regnier

Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low μg/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.

Retention model for high-performance ion-exchange chromatography☆

Abstract Protein retention on an ionic surface is the result of protein charge, surface charge and the charge characteristics of the surrounding medium. To date, a “net charge” model has been used to explain this phenomenon; however, retention mapping studies on strong ion-exchange columns showed it to be inadequate. Deviations may result from charge asymmetry, since it appears that only a fraction of the protein surface interacts with the stationary phase. Retention is also altered by the type of displacing salt. A non-mechanistic model has been developed which shows a positive correlation between protein retention and the number of charges associated with the adsorption-desorption process. Integration of these observations and concepts provides a deeper understanding of protein retention on ion-exchange surfaces.

Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography−Tandem Mass Spectrometry

The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies.

Ultramicro enzyme assays in a capillary electrophoretic system.

This paper describes an ultramicro method for achieving enzyme assays. Enzyme saturating concentrations of substrate, coenzyme when appropriate, and running buffer were mixed and used to fill a deactivated fused-silica capillary in a capillary zone electrophoresis apparatus. The enzyme glucose-6-phosphate dehydrogenase was injected by either electrophoresis or siphoning and mixed with the reagents in the capillary by electrophoretic mixing. Enzyme activity was assayed by electrophoresing the product, reduced nicotinamide adenine dinucleotide phosphate, to the detector where it was detected at 340 nm. Under constant potential, the transport velocity of enzyme and the product was generally different. This caused product to be separated from the enzyme after it was formed. Because product formation was much faster than the rate of enzyme-product separation, product accumulated. The amount of accumulated product was inversely related to operating potential. In the extreme case, the operating potential was zero. Zero potential assays were generally carried out by electrophoresing the enzyme partially through the capillary and then switching to zero potential. This capillary was left at zero potential for several minutes to allow additional product to accumulate. After this additional amplification step, potential was again applied and the product transported to the detector. Product formed under constant potential appears as a broad peak with a flat plateau. When the voltage is switched to zero at intermediate migration distance, a peak will be observed on top of this plateau. Either the eight of the plateau or the area of the peak may be used to determine enzyme concentration. The lower limit of detection was 4.6.10(-17) mol of glucose-6-phosphate dehydrogenase.

Polyethyleneimine-bonded phases in the separation of proteins by capillary electrophoresis

A hydrophilic, positively charged, durable coating has been developed for capillary electrophoresis of macromolecules. Polyethyleneimine is adsorbed to the inner wall of fused silica capillaries and the adsorbed coating cross-linked into a stable layer. Capillaries of polyethyleneimine-coated silica gave unique separations owing to the reversal of electro-osmotic flow caused by the positively charged coating. The resulting coating was stable from pH 2-12 and could be used over a wide pH range without substantial change in electro-osmotic flow. High-molecular-weight polymers were needed to give thick coatings which mask silanol groups on the wall. Proteins were resolved quickly and efficiently with good recovery using capillaries of 50 cm in length.

Retention model for proteins in reversed-phase liquid chromatography.

This paper presents a retention model for proteins on an reversed-phase chromatography support in which retention is a function of the number (Z) of solvent molecules required to displace the solute from the surface. An equation is derived that relates the capacity factor of a protein to the displacing agent concentration and the stoichiometry of solvent-solute displacement. Experimental tests of the model indicate that each protein has a unique Z value and that Z is directly proportional to the molecular weight of a series of proteins when 60% formic acid is used as the mobile phase additive. This relationship is attributed to a direct relationship between Z and the contact surface area between polypeptide solutes and the support. Desorption curves for proteins also become more convex with increasingly molecular weight, as predicted by the retention model. In the solvent series of methanol, ethanol, propanol, the Z number decreases from the C1 to C3 alcohol. The Z number for any particular solvent is also related to other mobile phase additives, such as acids, and the concentration of additives.

Perfusion chromatography packing materials for proteins and peptides

Abstract This paper describes the performance and properties of column packings for very high-speed, high-capacity and high-resolution separations of proteins and peptides by perfusion chromatography. Perfusion of mobile phase through the supports is achieved by using through-pores of 6000–8000 A. In addition, these large through-pores are combined with smaller (800–1500 A) pores which provide high-binding surface area. Media produced from poly(styrene-divinylbenzene) having such a pore structure plus a thin, crosslinked polymer coating allow perfusion chromatography to be performed in the reversed-phase, ion exchange, hydrophobic interaction, immobilized metal affinity and bioaffinity modes.

Sweetening the Pot: Adding Glycosylation to the Biomarker Discovery Equation

BACKGROUND Cancer has profound effects on gene expression, including a cells glycosylation machinery. Thus, tumors produce glycoproteins that carry oligosaccharides with structures that are markedly different from the same protein produced by a normal cell. A single protein can have many glycosylation sites that greatly amplify the signals they generate compared with their protein backbones. CONTENT In this article, we survey clinical tests that target carbohydrate modifications for diagnosing and treating cancer. We present the biological relevance of glycosylation to disease progression by highlighting the role these structures play in adhesion, signaling, and metastasis and then address current methodological approaches to biomarker discovery that capitalize on selectively capturing tumor-associated glycoforms to enrich and identify disease-related candidate analytes. Finally, we discuss emerging technologies--multiple reaction monitoring and lectin-antibody arrays--as potential tools for biomarker validation studies in pursuit of clinically useful tests. SUMMARY The future of carbohydrate-based biomarker studies has arrived. At all stages, from discovery through verification and deployment into clinics, glycosylation should be considered a primary readout or a way of increasing the sensitivity and specificity of protein-based analyses.

Preparation of a porous microparticulatee anion-exchange chromatography support for proteins

Abstract A hydrophilic, durable anion-exchange material has been developed for high- performance liquid chromatography of proteins. Polyethyleneimine and simpler amines are adsorbed to porous, microparticulate silicas so strongly that the adsorbed coatings may be crosslinked into a stable layer by a wide variety of reagents in organic solution. Epoxy resins, alkyl bromides, and nitro alcohols are the best cross- linkers. The resulting pellicular coating is more stable in aqueous media than the underlying silica. Porous glass, alumina, and titania can also be coated in this manner. The materials are quite reproducible and of high ion-exchange capacity: up to 2.7 mequiv./g. Columns of polyethyleneimine-coated silica resolve proteins quickly and efficiently with excellent recoveries of enzyme activity. Nucleotides are also well resolved. Efficiency does not change during a columns lifetime.

Strategy for qualitative and quantitative analysis in proteomics based on signature peptides

This paper describes a new analytical strategy for identifying proteins in concentration flux based on isotopic labeling peptides in tryptic digests. Primary amino groups in peptides from control and experimental samples were derivatized with acetate and trideuteroacetate, respectively. After mixing samples thus labeled from these two sources, the relative concentration of peptides was determined by isotope ratio analysis with MALDI and ESI mass spectrometry. More than a 100-fold difference in relative concentration could be detected. Simplification of complex tryptic digests prior to mass spectral analysis was achieved by selection of histidine-containing peptides with immobilized metal affinity sorbents or of glycopeptides by lectin columns. Because most of these peptides have sequences that are unique to a single protein, they are a signature of the protein from which they were derived; providing a facile route to protein analysis.