Paolo Lecchi

Size-exclusion chromatography in multidimensional separation schemes for proteome analysis.

Size-exclusion chromatography (SEC) is a separation technique with a relatively low resolving power, compared to those usually utilized in proteomics. Therefore, it is often overlooked in experimental protocols, when the main goal is resolving complex biological mixtures. In this report, we introduce innovative multidimensional schemes for proteomics analysis, in which SEC plays a practical role. Liquid isoelectric focusing (IEF) was combined with SEC, and experimental results were compared to those obtained by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), well-established techniques relying upon similar criteria for separation. Additional experiments were performed to evaluate the practical contribution of SEC in multidimensional chromatographic separations. Specifically, we evaluated the combination of SEC and ion exchange chromatography in an analytical scheme for the mass spectrometric analysis of protein-extracts obtained from bacterial cultures grown in stable isotope enriched media. Experimental conditions and practical considerations are discussed.

Application of high-performance liquid chromatography with isotope-ratio mass spectrometry for measuring low levels of enrichment of underivatized materials

Combining HPLC separations with an isotope-ratio mass spectrometric (IRMS) detection produces a device capable of measuring very low alterations in 13C abundance from analyte species that cannot be volatilized. Examples are presented showing proteins, carbohydrates, and nucleotides that are eluted from varying types of HPLC columns (reversed-phase, normal-phase, ion-exchange and size-exclusion). This wide range of chromatographic methods enables the analysis of compounds never before amenable to IRMS techniques and may lead to the development of many new assays.

In vitro performance of hemodialysis membranes after repeated processing

BACKGROUND Dialyzers reprocessed with chlorine-based solutions have been associated with increases in ultrafiltration coefficient and middle-molecule removal. Increased pore size has been hypothesized as the mechanism for the latter phenomenon. Dialyzers exposed to Amukin-D (Amuchina Int Inc, Gaithersburg, MD), a chlorine-based reprocessing agent, were evaluated for changes in molecular weight (MW) cutoff and ultrafiltration properties. METHODS In vitro MW cutoff studies were performed on Fresenius F-80A (Fresenius, Lexington, MA) and Gambro Polyflux 17 (Gambro, Lakewood, CO) hemodialyzers that were reprocessed 20 times using Amukin-D. Permeability (Uf-A), defined as the area from the ultrafiltered compartment (Uf) compared with the area from the equivalent arterial compartment (A), for dextran across the hemodialyzer membrane was determined after the initial use and after reuses 1, 5, 10, 15, and 20 by using size-exclusion chromatography. RESULTS Uf-A for dextran increased approximately 10-fold between hemodialyzer reuses 1 and 5. Thereafter, additional reprocessing did not increase the Uf-A ratio further. MW cutoff increased during these 5 washes and did not change thereafter. CONCLUSION Reprocessing with Amukin-D increased the MW cutoff and permeability of both hemodialyzers between reuses 1 and 5, resulting in a greater ultrafiltration rate and greater middle-molecule removal. After reuse 5, there were no further increases in MW cutoff with additional reprocessing in either hemodialyzer. This suggests that reprocessing and storage of each hemodialyzer with Amukin-D affects the permeability of dextran in a nonlinear fashion and to a finite level, such that subsequent reprocessing has no further effect on the MW cutoff of the membrane.

An innovative method for measuring hydrogen and deuterium: Chemical reaction interface mass spectrometry with nitrogen reactant gas

A new method for measuring deuterium isotopic enrichment with CRIMS (chemical reaction interface mass spectrometry) is described. Using nitrogen as the reactant gas in a chemical reaction interface generates molecular hydrogen that provides the H2 and HD from which the deuterium content can be analyzed with a benchtop quadrupole mass spectrometer. Samples of deuterated leucine in unlabeled leucine were used as the primary test species. Detection of deuterium enrichment was accurate, precise, and linear. We used this scheme to evaluate the results of a process to acetylate lysine residues in a peptide—neurotensin. With separation on a C18 column, we found a 61% yield of the desired monoethylated product that had a D/H ratio very close to the theoretical one. Isotope ratio monitoring for deuterated species will be important in metabolism studies where CRIMS generates a comprehensive and quantitative view of products of deuterated precursors. Where concerns about metabolic isotope effects of deuterium are absent, the use of deuterium will enable these studies to be performed with simpler syntheses and at less cost than if using 13C or 15N.