Comprehensive on-line RPLC-CZE-MS of peptides

Peptide standards and tryptic digests of ribonuclease B are separated by comprehensive two-dimensional reversed phase liquid chromatography (RPLC) and capillary zone electrophoresis (CZE) and detected on-line by electrospray mass spectrometry. The RPLC column is coupled to the CZE column by a transverse flow gating interface. A new rugged microelectrospray needle is described that combines high ionization efficiency, low flow rates, and a sheath flow. The result is a system combining the separation capabilities of both RPLC and CZE with on-line mass spectrometric detection, all in about 15 min.     

Energy spectra for decaying 2D turbulence in a bounded domain

We use results derived in the framework of the replica approach to study the liquid-glass thermodynamic transition. The main results are derived without using replicas and applied to the study of the Lennard-Jones binary mixture introduced by Kob and Andersen. We find that there is a phase transition due to the entropy crisis. We compute both analytically and numerically the value of the phase transition point T(K) and the specific heat in the low temperature phase.     

Peptide shifter: Enhancing separation reproducibility using correlated expression profiles

Chromatographic protein and peptide separation technologies enable comprehensive proteomic analysis of plasma and other complex biological samples by mass spectrometry. However, as the number of separations and/or fractions increases, so does the number of peptides split across fraction boundaries. Irreproducibility of peptide chromatographic separation results in peptides on or near the boundary moving partially or entirely into adjacent fractions. Peptide shifting across fraction boundaries increases the variability of measured peptide abundance, and so there is a trade-off between proteomic comprehensiveness using separation technologies and accurate quantitative proteomic measurements. In this paper, a method for detecting and correcting split peptides, called Peptide Shifter, is introduced and evaluated. An essential component of Peptide Shifter is a global peptide expression profile analysis that allows the inference of the underlying peptide shift pattern without the use of peptide labeling or internal standards. A controlled proteomic analysis of plasma samples demonstrates a 34% decrease in peptide intensity variability after the application of Peptide Shifter.     

Guidelines for the Routine Application of the Peptide Hits Technique

A set of guidelines has been developed for using the peptide hits technique (PHT) as a semi-quantitative screening tool for the identification of proteins that change in abundance in a complex mixture. The dataset that formed the basis for these experiments was created using a cell lysate derived from the yeast Saccharomyces cerevisiae, spiked at various levels with serum albumin (BSA), and analyzed by LC/MS/MS and SEQUEST. Knowing that the level of only one protein (BSA) actually changed in the mixture allowed for the development and refinement of the necessary bioinformatics and statistical analyses, e.g., principal component analysis (PCA), normalization, and analysis of variation (ANOVA). As expected, the number of BSA peptide hits changed in proportion to the amount of BSA added to the sample. PCA was able to clearly distinguish between the spiked samples and the untreated sample, indicating that PCA may be able to classify samples, e.g., healthy versus diseased, in future experiments. The use of an endogenous "housekeeping" protein was found to be superior to the use of total hits for data normalization prior to analysis. An ANOVA based model readily identified BSA as a protein of interest, that is, one likely to be changing from amongst the background proteins, indicating that an ANOVA model may be able to identify individual proteins in target or biomarker discovery experiments. General guidelines based on these combined observations are set forth for future analyses and the rapid screening for candidate proteins of interest.     

Electrochemical processes in electrospray ionization mass spectrometry

Editorial Comment Last month we presented, as a Special Feature, a set of five articles that constituted a Commentary on the fundamentals and mechanism of electrospray ionization (ESI). These articles produced some lively discussion among the authors on the role of electrochemistry in ESI. Six authors participated in a detailed exchange of views on this topic, the final results of which constitute this month's Special Feature. We particularly hope that younger scientists will find value in this month's Special Feature, not only for the science that it teaches but also what it reveals about the processes by which scientific conclusions are drawn. To a degree, the contributions part the curtains on these processes and show science in action. We sincerely thank the contributors to this discussion. The give and take of intellectual debate is not always easy, and to a remarkable extent this set of authors has maintained good humor and friendships, even when disagreeing strongly on substance. Graham Cooks and Richard Caprioli Copyright 2000 John Wiley & Sons, Ltd.     

Derivatization for electrospray ionization-mass spectrometry. 4. Alkenes and alkynes

A survey of derivatization strategies and prospective derivatization reactions for conversion of simple alkenes and alkynes to 'electrospray-active' species is presented. General synthetic strategies are discussed and illustrative examples of prospective derivatives prepared from model compounds are presented along with their electrospray ionization (ES) mass spectra. The identified derivatives of these neutral, nonpolar analytes are either ionic or are ionizable in solution through Bronsted acid/base chemistry, by Lewis acid/base chemistry, or by chemical or electrochemical electron-transfer chemistry. Once ionized, the derivatives are expected to be amenable to detection by electrospray ionization-mass spectrometry. Derivatives are identified for positive and negative ion analysis of both alkenes and alkynes. Copyright 2000 John Wiley & Sons, Ltd.