Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS.

The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34-80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics.

Intracellular adaptation of Brucella abortus.

Macrophages were infected with virulent Brucella abortus strain 2308 or attenuated strain 19. Intracellular bacteria were recovered at different times after infection and their proteomes compared. The virulent strain initially reduced most biosynthesis and altered its respiration; adaptations reversed later in infection. The attenuated strain was unable to match the magnitude of the virulent strain's adjustments. The results provide insight into mechanisms utilized by Brucella to establish intracellular infections.

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.

Protein depletion from blood plasma using a volatile buffer.

Removal of high abundance proteins is widely used in sample processing for proteomics studies of blood plasma. Immunoaffinity (IA) depletion is currently the most specific method for performing this step. Historically, IA depletion matrices have been designed to be used with inorganic buffers. However, the presence of salts in depleted samples presents a particular problem, and these must be removed in order to make samples compatible with post-depletion processing. Desalting (dialysis, ultrafiltration, size-exclusion, etc.) usually diminishes sample integrity due to labware associated losses. Moreover, these steps require additional labor, increasing the processing time and cost of analysis. In order to avoid these problems, we have developed an IA method using a volatile buffer that can be removed from depleted samples by lyophilization. This method allows the execution of reproducible and efficient depletion of blood plasma in a semi-automated manner.