In planta changes in protein phosphorylation induced by the plant hormone abscisic acid.

Abscisic acid (ABA) is a hormone that controls seed dormancy and germination as well as the overall plant response to important environmental stresses such as drought. Recent studies have demonstrated that the ABA-bound receptor binds to and inhibits a class of protein phosphatases. To identify more broadly the phosphoproteins affected by this hormone in vivo, we used (14)N/(15)N metabolic labeling to perform a quantitative untargeted mass spectrometric analysis of the Arabidopsis thaliana phosphoproteome following ABA treatment.

Protein quantitation using isotope-assisted mass spectrometry.

Genetic, chemical, and environmental perturbations can all induce large changes in cellular proteomes, and research aimed at quantifying these changes are an important part of modern biology. Although improvements in the hardware and software of mass spectrometers have produced increased throughput and accuracy of such measurements, new uses of heavy isotope internal standards that assist in this process have emerged. Surprisingly, even complex life forms such as mammals can be grown to near-complete replacement with heavy isotopes of common biological elements such as (15)N, and these isotopically labeled organisms provide excellent controls for isolating and identifying experimental variables such as extraction or fractionation efficiencies.

Quantitative improvements in peptide recovery at elevated chromatographic temperatures from microcapillary liquid chromatography-mass spectrometry analyses of brain using selected reaction monitoring.

Elevated chromatographic temperatures are well recognized to provide beneficial analytical effects. Previously, we demonstrated that elevated chromatographic temperature enhances the identification of hydrophobic peptides from enriched membrane samples. Here, we quantitatively assess and compare the recovery of peptide analytes from both simple and complex tryptic peptide matrices using selected reaction monitoring (SRM) mass spectrometry.

Molecular characterization of mutant Arabidopsis plants with reduced plasma membrane proton pump activity.

Arabidopsis mutants containing gene disruptions in AHA1 and AHA2, the two most highly expressed isoforms of the Arabidopsis plasma membrane H(+)-ATPase family, have been isolated and characterized. Plants containing homozygous loss-of-function mutations in either gene grew normally under laboratory conditions. Transcriptome and mass spectrometric measurements demonstrate that lack of lethality in the single gene mutations is not associated with compensation by increases in RNA or protein levels.

Quantitative strategies to fuel the merger of discovery and hypothesis-driven shotgun proteomics.

The ultimate goal of most shotgun proteomic pipelines is the discovery of novel biomarkers to direct the development of quantitative diagnostics for the detection and treatment of disease. Differential comparisons of biological samples identify candidate peptides that can serve as proxys of candidate proteins. While these discovery approaches are robust and fairly comprehensive, they have relatively low throughput.

MudPIT analysis: application to human heart tissue.

Although two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used as the standard proteomic approach for separating proteins in a complex mixture, this technique has many drawbacks. These include a limited molecular mass range, poor separation of highly acidic or basic proteins, and exclusion of the majority of membrane proteins from analysis. Considering the important functions of many membrane proteins, such as receptors, ion transporters, signal transducers, and cell adhesion proteins, it is increasingly important that these proteins are not excluded during the global proteomic analysis of cellular systems.

High quality catalog of proteotypic peptides from human heart.

Proteomics research is beginning to expand beyond the more traditional shotgun analysis of protein mixtures to include targeted analyses of specific proteins using mass spectrometry. Integral to the development of a robust assay based on targeted mass spectrometry is prior knowledge of which peptides provide an accurate and sensitive proxy of the originating gene product (i.e.