Improving Proteome Coverage by Reducing Sample Complexity via Chromatography.

High performance liquid chromatography (HPLC) is currently one of the most powerful analytical tools that has revolutionized the field of proteomics. Formerly known as high pressure liquid chromatography, this technique was introduced in the early 1960s to improve the efficiency of liquid chromatography separations using small stationary phase particles packed in columns. Since its introduction, continued advancements in column technology, development of different stationary phase materials and improved instrumentation has allowed the full potential of this technique to be realized.

Protein biomarkers on tissue as imaged via MALDI mass spectrometry: A systematic approach to study the limits of detection.

MALDI mass spectrometry imaging (MSI) is emerging as a tool for protein and peptide imaging across tissue sections. Despite extensive study, there does not yet exist a baseline study evaluating the potential capabilities for this technique to detect diverse proteins in tissue sections. In this study, we developed a systematic approach for characterizing MALDI-MSI workflows in terms of limits of detection, coefficients of variation, spatial resolution, and the identification of endogenous tissue proteins.

An autophosphorylation site database for leucine-rich repeat receptor-like kinases in Arabidopsis thaliana.

Leucine-rich repeat receptor-like kinases (LRR RLKs) form a large family of plant signaling proteins consisting of an extracellular domain connected by a single-pass transmembrane sequence to a cytoplasmic kinase domain. Autophosphorylation on specific Ser and/or Thr residues in the cytoplasmic domain is often critical for the activation of several LRR RLK family members with proven functional roles in plant growth regulation, morphogenesis, disease resistance, and stress responses. While identification and functional characterization of in vivo phosphorylation sites is ultimately required for a full understanding of LRR RLK biology and function, bacterial expression of recombinant LRR RLK cytoplasmic catalytic domains for identification of in vitro autophosphorylation sites provides a useful resource for further targeted identification and functional analysis of in vivo sites.

Advances in qualitative and quantitative plant membrane proteomics.

The membrane proteome consists of integral and membrane-associated proteins that are involved in various physiological and biochemical functions critical for cellular function. It is also dynamic in nature, where many proteins are only expressed during certain developmental stages or in response to environmental stress. These proteins can undergo post-translational modifications in response to these different conditions, allowing them to transiently associate with the membrane or other membrane proteins.

Isotope-labeling and affinity enrichment of phosphopeptides for proteomic analysis using liquid chromatography-tandem mass spectrometry.

The reversible phosphorylation of proteins is a dynamic process that plays a major role in many vital physiological processes by transmitting signals within cellular pathways and networks. Proteomic measurements using mass spectrometry are capable of characterizing the sites of protein phosphorylation and to quantify their change in abundance. However, the low stoichiometry of protein phosphorylation events often preclude mass spectrometry detection and require additional sample preparation steps to facilitate their characterization.

Tyrosine phosphorylation of the BRI1 receptor kinase emerges as a component of brassinosteroid signaling in Arabidopsis.

Brassinosteroids (BRs) are essential growth-promoting hormones that regulate many aspects of plant growth and development. Two leucine-rich repeat receptor-like kinases (LRR-RLKs) are involved in BR perception and signal transduction: brassinosteroid insensitive 1 (BRI1), which is the BR receptor, and its coreceptor BRI1-associated kinase 1 (BAK1). Both proteins are classified as serine/threonine protein kinases, but here we report that recombinant cytoplasmic domains of BRI1 and BAK1 also autophosphorylate on tyrosine residues and thus are dual-specificity kinases.

Sequential transphosphorylation of the BRI1/BAK1 receptor kinase complex impacts early events in brassinosteroid signaling.

Brassinosteroids (BRs) regulate plant development through a signal transduction pathway involving the BRI1 and BAK1 transmembrane receptor kinases. The detailed molecular mechanisms of phosphorylation, kinase activation, and oligomerization of the BRI1/BAK1 complex in response to BRs are uncertain. We demonstrate that BR-dependent activation of BRI1 precedes association with BAK1 in planta, and that BRI1 positively regulates BAK1 phosphorylation levels in vivo.

Characterization of a new family of protein kinases from Arabidopsis containing phosphoinositide 3/4-kinase and ubiquitin-like domains.

At least two of the genes predicted to encode type II PI4K (phosphoinositide 4-kinase) in Arabidopsis thaliana (thale cress), namely AtPI4Kgamma4 and AtPI4Kgamma7, encode enzymes with catalytic properties similar to those of members of the PIKK (phosphoinositide kinase-related kinase) family. AtPI4Kgamma4 and AtPI4Kgamma7 undergo autophosphorylation and phosphorylate serine/threonine residues of protein substrates, but have no detectable lipid kinase activity. AtPI4Kgamma4 and AtPI4Kgamma7 are members of a subset of five putative AtPI4Ks that contain N-terminal UBL (ubiquitin-like) domains.