We established robust, reliable protocols for ‘Differential Display (DD),’ an RNA fingerprinting method originally developed by Liang and Pardee [(1992) Science 257, 967–971] using RT‐PCR with arbitrary primers. Our are optimized so that DD analysis can be performed on a fluorescent DNA sequencer to ensure high throughput as well improved operational safety, compared the original one radioactive compounds. Such ‘Fluorescent Differential (FDD)’ techniques will accelerate identification of...

Abstract Background An ideal format to describe transcriptome would be its composition measured on the scale of absolute numbers individual mRNAs per cell. It help not only precisely grasp structure but also accelerate data exchange and integration. Results We conceived an idea competitive PCR between genomic DNA cDNA. Since former contains every gene exactly at same copy number, it can serve as normalization standard for latter obtain stoichiometric transcriptome. This then easily converted...

The ultimate overexpression of a protein could cause growth defects, which are known as the burden. However, expression limit at protein-burden effect is triggered still unclear. To estimate this limit, we systematically measured limits glycolytic proteins in Saccharomyces cerevisiae. some were up to 15% total cellular protein. These independent proteins’ catalytic activities, finding that was supported by an silico analysis. Some had low explained their localization and metabolic...

Mass spectrometry has served as a major tool for the discipline of proteomics to catalogue proteins in an unprecedented scale. With chemical and metabolic techniques stable isotope labeling developed over past decade, it is now routinely used method relative quantification provide valuable information on alteration protein abundance proteome-wide More recently, absolute or stoichiometric proteome becoming feasible, particular, with development strategies isotope-labeled standards composed...

Quantitative description of protein interactions is crucial to understand and model molecular systems regulating various cellular activities. Here, we developed a novel peptide-concatenated standard (PCS) strategy for accurate mass spectrometric quantification component stoichiometry multiprotein complexes. In this strategy, tryptic peptides suitable are selected with their natural flanking sequences from each complex concatenated into single synthetic called PCS. The concatenation...

Pattern recognition receptors on the plant cell surface mediate of microbe/damage-associated molecular patterns (MAMPs/DAMPs) and activate downstream immune signaling. Autophosphorylation signaling receptor-like kinases is a critical event for activation responses but function each phosphorylation site in regulation not well understood. In this study, 41 Ser/Thr/Tyr 15 Ser/Thr residues were identified as vitro vivo autophosphorylation sites Arabidopsis CERK1, which essential chitin...

The calcitic brachipod shells contain proteins that play pivotal roles in shell formation and are important understanding the evolution of biomineralization. Here, we performed a large-scale exploration matrix brachiopod Laqueus rubellus.A total 40 from were identified. Apart five proteins, i.e., ICP-1, MSP130, cysteine protease, superoxide dismutase, actin, all other identified had no homologues public databases. Among these unknown one protein was with domain architecture includes NAD(P)...

Over the past decade, many skeletal matrix proteins that are possibly related to calcification have been reported in various calcifying animals. Molluscs among most diverse animals and some gastropods adapted terrestrial ecological niches. Although shell (SMPs) already molluscs, reports focused on marine SMPs of snails remain unclear. In addition, stylommatophoran evolved an additional unique calcified character, called a "love dart," used for mating behavior. We identified 54 snail Euhadra...

Overproduction (op) of proteins triggers cellular defects. One the consequences overproduction is protein burden/cost, which produced by an overloading synthesis process. However, physiology cells under a burden not well characterized. We performed genetic profiling systematic analysis interactions between GFP-op, surveying both deletion and temperature-sensitive mutants in budding yeast. also with triple-GFP (tGFP), nuclear export signal-containing tGFP (NES-tGFP). The specifically...

Abstract Molluscan shell matrix proteins (SMPs) are essential in biomineralization. Here, we identify potentially important SMPs by exploiting the asymmetric growth snail, Lymnaea stagnalis . Asymmetric shells require bilaterally expression of SMP genes. We examined levels 35,951 transcripts expressed left and right sides mantle tissue pond This transcriptome dataset was used to 207 LC-MS/MS. 32 genes show patterns, which were further verified for 4 using quantitative PCR analysis. Among...

Ubiquitination plays various critical roles in eukaryotic cellular regulation and is mediated by a cascade of enzymes including ubiquitin protein ligase (E3). The Skp1-Cullin-F-box complex comprises the largest E3 family, each member which unique F-box binds its targets to define substrate specificity. Although genome sequencing uncovers growing number proteins, most them have remained as "orphans" because difficulties identification their substrates. To address this issue, we tested...

Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared proteome expression profiles four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, lactis) grown on glucose- or glycerol-containing media. Conserved changes all were observed only small proportion proteins differentially expressed between two growth conditions. Two species, both which exhibited high...

The accurate and precise absolute abundance of proteins can be determined using mass spectrometry by spiking the sample with stable isotope-labeled standards. In this study, we developed a strategy hierarchical use peptide-concatenated standards (PCSs) to quantify more over wider dynamic range. Multiple primary PCSs were used for quantification many target proteins. Unique "ID-tag peptides" introduced into individual PCSs, allowing us monitor exact amounts "secondary PCS" in which all...

Amino acid‐starved yeast activates the eIF2α kinase Gcn2p to suppress general translation and selectively derepress transcription factor Gcn4p, which induces various biosynthetic genes elicit amino acid control (GAAC). Well‐fed target of rapamycin (TOR) stimulate via eIF4F complex. A crosstalk was demonstrated between pathways for GAAC TOR signaling: TOR‐specific inhibitor Gcn2p. Here we demonstrate that, upon TOR‐inactivation, putative TOR‐regulated eIF4E‐associated protein Eap1p likely...

In budding yeast, a mother cell can produce finite number of daughter cells over its life. The accumulation variety types damaged components has an impact on the aging process. Asymmetrical inheritance during division causes these aberrant intracellular constituents to be retained in and prevents them from segregating cells. However, understanding asymmetrical individual proteins that are or old age, their relevance process, been limited. aim this study is propose proteomics strategy for...

Despite being a member of the shelled mollusks (Conchiferans), most members extant cephalopods have lost their external biomineralized shells, except for basally diverging Nautilids. Here, we report result our study to identify major Shell Matrix Proteins and domains in Nautilid Nautilus pompilius, order gain general insight into evolution Conchiferan Proteins. In do so, performed multiomics on shell N. by conducting transcriptomics its mantle tissue proteomics matrix. Analyses obtained data...

Abstract To discriminate between stable and dynamic protein–protein interactions, we propose a strategy in which cells with without tagged bait are differentially labeled isotope combined prior to complex purification. Mass‐spectrometric analysis of the purified complexes identifies components as those derived exclusively from both cells, respectively. We successfully applied this analyze two yeast protein complexes, eIF2B–eIF2 cyclin–Cdc28.

We developed a parallel affinity purification (PAP) procedure, in which ubiquitinated proteins are purified from the cells that coexpress two affinity-tagged ubiquitins by sequential use of chromatography specific to each tag. In contrast with previous procedures using single ubiquitin, PAP eliminates highly abundant ubiquitin monomers and monoubiquitinated selectively enrich bearing both affinity-tags, or poly- multiubiquitinated proteins. Accordingly, it would serve as powerful method...

Abstract Monocyte chemoattractant protein-1 (MCP-1) induces monocyte chemotaxisvia interaction with the MCP-1 receptor CCR2. We found that MCP-1binding to monocytic THP-1 cells was increased by pre-treatment withMCP-1. The amount of CCR2 mRNA and cell-surface expression CCR2were not affected stimuli. In contrast, MCP-1-treatedTHP-1 showed a sixfold increase in binding affinitycompared untreated cells. CCR2B-transfectedHEK-293 also enhanced MCP-1, affinity sixfold. both cell lines,...

Ubiquitination regulates not only the stability but localization and activity of substrate proteins involved in a plethora cellular processes. The Skp1-Cullin-F-box protein (SCF) complexes constitute major family ubiquitin ligases, each member which an F-box serves as variable component responsible for recognition, thereby defining function complex. Here we studied whether composition SCF is remodeled under different conditions. We exploited stable isotope labeling MS relative quantification...