Mice and cattle with genetic deficiencies in myostatin exhibit dramatic increases in skeletal muscle mass, suggesting that myostatin normally suppresses muscle growth. Whether this increased muscling results from prenatal or postnatal lack of myostatin activity is unknown. Here we show that myostatin circulates in the blood of adult mice in a latent form that can be activated by acid treatment. Systemic overexpression of myostatin in adult mice was found to induce profound muscle and fat loss analogous to that seen in human cachexia syndromes. These data indicate that myostatin acts systemically in adult animals and may be a useful pharmacologic target in clinical settings such as cachexia, where muscle growth is desired.

In this review we examine techniques, software, and statistical analyses used in label-free quantitative proteomics studies for area under the curve and spectral counting approaches. Recent advances in the field are discussed in an order that reflects a logical workflow design. Examples of studies that follow this design are presented to highlight the requirement for statistical assessment and further experiments to validate results from label-free quantitation. Limitations of label-free approaches are considered, label-free approaches are compared with labelling techniques, and forward-looking applications for label-free quantitative data are presented. We conclude that label-free quantitative proteomics is a reliable, versatile, and cost-effective alternative to labelled quantitation.

The majority of disulfide-linked cytosolic proteins are thought to be enzymes that transiently form disulfide bonds while catalyzing oxidation-reduction (redox) processes. Recent evidence indicates that reactive oxygen species can act as signaling molecules by promoting the formation of disulfide bonds within or between select redox-sensitive proteins. However, few studies have attempted to examine global changes in disulfide bond formation following reactive oxygen species exposure. Here we isolate and identify disulfide-bonded proteins (DSBP) in a mammalian neuronal cell line (HT22) exposed to various oxidative insults by sequential nonreducing/reducing two-dimensional SDS-PAGE combined with mass spectrometry. By using this strategy, several known cytosolic DSBP, such as peroxiredoxins, thioredoxin reductase, nucleoside-diphosphate kinase, and ribonucleotide-diphosphate reductase, were identified. Unexpectedly, a large number of previously unknown DSBP were also found, including those involved in molecular chaperoning, translation, glycolysis, cytoskeletal structure, cell growth, and signal transduction. Treatment of cells with a wide range of hydrogen peroxide concentrations either promoted or inhibited disulfide bonding of select DSBP in a concentration-dependent manner. Decreasing the ratio of reduced to oxidized glutathione also promoted select disulfide bond formation within proteins from cytoplasmic extracts. In addition, an epitope-tagged version of the molecular chaperone HSP70 forms mixed disulfides with both ␤4-spectrin and adenomatous polyposis coli protein in the cytosol. Our findings indicate that disulfide bond formation within families of cytoplasmic proteins is dependent on the nature of the oxidative insult and may provide a common mechanism used to control multiple physiological processes.Oxidative stress occurs when the rate of reactive oxygen species (ROS) 1 generation exceeds the detoxification abilities of the cell, and it has been implicated in many degenerative diseases. It is frequently argued that ROS cause relatively nonspecific damage to vital cellular components such as lipids, DNA, and proteins. However, emerging evidence indicates that ROS can cause specific protein modifications that may lead to a change in the activity or function of the oxidized protein (1, 2). Several major forms of oxidative modifications can occur on amino acid residue side chains including carbonylation, nitrosylation, and oxidation of methionine to methionine sulfoxide (3). Protein sulfhydryls can be oxidized to protein disulfides and sulfenic acids as well as more highly oxidized states such as the sulfinic and sulfonic acid forms of protein cysteines (4). Under non-stressed conditions, disulfide bond formation occurs primarily in the oxidizing environment of the endoplasmic reticulum (ER) in eukaryotic cells (5). The sulfhydryl groups in the vast majority of protein cysteine residues (Cys-SH) have a pK a Ͼ8.0 and, in the reducing environment of the cytoplasm, remain protonated at physiological pH. Thus,...

Abstract-Bovine aortic smooth muscle cell (BASMC) cultures undergo mineralization on addition of the organic phosphate donor, ␤-glycerophosphate (␤GP). Mineralization is characterized by apatite deposition on collagen fibrils and the presence of matrix vesicles, as has been described in calcified vascular lesions in vivo as well as in bone and teeth. In the present study, we used this model to investigate the molecular mechanisms driving vascular calcification. We found that BASMCs lost their lineage markers, SM22␣ and smooth muscle ␣-actin, within 10 days of being placed under calcifying conditions. Conversely, the cells gained an osteogenic phenotype as indicated by an increase in expression and DNA-binding activity of the transcription factor, core binding factor ␣1 (Cbfa1). Moreover, genes containing the Cbfa1 binding site, OSE2, including osteopontin, osteocalcin, and alkaline phosphatase were elevated. The relevance of these in vitro findings to vascular calcification in vivo was further studied in matrix GLA protein null (MGP Ϫ/Ϫ ) mice whose arteries spontaneously calcify. We found that arterial calcification was associated with a similar loss in smooth muscle markers and a gain of osteopontin and Cbfa1 expression. These data demonstrate a novel association of vascular calcification with smooth muscle cell phenotypic transition, in which several osteogenic proteins including osteopontin, osteocalcin, and the bone determining factor Cbfa1 are gained. The findings suggest a positive role for SMCs in promoting vascular calcification.

A systematic proteomic analysis of rice (Oryza sativa) leaf, root, and seed tissue using two independent technologies, two-dimensional gel electrophoresis followed by tandem mass spectrometry and multidimensional protein identification technology, allowed the detection and identification of 2,528 unique proteins, which represents the most comprehensive proteome exploration to date. A comparative display of the expression patterns indicated that enzymes involved in central metabolic pathways are present in all tissues, whereas metabolic specialization is reflected in the occurrence of a tissue-specific enzyme complement. For example, tissuespecific and subcellular compartment-specific isoforms of ADPglucose pyrophosphorylase were detected, thus providing proteomic confirmation of the presence of distinct regulatory mechanisms involved in the biosynthesis and breakdown of separate starch pools in different tissues. In addition, several previously characterized allergenic proteins were identified in the seed sample, indicating the potential of proteomic approaches to survey food samples with regard to the occurrence of allergens.

Diatoms are unicellular eucaryotic algae with cell walls containing silica, intricately and ornately structured on the nanometer scale. Overall silica structure is formed by expansion and molding of the membrane-bound silica deposition vesicle. Although molecular details of silica polymerization are being clarified, we have limited insight into molecular components of the silica deposition vesicle, particularly of membrane-associated proteins that may be involved in structure formation. To identify such proteins, we refined existing procedures to isolate an enriched cell wall fraction from the diatom Thalassiosira pseudonana, the first diatom with a sequenced genome. We applied tandem mass spectrometric analysis to this fraction, identifying 31 proteins for further evaluation. mRNA levels for genes encoding these proteins were monitored during synchronized progression through the cell cycle and compared with two previously identified silaffin genes (involved in silica polymerization) having distinct mRNA patterns that served as markers for cell wall formation. Of the 31 proteins identified, 10 had mRNA patterns that correlated with the silaffins, 13 had patterns that did not, and seven had patterns that correlated but also showed additional features. The possible involvements of these proteins in cell wall synthesis are discussed. In particular, glutamate acetyltransferase was identified, prompting an analysis of mRNA patterns for other genes in the polyamine biosynthesis pathway and identification of those induced during cell wall synthesis. Application of a specific enzymatic inhibitor for ornithine decarboxylase resulted in dramatic alteration of silica structure, confirming the involvement of polyamines and demonstrating that manipulation of proteins involved in cell wall synthesis can alter structure. To our knowledge, this is the first proteomic analysis of a diatom, and furthermore we identified new candidate genes involved in structure formation and directly demonstrated the involvement of one enzyme (and its gene) in the structure formation process. The silicified cell walls of the unicellular algae known as diatoms are fascinating products of biomineralization processes. Each diatom species constructs a uniquely structured cell wall called a frustule, consisting of valves and girdle bands (1, 2) composed of organic material and silica, which is often ornately and intricately shaped with fine features on the nanometer scale. Silica structures are made in the specialized membrane-bound intracellular compartment known as the silica deposition vesicle, or SDV 1 (3-6). The SDV is a dynamic organelle that is expanded and molded during frustule formation, and these movements are essential to produce structures characteristic of the valve and girdle bands (1, 7-10). Upon completion, the entire silica structure is exocytosed to form an integral part of the cell wall (1). A first step to understand structure formation at the molecular level would be to isolate a purified SDV fraction and analyze its protein comp...

Oryza meridionalis Ng. is a wild relative of Oryza sativa L. found throughout northern Australia where temperatures regularly exceed 35 °C in the monsoon growing season. Heat tolerance in O. meridionalis was established by comparing leaf elongation and photosynthetic rates at 45 °C with plants maintained at 27 °C. By comparison with O. sativa ssp. japonica cv. Amaroo, O. meridionalis was heat tolerant. Elongation rates of the third leaf of O. meridionalis declined by 47% over 24 h at 45 °C compared with a 91% decrease for O. sativa. Net photosynthesis was significantly higher in O. sativa at 27 °C whereas the two species had the same assimilation rates at 45 °C. The leaf proteome and expression levels of individual heat-responsive genes provided insight into the heat response of O. meridionalis. After 24 h of heat exposure, many enzymes involved in the Calvin Cycle were more abundant, while mRNA of their genes generally decreased. Ferredoxin-NADP(H) oxidoreductase, a key enzyme in photosynthetic electron transport had both reduced abundance and gene expression, suggesting light reactions were highly susceptible to heat stress. Rubisco activase was strongly up-regulated after 24 h of heat, with the large isoform having the largest relative increase in protein abundance and a significant increase in gene expression. The protective proteins Cpn60, Hsp90, and Hsp70 all increased in both protein abundance and gene expression. A thiamine biosynthesis protein (THI1), previously shown to act protectively against stress, increased in abundance during heat, even as thiamine levels fell in O. meridionalis.

Multiple testing corrections are a useful tool for restricting the FDR, but can be blunt in the context of low power, as we demonstrate by a series of simple simulations. Unfortunately, in proteomics experiments low power can be common, driven by proteomics-specific issues like small effects due to ratio compression, and few replicates due to reagent high cost, instrument time availability and other issues; in such situations, most multiple testing corrections methods, if used with conventional thresholds, will fail to detect any true positives even when many exist. In this low power, medium scale situation, other methods such as effect size considerations or peptide-level calculations may be a more effective option, even if they do not offer the same theoretical guarantee of a low FDR. Thus, we aim to highlight in this article that proteomics presents some specific challenges to the standard multiple testing corrections methods, which should be employed as a useful tool but not be regarded as a required rubber stamp. Keywords: FDR / Multiple testing corrections / Shot gun proteomics ViewpointMultiple testing corrections come in many "flavors." They are employed to limit the number of false positives occurring by chance when an analysis is repeated many times and thus to reduce the FDR at the analysis level. In proteomics they were initially borrowed from microarray research and other high throughput areas, where they quickly became the norm. The informative review by Diz [1] shows that multiple testing corrections were seldom used in quantitative proteomics until relatively recently, and recommends a sensible multimethod approach. Here, we suggest that one reason for the slower uptake of such methods in proteomics experiments, despite their ease of use and theoretical appeal, is that they remain a useful but blunt tool that is less effective in discovery proteomics than in, for example, the microarray environment. In this paper, we describe five key factors that combine to make multiple testing corrections less effective in proteomics: medium problem scale; lower effect size due to possible compression; lower analysis power due to high cost; percentage of data showing an effect; and data distribution quirks. We then discuss some simple alternatives that can help reduce, or at least understand the FDR in this medium scale, low power situation.