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In this chapter we describe the workflow used in our laboratory for label-free quantitative shotgun proteomics based on spectral counting. The main tools used are a series of R modules known collectively as the Scrappy program. We describe how to go from peptide to spectrum matching in a shotgun proteomics experiment using the XTandem algorithm, to simultaneous quantification of up to thousands of proteins, using normalized spectral abundance factors. The outputs of the software are described in detail, with illustrative examples provided for some of the graphical images generated. While it is not strictly within the scope of this chapter, some consideration is given to how best to extract meaningful biological information from quantitative shotgun proteomics data outputs.

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., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We describe in this communication a set of functional perl script utilities for use in peptide mass spectral database searching and proteomics experiments, known as the Wildcat Toolbox. These are all freely available for download from our laboratory Web site (http://proteomics.arizona.edu/toolbox.html) as a combined zip file, and can also be accessed via the Proteome Commons Web site (www.proteomecommons.org) in the tools section. We make them available to other potential users in the spirit of open source software development; we do not have the resources to provide any significant technical support for them, but we hope users will share both bugs and improvements with the community at large.

We describe the initial characterization of the wheat amyloplast proteome, consisting of the identification and classification of 171 proteins. Whole amyloplasts and purified amyloplast membranes were prepared from wheat (Triticum aestivum). Protein extracts were examined by one-dimensional and two-dimensional electrophoresis, followed by high performance liquid chromatography-tandem mass spectrometry of separated proteins. Tandem mass spectrometry data of individual peptides was then searched by SEQUEST, using a database containing known protein sequences from both wheat and other homologous cereal crops. Using this approach we identified 108 proteins from whole amyloplasts and 63 proteins from purified amyloplast membranes. The majority of protein identifications were derived from protein sequences from cereal crops other than wheat, for which relatively little gene sequence data is available. The highest percentage of protein identifications obtained from any individual species was 46% of the total number of proteins identified, using sequence data found in our proprietary rice (Oryza sativa) genome database.

Heat stress specifically affects fertility by impairing pollen viability but cotton wild relatives successfully reproduce in hot savannas where they evolved. An Australian arid‐zone cotton (Gossypium robinsonii) was exposed to heat events during pollen development then mature pollen was subjected to deep proteomic analysis using 57 023 predicted genes from a genomic database we assembled for the same species. Three stages of pollen development, including tetrads (TEs), uninucleate microspores (UNs) and binucleate microspores (BNs) were exposed to 36°C or 40°C for 5 days and the resulting mature pollen was collected at anthesis (p‐TE, p‐UN and p‐BN, respectively). Using the sequential windowed acquisition of all theoretical mass spectra proteomic analysis, 2704 proteins were identified and quantified across all pollen samples analysed. Proteins predominantly decreased in abundance at all stages in response to heat, particularly after exposure of TEs to 40°C. Functional enrichment analyses demonstrated that extreme heat increased the abundance of proteins that contributed to increased messenger RNA splicing via spliceosome, initiation of cytoplasmic translation and protein refolding in p‐TE40. However, other functional categories that contributed to intercellular transport were inhibited in p‐TE40, linked potentially to Rab proteins. We ascribe the resilience of reproductive processes in G. robinsonii at temperatures up to 40°C, relative to commercial cotton, to a targeted reduction in protein transport. Summary statement: Heat lowers pollen viability and yet a cotton relative (Gossypium robinsonii) reproduces in the arid north‐western of Australia. The first genomic database for this species was used to identify 2704 proteins from mature pollen. Distinctive proteins were identified after tetrads had been exposed to 40°C. [ABSTRACT FROM AUTHOR]

Grapes are an important crop plant which forms the basis of a globally important industry. Grape and wine production is particularly vulnerable to environmental and climatic fluctuations, which makes it essential for us to develop a greater understanding of the molecular level responses of grape plants to various abiotic stresses. The completion of the initial grape genome sequence in 2007 has led to a significant increase in research on grapes using proteomics approaches. In this article, we discuss some of the current research on abiotic stress in grapevines, in the context of abiotic stress research in other plant species. We also highlight some of the current limitations in grapevine proteomics and identify areas with promising scope for potential future research. [ABSTRACT FROM AUTHOR]

Ketimine reductase (E.C. 1.5.1.25) was purified to apparent homogeneity from lamb forebrain by means of a rapid multistep chromatography protocol. The purified enzyme was identified by MS/MS (mass spectrometry) as μ-crystallin. The identity was confirmed by heterologously expressing human μ-crystallin in Escherichia coli and subsequent chromatographic purification of the protein. The purified human μ-crystallin was confirmed to have ketimine reductase activity with a maximum specific activity similar to that of native ovine ketimine reductase, and was found to catalyse a sequential reaction. The enzyme substrates are putative neuromodulator/ transmitters. The thyroid hormone 3,5,3‱-L-triiodothyronine (T3) was found to be a strong reversible competitive inhibitor, and may have a novel role in regulating their concentrations. μ-Crystallin is also involved in intracellular T3 storage and transport. This research is the first to demonstrate an enzyme function for μ-crystallin. This newly demonstrated enzymatic activity identifies a new role for thyroid hormones in regulating mammalian amino acid metabolism, and a possible reciprocal role of enzyme activity regulating bioavailability of intracellular T3. [ABSTRACT FROM AUTHOR]

Examines the simultaneous detection and identification of O-GlcNAc-modified glycoproteins. Assay for glycoproteins carrying O-linked N-acetylglucosamine modification; Correlation of the molecular modification to cellular stimuli; Use of high-pressure liquid chromatography-tandem mass spectrometry.

Drought often compromises yield in non-irrigated crops such as rainfed rice, imperiling the communities that depend upon it as a primary food source. In this study, two cultivated species (Oryza sativa cv. Nipponbare and Oryza glaberrima cv. CG14) and an endemic, perennial Australian wild species (Oryza australiensis) were grown in soil at 40% field capacity for 7 d (drought). The hypothesis was that the natural tolerance of O. australiensis to erratic water supply would be reflected in a unique proteomic profile. Leaves from droughted plants and well-watered controls were harvested for label-free quantitative shotgun proteomics. Physiological and gene ontology analysis confirmed that O. australiensis responded uniquely to drought, with superior leaf water status and enhanced levels of photosynthetic proteins. Distinctive patterns of protein accumulation in drought were observed across the O. australiensis proteome. Photosynthetic and stress-response proteins were more abundant in drought-affected O. glaberrima than O. sativa, and were further enriched in O. australiensis. In contrast, the level of accumulation of photosynthetic proteins decreased when O. sativa underwent drought, while a narrower range of stress-responsive proteins showed increased levels of accumulation. Distinctive proteomic profiles and the accumulated levels of individual proteins with specific functions in response to drought in O. australiensis indicate the importance of this species as a source of stress tolerance genes. [ABSTRACT FROM AUTHOR]

Rice is a critically important food source but yields worldwide are vulnerable to periods of drought. We exposed eight genotypes of upland and lowland rice (Oryza sativa L. ssp. japonica and indica) to drought stress at the late vegetative stage, and harvested leaves for label-free shotgun proteomics. Gene ontology analysis was used to identify common drought-responsive proteins in vegetative tissues, and leaf proteins that are unique to individual genotypes, suggesting diversity in the metabolic responses to drought. Eight proteins were found to be induced in response to drought stress in all eight genotypes. A total of 213 proteins were identified in a single genotype, 83 of which were increased in abundance in response to drought stress. In total, 10 of these 83 proteins were of a largely uncharacterized function, making them candidates for functional analysis and potential biomarkers for drought tolerance. [ABSTRACT FROM AUTHOR]

Ancient proteomic analysis has been shown to have several advantages over ancient DNA studies, largely due to the ability of many proteins to survive over longer periods of time than their corresponding genes. High-resolution mass spectrometry allows for large scale data to be generated from small amounts of sample. Typically, sampling of bioarchaeological material for proteomics has involved the use of invasive tools such as drills and hammers, which presents an issue when working with museums and other heritage organisations. There exist a few studies in the published literature on the use of minimally invasive techniques including specifically manufactured sampling tapes for the minimally invasive analysis of paintings and frescoes, and recently an example of their application to mummified skin. Here we present the development of a novel minimally invasive sample preparation technique for application in the mass spectrometric analysis of bioarchaeological materials. The extraction protocol was developed by applying commercially available, dermatology grade skin sampling strips to modern skin surfaces as a surrogate, and then applied to cranial and bone fragments belonging to a 26th Dynasty Egyptian mummified individual from the coffin of a woman named Mer-Neith-it-es. Extracted proteins were subjected to electrophoretic separation and proteolytic digestion, resulting in peptides that were separated, fragmented and identified using nanoflow liquid chromatography - high resolution tandem mass spectrometry. We have identified a number of ancient intracellular proteins on the surfaces of the various cranial and bone fragments, without causing any significant damage to these valuable remains. • Skin sampling strips recover protein remnants adhering to ancient bones. • There is no visible or morphological damage to the bone structures. • Skin and neural tissue associated proteins are identified on skull fragments. • The method developed is applicable to a variety of ancient remains with hard surfaces. [ABSTRACT FROM AUTHOR]

Abstract: The current study uses proteomics to assess the effects of metal contamination on Sydney Rock oyster haemolymph. Saccostrea glomerata were exposed in aquaria for four days to three environmentally relevant metals (copper, lead or zinc). Oyster haemolymph proteins from metal-exposed oysters were then compared to haemolymph from non-exposed controls using 2-dimensional electrophoresis to identify proteins that differed significantly in intensity. These proteins were then subjected to tandem mass spectrometry so that putative protein identities could be assigned. The data suggest that there are unique protein expression profiles for each metal. Exposure to 100μg/l of copper, lead or zinc yielded a total of 25 differentially expressed proteins. However, only one of these protein spots exhibited altered intensities in response to all three metals. Eighteen of the 25 spots were significantly affected by just one of the three metals. Differentially expressed proteins were assigned to five different categories of biological function. Proteins affecting shell properties were the most common functional group accounting for 34% of the identified proteins. Cytoskeletal activities and metabolism/stress responses each accounted for a further 25% of the proteins. [Copyright &y& Elsevier]

The composition of the editosome, a multi-protein complex that catalyzes uridine insertion and deletion RNA editing to produce mature mitochondrial mRNAs in trypanosomes, was analyzed by mass spectrometry. The editosomes were isolated by column chromatography, glycerol gradient sedimentation, and monoclonal antibody affinity purifications. At least 16 proteins form the catalytic core of the editosome, and additional associated proteins were identified. Analyses of mitochondrial fractions identified several non-editosome proteins and multi-protein complexes. These studies contribute to the functional annotation of T. brucei genome. [Copyright &y& Elsevier]