Mehdi Mirzaei

Less label, more free: Approaches in label-free quantitative mass spectrometry

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.

Proteomic analysis of temperature stress in plants

In this review we examine current approaches used for proteomic analysis of temperature stress in plants. Rapid advances in this field in recent years are discussed, including metabolic, chemical and isotopic labeling, and label‐free quantitative techniques. These are compared and contrasted with well‐established methods such as 2‐DE approaches. Examples of applications of various methods are presented, and technical difficulties and limitations of each are also considered. Results of previous studies are examined in detail, and commonly occurring temperature stress response proteins are collated. We conclude that technical advances, and improvements in genome sequence availability, will have an ever increasing impact on our understanding of molecular mechanisms of stress response in plants.

Shotgun proteomic analysis of long-distance drought signaling in rice roots.

Rice (Oryza sativa L. cv. IR64) was grown in split-root systems to analyze long-distance drought signaling within root systems. This in turn underpins how root systems in heterogeneous soils adapt to drought. The approach was to compare four root tissues: (1) fully watered; (2) fully droughted and split-root systems where (3) one-half was watered and (4) the other half was droughted. This was specifically aimed at identifying how droughted root tissues altered the proteome of adjacent wet roots by hormone signals and how wet roots reciprocally affected dry roots hydraulically. Quantitative label-free shotgun proteomic analysis of four different root tissues resulted in identification of 1487 nonredundant proteins, with nearly 900 proteins present in triplicate in each treatment. Drought caused surprising changes in expression, most notably in partially droughted roots where 38% of proteins were altered in level compared to adjacent watered roots. Specific functional groups changed consistently in drought. Pathogenesis-related proteins were generally up-regulated in response to drought and heat-shock proteins were totally absent in roots of fully watered plants. Proteins involved in transport and oxidation-reduction reactions were also highly dependent upon drought signals, with the former largely absent in roots receiving a drought signal while oxidation-reduction proteins were strongly present during drought. Finally, two functionally contrasting protein families were compared to validate our approach, showing that nine tubulins were strongly reduced in droughted roots while six chitinases were up-regulated, even when the signal arrived remotely from adjacent droughted roots.

Proteomics study reveals the molecular mechanisms underlying water stress tolerance induced by Piriformospora indica in barley

UNLABELLEDnPiriformospora indica is a mutualistic root endophytic fungus, which transfers several benefits to hosts including enhance plant growth and increase yield under both normal and stress conditions. It has been shown that P. indica root-colonization enhances water stress tolerance based on general and non-specific plant-species mechanism. To better understand the molecular mechanism of P. indica-mediated drought stress tolerance, we designed a set of comparative experiments to study the impact of P. indica on barely plants cultivar Golden Promise grown under different drought levels [Filed capacity (F.C.) and 25% F.C.]. P. indica enhanced root and shoot biomass of colonized plants under both well-watered and water-deficit conditions. Proteome analysis of P. indica-colonized barley leaves under well-treated and water-deficit conditions resulted in detection of 726 reproducibly protein spots. Mass spectrometry analysis resulted in the identification of 45 differentially accumulated proteins involved in photosynthesis, reactive oxygen scavenging, metabolisms, signal transduction, and plant defense responses. Interestingly, P. indica increased the level of proteins involved in photosynthesis, antioxidative defense system and energy transport. We propose that P. indica-mediated drought stress tolerance in barely is through photosynthesis stimulation, energy releasing and enhanced antioxidative capacity in colonized plants.nnnBIOLOGICAL SIGNIFICANCEnPlant mutualistic symbionts offer long-term abiotic stress tolerance through the host adaptation to environmental stress. There have been a few published proteomic studies of plant symbionts to drought, and this is thought to be the first proteomic analysis, demonstrating the impact of endophyte on barley plant under drought stress. For some of identified proteins like TCTP and PCNA, a connection to physiological function in plants is novel, and can be the best candidates for sources of drought tolerance in future studies.

Differential regulation of aquaporins, small GTPases and V‐ATPases proteins in rice leaves subjected to drought stress and recovery

Mechanisms of drought tolerance are complex, interacting, and polygenic. This paper describes patterns of gene expression at precise physiological stages of drought in 35‐day‐old seedlings of Oryza sativa cv. Nipponbare. Drought was imposed gradually for up to 15 days, causing abscisic acid levels to rise and growth to cease, and plants were then re‐watered. Proteins were identified from leaf samples after moderate drought, extreme drought, and 3 and 6 days of re‐watering. Label‐free quantitative shotgun proteomics resulted in identification of 1548 non‐redundant proteins. More proteins were down‐regulated in early stages of drought but more were up‐regulated as severe drought developed. After re‐watering, there was notable down regulation, suggesting that stress‐related proteins were being degraded. Proteins involved in signalling and transport became dominant as severe drought took hold but decreased again on re‐watering. Most of the nine aquaporins identified were responsive to drought, with six decreasing rapidly in abundance as plants were re‐watered. Nine G‐proteins appeared in large amounts during severe drought and dramatically degraded once plants were re‐watered. We speculate that water transport and drought signalling are critical elements of the overall response to drought in rice and might be the key to biotechnological approaches to drought tolerance.

Comparative physiology and proteomic analysis of two wheat genotypes contrasting in drought tolerance.

UNLABELLEDnComparative physiology and proteomic analyses were conducted to monitor the stress response of two wheat genotypes (SERI M 82 (SE) and SW89.5193/kAu2 (SW)) with contrasting responses to drought stress. Under stress condition, the tolerant genotype (SE) produced higher shoot and root biomasses, longer roots and accumulated higher level of ABA in leaves. Physiological measurements suggested that the SE genotype was more efficient in water absorption and could preserve more water presumably by controlling stomata closure. Proteomic analysis showed an increased abundance of proteins related to defense and oxidative stress responses such as GLPs, GST, and SOD, and those related to protein processing such as small HSPs in roots of both genotypes in response to drought stress. Interestingly, the abundance of proteins such as endo-1,3-beta-glucosidase, peroxidase, SAMS, and MDH significantly increased in roots or leaves of the SE genotype and decreased in that of the SW one. In addition, an increased abundance of APX was detected in leaves and roots of the SE genotype and a decreased abundance of 14-3-3 and ribosomal proteins were noted in the SW one in response to drought stress. Our findings led to a better understanding about the integrated physiology and proteome responses of wheat genotypes with nearly contrasting responses to drought stress.nnnBIOLOGICAL SIGNIFICANCEnWe applied a comparative physiology and proteomic analysis to decipher the differential responses of two contrasting wheat genotypes to drought stress. Based on physiological measurements the tolerant genotype (SE) showed better drought response by developing deep root system, higher root and shoot biomasses, and higher level of ABA in leaves. Proteomic analysis showed an increased abundance of proteins related to defense and oxidative stress responses such as GLPs, GST, and SOD, and those related to protein processing such as small HSPs in roots of both genotypes in response to drought stress. In addition, the abundance of proteins such as glucan endo-1,3-beta-glucosidase, peroxidases, SAMS, and MDH increased in roots or leaves of the tolerant genotype (SE) and decreased in that of the sensitive genotype (SW). Overall, proteins related to oxidative stress, protein processing and photosynthesis showed decreased abundance to a greater extent in the sensitive genotype (SW).

Shotgun Proteomic Analysis of the Mexican Lime Tree Infected with “CandidatusPhytoplasma aurantifolia”

Infection of Mexican lime trees (Citrus aurantifolia L.) with the specialized bacterium CandidatusPhytoplasma aurantifolia causes witches broom disease. Witches broom disease has the potential to cause significant economic losses throughout western Asia and North Africa. We used label-free quantitative shotgun proteomics to study changes in the proteome of Mexican lime trees in response to infection by Ca. Phytoplasma aurantifolia. Of 990 proteins present in five replicates of healthy and infected plants, the abundances of 448 proteins changed significantly in response to phytoplasma infection. Of these, 274 proteins were less abundant in infected plants than in healthy plants, and 174 proteins were more abundant in infected plants than in healthy plants. These 448 proteins were involved in stress response, metabolism, growth and development, signal transduction, photosynthesis, cell cycle, and cell wall organization. Our results suggest that proteomic changes in response to infection by phytoplasmas might support phytoplasma nutrition by promoting alterations in the hosts sugar metabolism, cell wall biosynthesis, and expression of defense-related proteins. Regulation of defense-related pathways suggests that defense compounds are induced in interactions with susceptible as well as resistant hosts, with the main differences between the two interactions being the speed and intensity of the response.

Manipulating root water supply elicits major shifts in the shoot proteome

Substantial reductions in yield caused by drought stress can occur when parts of the root system experience water deficit even though other parts have sufficient access to soil water. To identify proteins associated to drought signaling, rice (Oryza sativa L. cv. IR64.) plants were transplanted into plastic pots with an internal wall dividing each pot into two equal compartments, allowing for equal distribution of soil and the root system between these compartments. The following treatments were applied: either both compartments were watered daily (wet roots), or water was withheld from both compartments (dry roots), or water was withheld from only one of the two compartments in each pot (wet and dry roots). The substantial differences in physiological parameters of different growth conditions were accompanied by differential changes in protein abundances. Label-free quantitative shotgun proteomics have resulted in identification of 1383 reproducible proteins across all three conditions. Differentially expressed proteins were categorized within 17 functional groups. The patterns observed were interesting in that in some categories such as protein metabolism and oxidation-reduction, substantial numbers of proteins were most abundant when leaves were receiving signals from wet and dry roots. In yet other categories such as transport, several key transporters were surprisingly abundant in leaves supported by partially or completely droughted root systems, especially plasma membrane and vacuolar transporters. Stress-related proteins behaved very consistently by increasing in droughted plants but notably some proteins were most abundant when roots of the same plant were growing in both wet and dry soils. Changes in carbohydrate-processing proteins were consistent with the passive accumulation of soluble sugars in shoots under drought, with hydrolysis of sucrose and starch synthesis both enhanced. These results suggest that drought signals are complex interactions and not simply the additive effect of water supply to the roots.

A fresh look at the male-specific region of the human Y chromosome.

The Chromosome-centric Human Proteome Project (C-HPP) aims to systematically map the entire human proteome with the intent to enhance our understanding of human biology at the cellular level. This project attempts simultaneously to establish a sound basis for the development of diagnostic, prognostic, therapeutic, and preventive medical applications. In Iran, current efforts focus on mapping the proteome of the human Y chromosome. The male-specific region of the Y chromosome (MSY) is unique in many aspects and comprises 95% of the chromosomes length. The MSY continually retains its haploid state and is full of repeated sequences. It is responsible for important biological roles such as sex determination and male fertility. Here, we present the most recent update of MSY protein-encoding genes and their association with various traits and diseases including sex determination and reversal, spermatogenesis and male infertility, cancers such as prostate cancers, sex-specific effects on the brain and behavior, and graft-versus-host disease. We also present information available from RNA sequencing, protein-protein interaction, post-translational modification of MSY protein-coding genes and their implications in biological systems. An overview of Human Y chromosome Proteome Project is presented and a systematic approach is suggested to ensure that at least one of each predicted protein-coding genes major representative proteins will be characterized in the context of its major anatomical sites of expression, its abundance, and its functional relevance in a biological and/or medical context. There are many technical and biological issues that will need to be overcome in order to accomplish the full scale mapping.

PloGO: plotting gene ontology annotation and abundance in multi-condition proteomics experiments.

We describe the PloGO R package, a simple open‐source tool for plotting gene ontology (GO) annotation and abundance information, which was developed to aid with the bioinformatics analysis of multi‐condition label‐free proteomics experiments using quantitation based on spectral counting. PloGO can incorporate abundance (raw spectral counts) or normalized spectral abundance factors (NSAF) data in addition to the GO annotation, as well as handle multiple files and allow for a targeted collection of GO categories of interest. Our main aims were to help identify interesting subsets of proteins for further analysis such as those arising from a protein data set partition based on the presence and absence or multiple pair‐wise comparisons, as well as provide GO summaries that can be easily used in subsequent analyses. Though developed with label‐free proteomics experiments in mind it is not specific to that approach and can be used for any multi‐condition experiment for which GO information has been generated.