Craig Dufresne

Interlaboratory studies and initiatives developing standards for proteomics

Proteomics is a rapidly transforming interdisciplinary field of research that embraces a diverse set of analytical approaches to tackle problems in fundamental and applied biology. This viewpoint article highlights the benefits of interlaboratory studies and standardization initiatives to enable investigators to address many of the challenges found in proteomics research. Among these initiatives, we discuss our efforts on a comprehensive performance standard for characterizing PTMs by MS that was recently developed by the Association of Biomolecular Resource Facilities (ABRF) Proteomics Standards Research Group (sPRG).

The proteome of human retina

The retina is a delicate tissue that detects light, converts photochemical energy into neural signals, and transmits the signals to the visual cortex of the brain. A detailed protein inventory of the proteome of the normal human eye may provide a foundation for new investigations into both the physiology of the retina and the pathophysiology of retinal diseases. To provide an inventory, proteins were extracted from five retinas of normal eyes and fractionated using SDS‐PAGE. After in‐gel digestion, peptides were analyzed in duplicate using LC–MS/MS on an Orbitrap Elite mass spectrometer. A total of 3436 nonredundant proteins were identified in the human retina, including 20 unambiguous protein isoforms, of which eight have not previously been demonstrated to exist at the protein level. The proteins identified in the retina included most of the enzymes involved in the visual cycle and retinoid metabolism. One hundred and fifty‐eight proteins that have been associated with age‐related macular degeneration were identified in the retina. The MS proteome database of the human retina may serve as a valuable resource for future investigations of retinal biology and disease. All MS data have been deposited in the ProteomeXchange with identifier PXD001242 (http://proteomecentral.proteomexchange.org/dataset/PXD001242).

Integrated proteomics and metabolomics of Arabidopsis acclimation to gene-dosage dependent perturbation of isopropylmalate dehydrogenases.

Maintaining metabolic homeostasis is critical for plant growth and development. Here we report proteome and metabolome changes when the metabolic homeostasis is perturbed due to gene-dosage dependent mutation of Arabidopsis isopropylmalate dehydrogenases (IPMDHs). By integrating complementary quantitative proteomics and metabolomics approaches, we discovered that gradual ablation of the oxidative decarboxylation step in leucine biosynthesis caused imbalance of amino acid homeostasis, redox changes and oxidative stress, increased protein synthesis, as well as a decline in photosynthesis, which led to rearrangement of central metabolism and growth retardation. Disruption of IPMDHs involved in aliphatic glucosinolate biosynthesis led to synchronized increase of both upstream and downstream biosynthetic enzymes, and concomitant repression of the degradation pathway, indicating metabolic regulatory mechanisms in controlling glucosinolate biosynthesis.

Defining the proteome of human iris, ciliary body, retinal pigment epithelium, and choroid

The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS‐PAGE. After in‐gel digestion, peptides were analyzed using LC‐MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false‐positive rates of <0.1% and <1%, respectively. Forty‐three unambiguous protein isoforms were identified in iris, ciliary body, and RPE/choroid. Four “missing proteins” were identified in ciliary body based on ≥2 proteotypic peptides. The mass spectrometric proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194.

New nodes and edges in the glucosinolate molecular network revealed by proteomics and metabolomics of Arabidopsis myb28/29 and cyp79B2/B3 glucosinolate mutants

UNLABELLED Glucosinolates present in Brassicales are important for human health and plant defense against insects and pathogens. Here we investigate the proteomes and metabolomes of Arabidopsis myb28/29 and cyp79B2/B3 mutants deficient in aliphatic glucosinolates and indolic glucosinolates, respectively. Quantitative proteomics of the myb28/29 and cyp79B2/B3 mutants led to the identification of 2785 proteins, of which 142 proteins showed significant changes in the two mutants compared to wild type (WT). By mapping the differential proteins using STRING, we detected 59 new edges in the glucosinolate metabolic network. These connections can be classified as primary with direct roles in glucosinolate metabolism, secondary related to plant stress responses, and tertiary involved in other biological processes. Gene Ontology analysis of the differential proteins showed high level of enrichment in the nodes belonging to metabolic process including glucosinolate biosynthesis and response to stimulus. Using metabolomics, we quantified 292 metabolites covering a broad spectrum of metabolic pathways, and 89 exhibited differential accumulation patterns between the mutants and WT. The changing metabolites (e.g., γ-glutamyl amino acids, auxins and glucosinolate hydrolysis products) complement our proteomics findings. This study contributes toward engineering and breeding of glucosinolate profiles in plants in efforts to improve human health, crop quality and productivity. BIOLOGICAL SIGNIFICANCE Glucosinolates in Brassicales constitute an important group of natural metabolites important for plant defense and human health. Its biosynthetic pathways and transcriptional regulation have been well-studied. Using Arabidopsis mutants of important genes in glucosinolate biosynthesis, quantitative proteomics and metabolomics led to identification of many proteins and metabolites that are potentially related to glucosinolate metabolism. This study provides a comprehensive insight into the molecular networks of glucosinolate metabolism, and will facilitate efforts toward engineering and breeding of glucosinolate profiles for enhanced crop defense, and nutritional value.

The Human Skeletal Muscle Proteome Project: a reappraisal of the current literature

Skeletal muscle is a large organ that accounts for up to half the total mass of the human body. A progressive decline in muscle mass and strength occurs with ageing and in some individuals configures the syndrome of ‘sarcopenia’, a condition that impairs mobility, challenges autonomy, and is a risk factor for mortality. The mechanisms leading to sarcopenia as well as myopathies are still little understood. The Human Skeletal Muscle Proteome Project was initiated with the aim to characterize muscle proteins and how they change with ageing and disease. We conducted an extensive review of the literature and analysed publically available protein databases. A systematic search of peer‐reviewed studies was performed using PubMed. Search terms included ‘human’, ‘skeletal muscle’, ‘proteome’, ‘proteomic(s)’, and ‘mass spectrometry’, ‘liquid chromatography‐mass spectrometry (LC‐MS/MS)’. A catalogue of 5431 non‐redundant muscle proteins identified by mass spectrometry‐based proteomics from 38 peer‐reviewed scientific publications from 2002 to November 2015 was created. We also developed a nosology system for the classification of muscle proteins based on localization and function. Such inventory of proteins should serve as a useful background reference for future research on changes in muscle proteome assessed by quantitative mass spectrometry‐based proteomic approaches that occur with ageing and diseases. This classification and compilation of the human skeletal muscle proteome can be used for the identification and quantification of proteins in skeletal muscle to discover new mechanisms for sarcopenia and specific muscle diseases that can be targeted for the prevention and treatment.

The Human Skeletal Muscle Proteome Project: A reappraisal of the current literature

Skeletal muscle is a large organ that accounts for up to half the total mass of the human body. A progressive decline in muscle mass and strength occurs with ageing and in some individuals configures the syndrome of ‘sarcopenia’, a condition that impairs mobility, challenges autonomy, and is a risk factor for mortality. The mechanisms leading to sarcopenia as well as myopathies are still little understood. The Human Skeletal Muscle Proteome Project was initiated with the aim to characterize muscle proteins and how they change with ageing and disease. We conducted an extensive review of the literature and analysed publically available protein databases. A systematic search of peer‐reviewed studies was performed using PubMed. Search terms included ‘human’, ‘skeletal muscle’, ‘proteome’, ‘proteomic(s)’, and ‘mass spectrometry’, ‘liquid chromatography‐mass spectrometry (LC‐MS/MS)’. A catalogue of 5431 non‐redundant muscle proteins identified by mass spectrometry‐based proteomics from 38 peer‐reviewed scientific publications from 2002 to November 2015 was created. We also developed a nosology system for the classification of muscle proteins based on localization and function. Such inventory of proteins should serve as a useful background reference for future research on changes in muscle proteome assessed by quantitative mass spectrometry‐based proteomic approaches that occur with ageing and diseases. This classification and compilation of the human skeletal muscle proteome can be used for the identification and quantification of proteins in skeletal muscle to discover new mechanisms for sarcopenia and specific muscle diseases that can be targeted for the prevention and treatment.

Redox proteomics of tomato in response to Pseudomonas syringae infection.

Unlike mammals with adaptive immunity, plants rely on their innate immunity based on pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) for pathogen defense. Reactive oxygen species, known to play crucial roles in PTI and ETI, can perturb cellular redox homeostasis and lead to changes of redox-sensitive proteins through modification of cysteine sulfhydryl groups. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, little is known about redox proteins and how they function in PTI and ETI. In this study, cysTMT proteomics technology was used to identify similarities and differences of protein redox modifications in tomato resistant (PtoR) and susceptible (prf3) genotypes in response to Pseudomonas syringae pv tomato (Pst) infection. In addition, the results of the redox changes were compared and corrected with the protein level changes. A total of 90 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, biosynthesis of cysteine, sucrose and brassinosteroid, cell wall biogenesis, polysaccharide/starch biosynthesis, cuticle development, lipid metabolism, proteolysis, tricarboxylic acid cycle, protein targeting to vacuole, and oxidation–reduction. This inventory of previously unknown protein redox switches in tomato pathogen defense lays a foundation for future research toward understanding the biological significance of protein redox modifications in plant defense responses.

Identification of thioredoxin targets in guard cell enriched epidermal peels using cysTMT proteomics.

UNLABELLED Thioredoxins (Trx) play central roles in cellular redox regulation. Although hundreds of Trx targets have been identified using different approaches, the capture of targets in a quantitative and efficient manner is challenging. Here we report a high-throughput method using cysteine reactive tandem mass tag (cysTMT) labeling followed by liquid chromatography (LC)-mass spectrometry (MS) to screen for Trx targets. Compared to existing methods, this approach allows for i) three replicates of pairwise comparison in a single LC-MS run to reduce run-to-run variation; ii) efficient enrichment of cysteine-containing peptides that requires low protein input; and iii) accurate quantification of the cysteine redox status and localization of the Trx targeted cysteine residues. Application of this method in guard cell-enriched epidermal peels from Brassica napus revealed 80 Trx h targets involved in a broad range of processes, including photosynthesis, stress response, metabolism and cell signaling. The adaption of this protocol in other systems will greatly improve our understanding of the Trx function in regulating cellular redox homeostasis. BIOLOGICAL SIGNIFICANCE Redox homeostasis is tightly regulated for proper cellular activities. Specific protein-protein interactions between redox active molecules such as thioredoxin (Trx) and target proteins constitute the basis for redox-regulated biological processes. The use of cysTMT quantitative proteomics for studying Trx reactions enabled identification of potential Trx targets that provide important insights into the redox regulation in guard cells, a specialized plant cell type responsible for sensing of environmental signals, gas exchange and plant productivity.

Bicarbonate Induced Redox Proteome Changes in Arabidopsis Suspension Cells

Climate change as a result of increasing atmospheric CO2 affects plant growth and productivity. CO2 is not only a carbon donor for photosynthesis but also an environmental signal that can perturb cellular redox homeostasis and lead to modifications of redox-sensitive proteins. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, protein redox modifications and how they function in plant CO2 response remain unclear. Here a new iodoTMTRAQ proteomics technology was employed to analyze changes in protein redox modifications in Arabidopsis thaliana suspension cells in response to bicarbonate (mimic of elevated CO2) in a time-course study. A total of 47 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, transport, ROS scavenging, cell structure modulation and protein turnover. This inventory of previously unknown redox responsive proteins in Arabidopsis bicarbonate responses lays a foundation for future research toward understanding the molecular mechanisms underlying plant CO2 responses.