Difference gel electrophoresis Timms, John F.; Cramer, Rainer
Proteomics (Weinheim),
12/2008, Letnik:
8, Številka:
23-24
Journal Article
Recenzirano
DIGE is a protein labelling and separation technique allowing quantitative proteomics of two or more samples by optical fluorescence detection of differentially labelled proteins that are ...electrophoretically separated on the same gel. DIGE is an alternative to quantitation by MS‐based methodologies and can circumvent their analytical limitations in areas such as intact protein analysis, (linear) detection over a wide range of protein abundances and, theoretically, applications where extreme sensitivity is needed. Thus, in quantitative proteomics DIGE is usually complementary to MS‐based quantitation and has some distinct advantages. This review describes the basics of DIGE and its unique properties and compares it to MS‐based methods in quantitative protein expression analysis.
Protein effectors of pathogenicity are instrumental in modulating host immunity and disease resistance. The powdery mildew pathogen of grasses Blumeria graminis causes one of the most important ...diseases of cereal crops. B. graminis is an obligate biotrophic pathogen and as such has an absolute requirement to suppress or avoid host immunity if it is to survive and cause disease.
Here we characterise a superfamily predicted to be the full complement of Candidates for Secreted Effector Proteins (CSEPs) in the fungal barley powdery mildew parasite B. graminis f.sp. hordei. The 491 genes encoding these proteins constitute over 7% of this pathogen's annotated genes and most were grouped into 72 families of up to 59 members. They were predominantly expressed in the intracellular feeding structures called haustoria, and proteins specifically associated with the haustoria were identified by large-scale mass spectrometry-based proteomics. There are two major types of effector families: one comprises shorter proteins (100-150 amino acids), with a high relative expression level in the haustoria and evidence of extensive diversifying selection between paralogs; the second type consists of longer proteins (300-400 amino acids), with lower levels of differential expression and evidence of purifying selection between paralogs. An analysis of the predicted protein structures underscores their overall similarity to known fungal effectors, but also highlights unexpected structural affinities to ribonucleases throughout the entire effector super-family. Candidate effector genes belonging to the same family are loosely clustered in the genome and are associated with repetitive DNA derived from retro-transposons.
We employed the full complement of genomic, transcriptomic and proteomic analyses as well as structural prediction methods to identify and characterize the members of the CSEPs superfamily in B. graminis f.sp. hordei. Based on relative intron position and the distribution of CSEPs with a ribonuclease-like domain in the phylogenetic tree we hypothesize that the associated genes originated from an ancestral gene, encoding a secreted ribonuclease, duplicated successively by repetitive DNA-driven processes and diversified during the evolution of the grass and cereal powdery mildew lineage.
Quantitation is an inherent requirement in comparative proteomics and there is no exception to this for plant proteomics. Quantitative proteomics has high demands on the experimental workflow, ...requiring a thorough design and often a complex multi-step structure. It has to include sufficient numbers of biological and technical replicates and methods that are able to facilitate a quantitative signal read-out. Quantitative plant proteomics in particular poses many additional challenges but because of the nature of plants it also offers some potential advantages. In general, analysis of plants has been less prominent in proteomics. Low protein concentration, difficulties in protein extraction, genome multiploidy, high Rubisco abundance in green tissue, and an absence of well-annotated and completed genome sequences are some of the main challenges in plant proteomics. However, the latter is now changing with several genomes emerging for model plants and crops such as potato, tomato, soybean, rice, maize and barley. This review discusses the current status in quantitative plant proteomics (MS-based and non-MS-based) and its challenges and potentials. Both relative and absolute quantitation methods in plant proteomics from DIGE to MS-based analysis after isotope labeling and label-free quantitation are described and illustrated by published studies. In particular, we describe plant-specific quantitative methods such as metabolic labeling methods that can take full advantage of plant metabolism and culture practices, and discuss other potential advantages and challenges that may arise from the unique properties of plants.
Growing interest in food quality and traceability by regulators as well as consumers demands advances in more rapid, versatile and cost-effective analytical methods. Milk, as most food matrices, is a ...heterogeneous mixture composed of metabolites, lipids and proteins. One of the major challenges is to have simultaneous, quantitative detection (profiling) of this panel of biomolecules to gather valuable information for assessing food quality, traceability and safety. Here, for milk analysis, atmospheric pressure matrix-assisted laser desorption/ionization employing homogenous liquid sample droplets was used on a Q-TOF mass analyzer. This method has the capability to produce multiply charged proteinaceous ions as well as highly informative profiles of singly charged lipids/metabolites. In two examples, this method is coupled with user-friendly machine-learning software. First, rapid speciation of milk (cow, goat, sheep and camel) is demonstrated with 100% classification accuracy. Second, the detection of cow milk as adulterant in goat milk is shown at concentrations as low as 5% with 92.5% sensitivity and 94.5% specificity.
Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS) is a highly versatile and sensitive analytical technique, which is known for its soft ionisation of biomolecules such as ...peptides and proteins. Generally, MALDI MS analysis requires little sample preparation, and in some cases like MS profiling it can be automated through the use of robotic liquid-handling systems. For more than a decade now, MALDI MS has been extensively utilised in the search for biomarkers that could aid clinicians in diagnosis, prognosis, and treatment decision making. This review examines the various MALDI-based MS techniques like MS imaging, MS profiling and proteomics in-depth analysis where MALDI MS follows fractionation and separation methods such as gel electrophoresis, and how these have contributed to prostate cancer biomarker research. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.
•MALDI MS is a versatile and sensitive analytical technique used in PCa research.•MALDI MS imaging enables the direct analysis of fresh and FFPE prostate tissue.•Previous mistakes in MALDI MS profiling need to be addressed with SOPs.•The stages and process of biomarker discovery to clinical utility are described.•Perspectives on the future use of MALDI MS in PCa biomarker discovery are given.
MS-based proteomics was applied to the analysis of the medicinal plant Artemisia annua, exploiting a recently published contig sequence database (Graham et al. (2010) Science 327, 328-331) and other ...genomic and proteomic sequence databases for comparison. A. annua is the predominant natural source of artemisinin, the precursor for artemisinin-based combination therapies (ACTs), which are the WHO-recommended treatment for P. falciparum malaria.
The comparison of various databases containing A. annua sequences (NCBInr/viridiplantae, UniProt/viridiplantae, UniProt/A. annua, an A. annua trichome Trinity contig database, the above contig database and another A. annua EST database) revealed significant differences in respect of their suitability for proteomic analysis, showing that an organism-specific database that has undergone extensive curation, leading to longer contig sequences, can greatly increase the number of true positive protein identifications, while reducing the number of false positives. Compared to previously published data an order-of-magnitude more proteins have been identified from trichome-enriched A. annua samples, including proteins which are known to be involved in the biosynthesis of artemisinin, as well as other highly abundant proteins, which suggest additional enzymatic processes occurring within the trichomes that are important for the biosynthesis of artemisinin.
The newly gained information allows for the possibility of an enzymatic pathway, utilizing peroxidases, for the less well understood final stages of artemisinin's biosynthesis, as an alternative to the known non-enzymatic in vitro conversion of dihydroartemisinic acid to artemisinin. Data are available via ProteomeXchange with identifier PXD000703.
New advances in laser-based MS with respect to analytical depth, signal robustness and sample flexibility in combination with its intrinsically high sample analysis speed can fill many of the gaps in ...the field of proteomics that can currently only be served with severe limitations by the commonly employed but much slower ESI-based proteomic tools, thus arguably allowing for the analysis of ≥1M samples/day on a single platform for analyses that do not require deep proteome coverage.
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Highlights
•Future proteomic analyses for longitudinal studies and P4 medicine arguably require ≥1M samples/day.•Proteome depth/coverage is commonly the focus whereas analytical speed is typically neglected.•A compromise between analytical depth and speed is needed for future large-scale studies.•Ultrahigh-speed ‘omic’ analyses require tools that are intrinsically fast such as laser-based MS.
High-speed analysis of large (prote)omics sample sets at the rate of thousands or millions of samples per day on a single platform has been a challenge since the beginning of proteomics. For many years, ESI-based MS methods have dominated proteomics because of their high sensitivity and great depth in analyzing complex proteomes. However, despite improvements in speed, ESI-based MS methods are fundamentally limited by their sample introduction, which excludes off-line sample preparation/fractionation because of the time required to switch between individual samples/sample fractions, and therefore being dependent on the speed of on-line sample preparation methods such as liquid chromatography. Laser-based ionization methods have the advantage of moving from one sample to the next without these limitations, being mainly restricted by the speed of modern sample stages, i.e. 10 ms or less between samples. This speed matches the data acquisition speed of modern high-performing mass spectrometers whereas the pulse repetition rate of the lasers (>1 kHz) provides a sufficient number of desorption/ionization events for successful ion signal detection from each sample at the above speed of the sample stages. Other advantages of laser-based ionization methods include the generally higher tolerance to sample additives and contamination compared with ESI MS, and the contact-less and pulsed nature of the laser used for desorption, reducing the risk of cross-contamination. Furthermore, new developments in MALDI have expanded its analytical capabilities, now being able to fully exploit high-performing hybrid mass analyzers and their strengths in sensitivity and MS/MS analysis by generating an ESI-like stable yield of multiply charged analyte ions. Thus, these new developments and the intrinsically high speed of laser-based methods now provide a good basis for tackling extreme sample analysis speed in the omics.
Powdery mildews are biotrophic pathogens causing fungal diseases in many economically important crops, including cereals, which are affected by
Blumeria graminis
. Powdery mildews only invade the ...epidermal cell layer of leaf tissues, in which they form haustorial structures. Haustoria are at the center of the biotrophic interaction by taking up nutrients from the host and by delivering effectors in the invaded cells to jeopardize plant immunity. Haustoria are composed of a fungal core delimited by a haustorial plasma membrane and cell wall. Surrounding these is the extrahaustorial complex, of which the extrahaustorial membrane is of plant origin. Although haustoria transcriptomes and proteomes have been investigated for
Blumeria
, the proteomes of barley epidermis upon infection and the barley components of the extrahaustorial complex remains unexplored. When comparing proteomes of infected and non-infected epidermis, several classical pathogenesis-related (PR) proteins were more abundant in infected epidermis. These included peroxidases, chitinases, cysteine-rich venom secreted proteins/PR1 and two thaumatin-like PR5 protein isoforms, of which TLP5 was previously shown to interact with the
Blumeria
effector BEC1054 (CSEP0064). Against expectations, transient
TLP5
gene silencing suggested that TLP5 does not contribute to resistance but modulates susceptibility towards
B. graminis
. In a second proteomics comparison, haustorial structures were enriched from infected epidermal strips to identify plant proteins closely associated with the extrahaustorial complex. In these haustoria-enriched samples, relative abundances were higher for several V-type ATP synthase/ATPase subunits, suggesting the generation of proton gradients in the extrahaustorial space. Other haustoria-associated proteins included secreted or membrane proteins such as a PIP2 aquaporin, an early nodulin-like protein 9, an aspartate protease and other proteases, a lipase, and a lipid transfer protein, all of which are potential modulators of immunity, or the targets of pathogen effectors. Moreover, the ER BIP-like HSP70, may link ER stress responses and the idea of ER-like properties previously attributed to the extrahaustorial membrane. This initial investigation exploring the barley proteomes of
Blumeria
-infected tissues and haustoria, associated with a transient gene silencing approach, is invaluable to gain first insight of key players of resistance and susceptibility.
Omics analysis by mass spectrometry (MS) is a vast field, with proteomics, metabolomics and lipidomics dominating recent research by exploiting biological MS ionisation techniques. Traditional MS ...ionisation techniques such as electrospray ionisation have limitations in analyte‐specific sensitivity, modes of sampling and throughput, leading to many researchers investigating new ionisation methods for omics research. In this review, we examine the current landscape of these new ionisation techniques, divided into the three groups of (electro)spray‐based, laser‐based and other miscellaneous ionisation techniques. Due to the wide range of new developments, this review can only provide a starting point for further reading on each ionisation technique, as each have unique benefits, often for specialised applications, which promise beneficial results for different areas in the omics world.
MALDI-MS imaging in combination with cryo-SEM-EDX analysis demonstrated that glucosinolates are accumulated differentially in specific cells of reproductive organs in Arabidopsis thaliana. Display ...omitted
► Three glucosinolates were mapped by MALDI-MSI in flower buds, sepals and siliques. ► Two sites of accumulation of glucosinolates were found: S-cells and epidermis of sepals. ► Accumulation of sulphur compounds in S-cells was confirmed by cryo-SEM-EDX.
Glucosinolates are multi-functional plant secondary metabolites which play a vital role in plant defence and are, as dietary compounds, important to human health and livestock well-being. Knowledge of the tissue-specific regulation of their biosynthesis and accumulation is essential for plant breeding programs. Here, we report that in Arabidopsis thaliana, glucosinolates are accumulated differentially in specific cells of reproductive organs. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), distribution patterns of three selected compounds, 4-methylsulfinylbutyl (glucoraphanin), indol-3-ylmethyl (glucobrassicin), and 4-benzoyloxybutyl glucosinolates, were mapped in the tissues of whole flower buds, sepals and siliques. The results show that tissue localization patterns of aliphatic glucosinolate glucoraphanin and 4-benzoyloxybutyl glucosinolate were similar, but indole glucosinolate glucobrassicin had different localisation, indicating a possible difference in function. The high resolution images obtained by a complementary approach, cryo-SEM Energy Dispersive X-ray analysis (cryo-SEM-EDX), confirmed increased concentration of sulphur in areas with elevated amounts of glucosinolates, and allowed identifying the cell types implicated in accumulation of glucosinolates. High concentration of sulphur was found in S-cells adjacent to the phloem in pedicels and siliques, indicating the presence of glucosinolates. Moreover, both MALDI MSI and cryo-SEM-EDX analyses indicated accumulation of glucosinolates in cells on the outer surface of the sepals, suggesting that a layer of glucosinolate-accumulating epidermal cells protects the whole of the developing flower, in addition to the S-cells, which protect the phloem. This research demonstrates the high potential of MALDI MSI for understanding the cell-specific compartmentation of plant metabolites and its regulation.
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