6 research outputs found
Hydroponic isotope labeling of entire plants (HILEP) for quantitative plant proteomics
Quantitative analysis by mass spectrometry (MS) is a major challenge in
proteomics as the correlation between analyte concentration and signal
intensity is often poor due to varying ionisation efficiencies in the
presence of molecular competitors. However, relative quantitation
methods that utilise differential stable isotope labelling and mass
spectrometric detection are available. Many drawbacks inherent to
chemical labelling methods (ICAT, iTRAQ) can be overcome by
metabolic labelling with amino acids containing stable isotopes (e.g. 13C
and/or 15N) in methods such as Stable Isotope Labelling with Amino
acids in Cell culture (SILAC). SILAC has also been used for labelling of
proteins in plant cell cultures (1) but is not suitable for whole plant
labelling. Plants are usually autotrophic (fixing carbon from atmospheric
CO2) and, thus, labelling with carbon isotopes becomes impractical. In
addition, SILAC is expensive.
Recently, Arabidopsis cell cultures were labelled with 15N in a medium
containing nitrate as sole nitrogen source. This was shown to be suitable
for quantifying proteins and nitrogen-containing metabolites from this cell
culture (2,3).
Labelling whole plants, however, offers the advantage of studying
quantitatively the response to stimulation or disease of a whole multicellular
organism or multi-organism systems at the molecular level.
Furthermore, plant metabolism enables the use of inexpensive labelling
media without introducing additional stress to the organism. And finally,
hydroponics is ideal to undertake metabolic labelling under extremely
well-controlled conditions.
We demonstrate the suitability of metabolic 15N hydroponic isotope
labelling of entire plants (HILEP) for relative quantitative proteomic
analysis by mass spectrometry. To evaluate this methodology,
Arabidopsis plants were grown hydroponically in 14N and 15N media and subjected to oxidative stress
In planta proteomics and proteogenomics of the biotrophic barley fungal pathogen blumeria f.sp. hordei
Whilst there is increasing evidence tht the outcome of the interation between a pathogen and a host is dependent on protein-protein interactions, very little information is available on in planta proteomics of biotrophic plant pathogens. Here a proteogenomic approach has been employed to supplement the annotation of the recently sequenced genome and to cast light on the biology of the infection process of the economically important barley powdery mildew pathogen, Blumeria graminis f.sp horde
First-dimension separation with the MicroRotoforTM cell prior to SDS-PAGE and LC-MS/MS analysis
Free-flow isoelectric focusing (IEF) is a gel-free method for separating proteins based on their isoelectric point (pl) in a liquid environment and in the presence of carrier ampholytes. this method has been used with the RotoforTM cell at the preparative scale to fractionate proteins from samples containing several hundred milligrams of protein; see the refeences listed in Bio-Rad bulletin 3152. the MicroRotofor cell applies the same method to much sl=maller protein samples without dilution, separating and recoverng milligram quantities of protein in a total volume of about 2 ml
Comparison of bottom-up protein identification methods
With the rapid development of proteomics, a number of different methods appeared for the basic task of protein identification. We made a simple comparison between a common liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow using an ion trap mass spectrometer and a combined LC-MS and LC-MS/MS method using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and accurate peptide masses.
To compare the two methods for protein identification, we grew and extracted proteins from E. coli using established protocols. Cystines were reduced and alkylated, and proteins digested by trypsin. The resulting peptide mixtures were separated by reversed-phase liquid chromatography using a 4 h gradient from 0 to 50% acetonitrile over a C18 reversed-phase column. The LC separation was coupled on-line to either a Bruker Esquire HCT ion trap or a Bruker 7 tesla APEX-Qe Qh-FTICR hybrid mass spectrometer.
Data-dependent Qh-FTICR-MS/MS spectra were acquired using the quadrupole mass filter and collisionally induced dissociation into the external hexapole trap. Proteins were in both schemes identified by Mascot MS/MS ion searches and the peptides identified from these proteins in the FTICR MS/MS data were used for automatic internal calibration of the FTICR-MS data, together with ambient polydimethylcyclosiloxane ions
Translational plant proteomics: a perspective
Translational proteomics is an emerging sub-discipline of the proteomics field in the biological sciences. Translational plant proteomics aims to integrate knowledge from basic sciences to translate it into field applications to solve issues related but not limited to the recreational and economic values of plants, food security and safety, and energy sustainability. In this review, we highlight the substantial progress reached in plant proteomics during the past decade which has paved the way for translational plant proteomics. Increasing proteomics knowledge in plants is not limited to model and non-model plants, proteogenomics, crop improvement, and food analysis, safety, and nutrition but to many more potential applications. Given the wealth of information generated and to some extent applied, there is the need for more efficient and broader channels to freely disseminate the information to the scientific community
Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism
Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent
on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We
present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well
as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These
genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The
missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and
transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248
candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a
core set conserved in all three mildews, suggesting thatmost effectors represent species-specific adaptations