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The in-gel digestion of proteins for analysis by liquid chromatograph mass spectrometry has been used since the early 1990s. Although several improvements have contributed to increasing the quality of the data obtained, many recent publications still use sub-optimal approaches. Updates of the in-gel digestion protocol has been presented in the study. It has been shown that alternative reducing, alkylating agent reactions, and tryptic digestion buffers increase peptide and protein identification and reduce incubation times. The results indicate that a simultaneous and short, high temperature reduction and alkylation reaction using Tris(2-carboxyethyl)phosphine hydrochloride and chloroacetamide with a subsequent gel wash improve protein identification and sequence coverage, and diminish peptide side reactions. Additionally, use of 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid buffer allows a significant reduction in the digestion time improving trypsin performance and increasing the peptide recovery. The updates of the in-gel digestion protocol described here are efficient and offer flexibility to be incorporated in any proteomic laboratory., (© 2018 University of Warwick. Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The cell's endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrin-coated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein markers of the endomembrane system., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mass spectrometry (MS) has become the method of choice to identify and quantify proteins, typically by fragmenting peptides and inferring protein identification by reference to sequence databases. Well-established programs have largely solved the problem of identifying peptides in complex mixtures. However, to prevent the search space from becoming prohibitively large, most search engines need a list of expected modifications. Therefore, unexpected modifications limit both the identification of proteins and peptide-based quantification. We developed mass spectrometry-peak shift analysis (MS-PSA) to rapidly identify related spectra in large data sets without reference to databases or specified modifications. Peptide identifications from established tools, such as MASCOT or SEQUEST, may be propagated onto MS-PSA results. Modification of a peptide alters the mass of the precursor ion and some of the fragmentation ions. MS-PSA identifies characteristic fragmentation masses from MS/MS spectra. Related spectra are identified by pattern matching of unchanged and mass-shifted fragment ions. We illustrate the use of MS-PSA with simple and complex mixtures with both high and low mass accuracy data sets. MS-PSA is not limited to the analysis of peptides but can be used for the identification of related groups of spectra in any set of fragmentation patterns.

Proteomics has become a critical tool in the functional understanding of plant processes at the molecular level. Proteomics-based studies have also contributed to the ever-expanding array of data in modern biology, with many generating Web portals and online resources that contain incrementally expanding and updated information. Many of these resources reflect specialist research areas with significant and novel information that is not currently captured by centralized repositories. The Arabidopsis (Arabidopsis thaliana) community is well served by a number of online proteomics resources that hold an abundance of functional information. These sites can be difficult to locate among a multitude of online resources. Furthermore, they can be difficult to navigate in order to identify specific features of interest without significant technical knowledge. Recently, members of the Arabidopsis proteomics community involved in developing many of these resources decided to develop a summary aggregation portal that is capable of retrieving proteomics data from a series of online resources on the fly. The Web portal is known as the MASCP Gator and can be accessed at the following address: http://gator.masc-proteomics.org/. Significantly, proteomics data displayed at this site retrieve information from the data repositories upon each request. This means that information is always up to date and displays the latest data sets. The site also provides hyperlinks back to the source information hosted at each of the curated databases to facilitate more in-depth analysis of the primary data.

The identification of phosphorylation on proteins has become practicable for many laboratories in recent years, largely due to improvements in mass spectrometry (MS) and the development of methods to selectively enrich for phosphorylated peptides and proteins. However, phosphorylation is a dynamic and reversible modification which plays a central role in many biological processes including intracellular signalling. Therefore, the quantitative analysis of phosphorylated proteins and peptides is a subject of intense interest. We discuss three applications of isobaric tags for relative and absolute quantitation (iTRAQ) to the analysis of phosphopeptides from a variety of sample materials.

Plant vacuoles play essential roles in many physiological processes, particularly in mineral nutrition, turgor provision and cellular signalling. The vacuolar membrane, the tonoplast, contains many membrane transporters that are critical in the execution of these processes. However, although increasing knowledge is available about the identity of proteins involved in these processes very little is known about the regulation of tonoplast transporters. By studying the phosphoproteome of tonoplast-enriched membranes, we identified 66 phosphorylation sites on 58 membrane proteins. Amongst these, 31 sites were identified in 28 membrane transporters of various families including tonoplast anion transporters of the CLC family, potassium transporters of the KUP family, tonoplast sugar transporters and ABC transporters. In a number of cases, the detected sites were well conserved across isoforms of one family pointing to common mechanisms of regulation. In other cases, isoform-unique sites were present, suggesting regulatory mechanisms tailored to the function of individual proteins. These results provide the basis for future studies to elucidate the mechanistic regulation of tonoplast membrane transporters.

Advances in proteomic techniques have allowed the large-scale identification of phosphorylation sites in complex protein samples, but new biological insight requires an understanding of their in vivo dynamics. Here, we demonstrate the use of a stable isotope-based quantitative approach for pathway discovery and structure-function studies in Arabidopsis cells treated with the bacterial elicitor flagellin. The quantitative comparison identifies individual sites on plasma membrane (PM) proteins that undergo rapid phosphorylation or dephosphorylation. The data reveal both divergent dynamics of different sites within one protein and coordinated regulation of homologous sites in related proteins, as found for the PM H(+)-ATPases AHA1, 2 and 3. Strongly elicitor-responsive phosphorylation sites may reflect direct regulation of protein activity. We confirm this prediction for RbohD, an NADPH oxidase that mediates the rapid production of reactive oxygen species (ROS) in response to elicitors and pathogens. Plant NADPH oxidases are structurally distinct from their mammalian homologues, and regulation of the plant enzymes is poorly understood. On RbohD, we found both unchanging and strongly induced phosphorylation sites. By complementing an RbohD mutant plant with non-phosphorylatable forms of RbohD, we show that only those sites that undergo differential regulation are required for activation of the protein. These experiments demonstrate the potential for use of quantitative phosphoproteomics to determine regulatory mechanisms at the molecular level and provide new insights into innate immune responses.

Alterations in the proteome of Arabidopsis thaliana leaves during early responses to challenge by Pseudomonas syringae pv. tomato DC3000 (DC3000) were analysed using two-dimensional (2D) gel electrophoresis. Protein changes characteristic of the establishment of basal resistance and R-gene mediated resistance were examined by comparing responses to DC3000, a hrp mutant and DC3000 expressing avrRpm1 respectively. The abundance of selected transcripts was also analysed in GeneChip experiments. Here we present data from the soluble fraction of leaf protein, highlighting changes in two antioxidant enzyme groups; the glutathione S-transferases (GSTs F2, F6, F7 and F8) and peroxiredoxins (PrxA, B and IIE). Members of both enzyme groups showed signs of specific post-translational modifications, represented by multiple spots on gels. We suggest that oxidation of specific residues is responsible for some of the spot shifts. All forms of the GST proteins identified here increased following inoculation with bacteria. GSTF8 showed particularly dynamic responses to pathogen challenge, the corresponding transcript was significantly up-regulated by 2 h after inoculation, and the protein showed post-translational modifications specific to an incompatible interaction. Differential changes were observed with the peroxiredoxin proteins; PrxIIE and to a lesser extent PrxB, no change was observed with PrxA, but a truncated form PrxA-L was greatly reduced in abundance following bacterial challenges. Our data suggest that bacterial challenge generally induces Prxs and the antioxidants GSTs, however individual members of these families may be specifically modified dependent upon the virulence of the DC3000 strain and outcome of the interaction. Finally, proteomic and transcriptomic data derived from the same inoculation system are compared and the advantages offered by 2D gel analysis discussed in light of our results.

Selected reaction monitoring (SRM), sometimes called multiple reaction monitoring (MRM), is becoming the tool of choice for targeted quantitative proteomics in the plant science community. Key to a successful SRM experiment is prior identification of the distinct peptides for the proteins of interest and the determination of the so-called transitions that can be programmed into an LC-MS/MS to monitor those peptides. The transition for a given peptide comprises the intact peptide m/z and a high intensity product ion that can be monitored at a characteristic retention time (RT).To aid the design of SRM experiments, several online tools and databases have been produced to help researchers select transitions for their proteins of interest, but many of these tools are limited to the most popular model organisms such as human, yeast, and mouse or require the experimental acquisition of local spectral libraries. In this paper we present MRMaid1, a web-based SRM assay design tool whose transitions are generated by mining the millions of identified peptide spectra held in the EBI's PRIDE database. By using data from this large public repository, MRMaid is able to cover a wide range of species that can increase as the coverage of PRIDE grows. In this paper MRMaid transitions for 25 Arabidopsis thaliana proteins are evaluated experimentally, and found capable of quantifying 23 of these proteins. This performance was found to be comparable with the more time consuming approach of designing transitions using locally acquired orbitrap data, indicating that MRMaid is a valuable tool for targeted Arabidopsis proteomics. [ABSTRACT FROM AUTHOR]

Translation of most mRNAs is performed in a cap-dependent manner, requiring a protein complex, the cap complex, to regulate the accessibility of the message to the 40S ribosome. The cap complex initiates protein translation by binding to the 5′ cap of an mRNA and recruiting ribosomes to begin translation. Compared to animals and yeast, there are significant plant-specific differences in the regulation of cap-dependent mRNA translation, but these are poorly understood. Here, we purified proteins that bind to the 5′ cap during the Arabidopsis growth cycle. The protein profile of the cap-binding complexes varies during the various stages of the growth cycle in suspension culture cells. Using Western blotting, the cap complexes of quiescent cells were found to be composed of only three major proteins: eIF4isoE, which is primarily a cytoplasmic protein, and eIF4E and CBP80, which accumulate in the nucleus. However, when cells proliferate, at least 10 major proteins bind directly or indirectly to the 5′ cap. Proteomic, Western blotting and immunoprecipitation data establish that the spectrum of RNA helicases in the cap complexes also changes during the growth cycle. Cap complexes from proliferating cultures mainly contain eIF4A, which associates with at least four cap complexes, but eIF4A is replaced by additional helicases in quiescent cells. These findings suggest that the dynamic and selective recruitment of various proteins to mRNA 5′ cap complexes could play an important role in the regulation of gene expression. [ABSTRACT FROM AUTHOR]

Site-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, using a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA. Here, we report an expansion of the zebrafish genetic code to incorporate the UNAAs, azido-lysine (AzK), bicyclononyne-lysine (BCNK), and diazirine-lysine (AbK) into green fluorescent protein (GFP) and glutathione-s-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of AzK, BCNK, and AbK introduction into proteins as a means to investigate and engineer their function in zebrafish. [ABSTRACT FROM AUTHOR]