Analysis of soybean tissue culture protein dynamics using difference gel electrophoresis.

UNLABELLED: Excised hypocotyls from developing soybean (Glycine max (L.) merr. cv. Jack) were cultivated on agar-solidified medium until callus formed. The calli were then propagated in liquid medium until stable, relatively uniform, finely-divided suspension cultures were obtained. Cells were typically transferred to fresh medium at 7-day intervals. Cultures were harvested by filtration five days (early log phase) or eight days (late log phase) after transfer. In order to evaluate dynamic changes, both intracellular and extracellular proteins were analyzed by 2-dimensional difference gel electrophoresis. Selected spots were subjected to in-gel tryptic-digestion and the resultant peptides were analyzed by nLC-MS/MS. In follow-up studies gel-free shot-gun analyses led to identification of 367 intracellular proteins and 188 extracellular proteins. SIGNIFICANCE: The significance of the described research is two-fold. First a gel-based proteomics method was applied to the study of the dynamics of the secretome (extracellular proteins). Second, results of a shot-gun non-gel based proteomic survey of both cellular and extracellular proteins are presented.

A novel regulatory mechanism based upon a dynamic core structure for the mitochondrial pyruvate dehydrogenase complex?

The Arabidopsis thaliana genome includes three genes for mitochondrial dihydrolipoamide acetyltransferase, the E2-component of the mitochondrial pyruvate dehydrogenase complex (PDC). Two genes encode E2-proteins with a single lipoyl domain, while the third has a two-lipoyl domain structure. Transcripts for each E2 protein were expressed in all plant organs. Each recombinant AtmtE2 can individually form an icosahedral PDC core structure, and results from bimolecular fluorescence complementation assays are consistent with formation of hetero-core structures from all permutations of the AtmtE2 proteins. We propose a unique regulatory mechanism involving dynamic formation of hetero-core complexes that include both mono- and di-lipoyl forms of AtmtE2.

Initial description of the developing soybean seed protein Lys-N(ε)-acetylome.

Characterization of the myriad protein posttranslational modifications (PTM) is a key aspect of proteome profiling. While there have been previous studies of the developing soybean seed phospho-proteome, herein we present the first analysis of non-histone lysine-N(Ɛ)-acetylation in this system. In recent years there have been reports that lysine acetylation is widespread, affecting thousands of proteins in diverse species from bacteria to mammals. Recently preliminary descriptions of the protein lysine acetylome from the plants Arabidopsis thaliana and Vitis vinifera have been reported. Using a combination of immunoenrichment and mass spectrometry-based techniques, we have identified over 400 sites of lysine acetylation in 245 proteins from developing soybean (Glycine max (L.) Merr., cv. Jack) seeds, which substantially increases the number of known plant N(Ɛ)-lysine-acetylation sites. Results of functional annotation indicate acetyl-proteins are involved with a host of cellular activities. In addition to histones, and other proteins involved in RNA synthesis and processing, acetyl-proteins participate in signaling, protein folding, and a plethora of metabolic processes. Results from in silico localization indicate that lysine-acetylated proteins are present in all major subcellular compartments. In toto, our results establish developing soybean seeds as a physiologically distinct addendum to Arabidopsis thaliana seedlings for functional analysis of protein Lys-N(Ɛ)-acetylation. BIOLOGICAL SIGNIFICANCE: Several modes of peptide fragmentation and database search algorithms are incorporated to identify, for the first time, sites of lysine acetylation on a plethora of proteins from developing soybean seeds. The contributions of distinct techniques to achieve increased coverage of the lysine acetylome are compared, providing insight to their respective benefits. Acetyl-proteins and specific acetylation sites are characterized, revealing intriguing similarities as well as differences with those previously identified in other plant and non-plant species.

Proteomic analysis of the testa from developing soybean seeds.

Soybean (Glycine max (L.) Merr. cv Jack) seed development was separated into nine defined stages (S1 to S9). Testa (seed coats) were removed from developing seeds at stages S2, 4, 6, 8, and 9, and subjected to shotgun proteomic profiling. For each stage "total proteins" were isolated from 150 mg dry weight of seed coat using a phenol-based method, then reduced, alkylated, and digested with trypsin. The tryptic peptides were separated using a C18-reversed phase matrix, then analyzed using an LTQ Orbitrap Mass Spectrometer. Spectra were searched against the Phytozome G. max DB using the Sorcerer 2 IDA Sequest-based search algorithm. Identities were verified using Scaffold 3. A total of 306 (S2), 328 (S4), 273 (S6), 193 (S8), and 272 (S9) proteins were identified in three out of three biological replicates, and sorted into 11 functional groups: Primary Metabolism, Secondary Metabolism, Cellular Structure, Stress Responses, Nucleic Acid metabolism, Protein Synthesis, Protein Folding, Protein Targeting, Hormones and Signaling, Seed Storage Proteins, and Proteins of Unknown Function. In selected instances, individual seed coat proteins were quantified by spectral counting. The number of proteins involved in intermediary metabolism, flavonoid biosynthesis, protein folding and degradation are discussed as they relate to seed coat function. BIOLOGICAL SIGNIFICANCE: Most previous analyses of seed coats have either targeted individual enzymes or used the results from high-throughput transcript profiling to infer biological function. Because there is seldom a linear correlation between transcript and protein levels, we have undertaken a shotgun proteomics-based description of soybean (G. max (L.) Merr. cv Jack) seed coats, as a function of development, in order to bridge this gap and to establish the baseline for a more comprehensive understanding of seed biology.

Using multiplex-staining to study changes in the maize leaf phosphoproteome in response to mechanical wounding.

Mechanical wounding of 2-week-old maize (Zea mays L.) leaves, one of the first steps in both pathogen infection and herbivore attack, stimulates metabolism and activates signal transduction pathways dedicated to defense and recovery. The signaling pathways include reversible protein phosphorylation which can modulate protein activities, and transmit signals within cellular pathways and networks. We have used multiplex-staining of high-resolution 2D gels for protein (Sypro Ruby) and phosphorylation (Pro-Q Diamond) as a strategy for quantifying changes in the stoichiometry of phosphorylation after wounding for 270 protein spots. Rigorous statistical analysis of the time-index data allowed us to accept patterns of change in 125 of the spots as non-random, and these patterns were assigned to five clusters. A reliable identity was assigned to 21 selected proteins, most of which have been previously described as phospho-proteins. The results suggest that analysis of protein spots from high-resolution 2D gels by multiplex-staining for protein plus phosphorylation is a strategy that can be broadly useful for study of how the phospho-proteome responds to abiotic stress.

Chemical cross-linking immobilized concanavalin A for use in proteomic analyses.

Lectin affinity chromatography was used to reduce the amount of the abundant glycoprotein beta-conglycinin in total protein samples prepared from developing soybean (Glycine max L. Merrill cv. Jack) seeds. Electrophoretic analysis of both the concanavalin A-Sepharose binding and non-binding fraction revealed an abundant protein band at Mr 26,000. The amount of this protein was greatly increased when concanavalin A-Sepharose was used with urea-containing buffers. Peptide mass fingerprint analysis of this abundant protein band unequivocally identified it as concanavalin A (con A). A simple and gentle method was used to chemically cross-link the con A subunits so that the lectin-Sepharose retained the ability to bind high-mannose type glycoproteins. The chemically cross-linked con A-Sepharose was stable in buffers that contained up to 8M urea, making this an affinity matrix suitable for use in electrophoresis-based proteomic analyses.

Developmental expression of the mitochondrial pyruvate dehydrogenase complex in pea (Pisum sativum) seedlings.

In order to better understand control of the mitochondrial pyruvate dehydrogenase complex (PDC), total catalytic activity was determined during development of the primary leaves of pea (Pisum sativum L.) seedlings, as well as in each leaf pair of 21-day-old plants. Activity of the PDC in clarified homogenates was highest in the youngest organs and then dropped dramatically as the leaves matured and became photosynthetically competent. As leaves began to senesce, total PDC activity dropped to zero. Steady-state mRNA levels were determined using E1 and E3 cDNA probes. The overall pattern of transcript abundance matched the pattern observed for total PDC activity; transcript levels for E1alpha and E1beta approached zero during senescence. Levels of the E1alpha, E1beta, E2 and E3 subunits of the PDC were analyzed in the same samples, using specific antibodies. Quantitation of the immunoblotting results throughout this developmental series showed a pattern in parallel with that of catalytic activity and mRNA levels, although the relative changes in subunit protein levels were not as extreme as the changes in activity. The exception to the global pattern was that of the E3 subunit: lipoamide dehydrogenase. Expression of this enzyme was highest in mature, fully expanded leaves, which were active in photosynthesis and photorespiration, reflecting the additional role of E3 as a component of glycine decarboxylase.