Oligonucleotide microarrays, also called "DNA chips," are currently made by a light-directed chemistry that requires a large number of photolithographic masks for each chip. Here we describe a maskless array synthesizer (MAS) that replaces the chrome masks with virtual masks generated on a computer, which are relayed to a digital micromirror array. A 1:1 reflective imaging system forms an ultraviolet image of the virtual mask on the active surface of the glass substrate, which is mounted in a flow cell reaction chamber connected to a DNA synthesizer. Programmed chemical coupling cycles follow light exposure, and these steps are repeated with different virtual masks to grow desired oligonucleotides in a selected pattern. This instrument has been used to synthesize oligonucleotide microarrays containing more than 76,000 features measuring 16 microm 2. The oligonucleotides were synthesized at high repetitive yield and, after hybridization, could readily discriminate single-base pair mismatches. The MAS is adaptable to the fabrication of DNA chips containing probes for thousands of genes, as well as any other solid-phase combinatorial chemistry to be performed in high-density microarrays.

We applied 15 N labeling approaches to leaves of the Arabidopsis thaliana rosette to characterize their protein degradation rate and understand its determinants. The progressive labeling of new peptides with 15 N and measuring the decrease in the abundance of >60,000 existing peptides over time allowed us to define the degradation rate of 1228 proteins in vivo. We show that Arabidopsis protein half-lives vary from several hours to several months based on the exponential constant of the decay rate for each protein. This rate was calculated from the relative isotope abundance of each peptide and the fold change in protein abundance during growth. Protein complex membership and specific protein domains were found to be strong predictors of degradation rate, while N-end amino acid, hydrophobicity, or aggregation propensity of proteins were not. We discovered rapidly degrading subunits in a variety of protein complexes in plastids and identified the set of plant proteins whose degradation rate changed in different leaves of the rosette and correlated with leaf growth rate. From this information, we have calculated the protein turnover energy costs in different leaves and their key determinants within the proteome.

Proteases usually cleave peptides, but under some conditions, they can ligate them. Seeds of the common sunflower contain the 14-residue, backbone-macrocyclic peptide sunflower trypsin inhibitor 1 (SFTI-1) whose maturation from its precursor has a genetic requirement for asparaginyl endopeptidase (AEP). To provide more direct evidence, we developed an in situ assay and used (18)O-water to demonstrate that SFTI-1 is excised and simultaneously macrocyclized from its linear precursor. The reaction is inefficient in situ, but a newfound breakdown pathway can mask this inefficiency by reducing the internal disulfide bridge of any acyclic-SFTI to thiols before degrading it. To confirm AEP can directly perform the excision/ligation, we produced several recombinant plant AEPs in E. coli, and one from jack bean could catalyze both a typical cleavage reaction and cleavage-dependent, intramolecular transpeptidation to create SFTI-1. We propose that the evolution of ligating endoproteases enables plants like sunflower and jack bean to stabilize bioactive peptides.

Protein turnover is a key component in cellular homeostasis; however, there is little quantitative information on degradation kinetics for individual plant proteins. We have used 15 N labeling of barley (Hordeum vulgare) plants and gas chromatography-mass spectrometry analysis of free amino acids and liquid chromatography-mass spectrometry analysis of proteins to track the enrichment of 15 N into the amino acid pools in barley leaves and then into tryptic peptides derived from newly synthesized proteins. Using information on the rate of growth of barley leaves combined with the rate of degradation of 14 N-labeled proteins, we calculate the turnover rates of 508 different proteins in barley and show that they vary by more than 100-fold. There was approximately a 9-h lag from label application until 15 N incorporation could be reliably quantified in extracted peptides. Using this information and assuming constant translation rates for proteins during the time course, we were able to quantify degradation rates for several proteins that exhibit half-lives on the order of hours. Our workflow, involving a stringent series of mass spectrometry filtering steps, demonstrates that 15 N labeling can be used for large-scale liquid chromatography-mass spectrometry studies of protein turnover in plants. We identify a series of abundant proteins in photosynthesis, photorespiration, and specific subunits of chlorophyll biosynthesis that turn over significantly more rapidly than the average protein involved in these processes. We also highlight a series of proteins that turn over as rapidly as the well-known D1 subunit of photosystem II. While these proteins need further verification for rapid degradation in vivo, they cluster in chlorophyll and thiamine biosynthesis.

In recent years there has been growing interest in the posttranslational regulation of P-type ATPases by protein kinasemediated phosphorylation. Pma1 H ؉ -ATPase, which is responsible for H ؉ -dependent nutrient uptake in yeast (Saccharomyces cerevisiae), is one such example, displaying a rapid 5-10-fold increase in activity when carbon-starved cells are exposed to glucose. Activation has been linked to Ser/Thr phosphorylation in the C-terminal tail of the ATPase, but the specific phosphorylation sites have not previously been mapped. The present study has used nanoflow high pressure liquid chromatography coupled with electrospray electron transfer dissociation tandem mass spectrometry to identify Ser-911 and Thr-912 as two major phosphorylation sites that are clearly related to glucose activation. In carbon-starved cells with low Pma1 activity, peptide 896 -918, which was derived from the C terminus upon Lys-C proteolysis, was found to be singly phosphorylated at Thr-912, whereas in glucose-metabolizing cells with high ATPase activity, the same peptide was doubly phosphorylated at Ser-911 and Thr-912. Reciprocal 14 N/ 15 N metabolic labeling of cells was used to measure the relative phosphorylation levels at the two sites. The addition of glucose to carbon-starved cells led to a 3-fold reduction in the singly phosphorylated form and an 11-fold increase in the doubly phosphorylated form. These results point to a mechanism in which the stepwise phosphorylation of two tandemly positioned residues near the C terminus mediates glucose-dependent activation of the H ؉ -ATPase. Pma1 H ϩ

Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intronexon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions ''antisense'' to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage.higher plant ͉ transcriptome ͉ maskless array synthesizer

We report the first metabolic labeling of Arabidopsis thaliana for proteomic investigation, demonstrating efficient and complete labeling of intact plants. Using a reversed-database strategy, we evaluate the performance of the MASCOT search engine in the analysis of combined natural abundance and 15N-labeled samples. We find that 15N-metabolic labeling appears to increase the ambiguity associated with peptide identifications due in part to changes in the number of isobaric amino acids when the isotopic label is introduced. This is reflected by changes in the distributions of false positive identifications with respect to MASCOT score. However, by determining the nitrogen count from each pair of labeled and unlabeled peptides we may improve our confidence in both heavy and light identifications.

The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a The growth and development of plant tissues is associated with an ordered succession of cellular processes that are dictated by the appearance and disappearance of proteins and the transcripts that encode them (1-4). The ratio of the synthesis and degradation rates of these molecules, whether they are in quasi-steady state or are rapidly changing in abundance, defines both the net turnover rate and the abundance of each (5). The control of the kinetics of these processes is central to how plants can rapidly alter specific protein abundance and thus molecular function to respond to environmental or developmental cues.Genome wide analysis of Arabidopsis mRNA turnover rates has confirmed that knowledge of transcript decay rates can provide insights into diverse biological processes (6). For example, the number of introns and sequence elements in the 3Ј-untranslated region and subcellular localization of the encoded protein affect the turnover rate of transcripts in Arabidopsis (6). Analysis of plant proteome synthesis and degradation has lagged considerably from our understanding of these processes in the transcriptome.Many methods have been developed to measure protein turnover in other organisms. Some are direct measurements of endogenous proteins using isotope labeling methods including both radioactive and stable isotope labeling (5, 7-10), whereas others use stable or transient transgenic techniques and a range of tags and markers (11,12). The clearest advantage of isotope labeling approaches is that the tags are very subtle with little or no impact on cellular processes and allow the fully functional proteins being assessed to be produced and distributed within cells in a normal context. The advent of mass spectrometry as a key tool in proteomics has provided a means to use enrichment of the natural abundance of stable isotopes to provide mass rather than radio decay signals to track the synthesis of new proteins. The ratio between light and heavy isotopes and the degrees of enrichment provided by mass spectrometry provides a powerful means to measure synthesis and degradation rates of individual proteins (5, 13).Stable isotope labeling using individual amino acids (SILAC) 1 has proven highly successful in mammalian cell culture systems (14). SILAC has also been used to measure protein turnover in yeast but required the use of auxotrophic mutants (5). However, N-methyl-N-(tert-butyldimethylsilyl...