AutoCCS: automated collision cross-section calculation software for ion mobility spectrometry-mass spectrometry.

MOTIVATION: Ion mobility spectrometry (IMS) separations are increasingly used in conjunction with mass spectrometry (MS) for separation and characterization of ionized molecular species. Information obtained from IMS measurements includes the ion's collision cross section (CCS), which reflects its size and structure and constitutes a descriptor for distinguishing similar species in mixtures that cannot be separated using conventional approaches. Incorporating CCS into MS-based workflows can improve the specificity and confidence of molecular identification. At present, there is no automated, open-source pipeline for determining CCS of analyte ions in both targeted and untargeted fashion, and intensive user-assisted processing with vendor software and manual evaluation is often required. RESULTS: We present AutoCCS, an open-source software to rapidly determine CCS values from IMS-MS measurements. We conducted various IMS experiments in different formats to demonstrate the flexibility of AutoCCS for automated CCS calculation: (i) stepped-field methods for drift tube-based IMS (DTIMS), (ii) single-field methods for DTIMS (supporting two calibration methods: a standard and a new enhanced method) and (iii) linear calibration for Bruker timsTOF and non-linear calibration methods for traveling wave based-IMS in Waters Synapt and Structures for Lossless Ion Manipulations. We demonstrated that AutoCCS offers an accurate and reproducible determination of CCS for both standard and unknown analyte ions in various IMS-MS platforms, IMS-field methods, ionization modes and collision gases, without requiring manual processing. AVAILABILITY AND IMPLEMENTATION: https://github.com/PNNL-Comp-Mass-Spec/AutoCCS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. Demo datasets are publicly available at MassIVE (Dataset ID: MSV000085979).

Top-down mass spectrometry of histone modifications in sorghum reveals potential epigenetic markers for drought acclimation.

Sorghum [Sorghum bicolor (L.) Moench] is an important cereal crop noted for its ability to survive water-limiting conditions. Herein, we present an analytical workflow to explore the changes in histone modifications through plant developmental stages and two drought stresses in two sorghum genotypes that differ in their response to drought. Top-down mass spectrometry (MS) is an ideal method to profile histone modifications and distinguish closely related histone proteoforms. We analyzed leaves of 48 plants and identified 26 unique histone proteins and 677 unique histone proteoforms (124 full-length and 553 truncated proteoforms). We detected trimethylation on nearly all H2B N-termini where acetylation is commonly expected. In addition, an unexpected modification on H2A histones was assigned to N-pyruvic acid 2-iminylation based on its unique neutral loss of CO2. Interestingly, some of the truncated histones, in particular H4 and H3.2, showed significant changes that correlated with the growth and water conditions. The histone proteoforms could serve as targets in search of chromatin modifiers and ultimately have important ramifications in future attempts of studying plant epigenetic reprogramming under stress.

Drought delays development of the sorghum root microbiome and enriches for monoderm bacteria.

Drought stress is a major obstacle to crop productivity, and the severity and frequency of drought are expected to increase in the coming century. Certain root-associated bacteria have been shown to mitigate the negative effects of drought stress on plant growth, and manipulation of the crop microbiome is an emerging strategy for overcoming drought stress in agricultural systems, yet the effect of drought on the development of the root microbiome is poorly understood. Through 16S rRNA amplicon and metatranscriptome sequencing, as well as root metabolomics, we demonstrate that drought delays the development of the early sorghum root microbiome and causes increased abundance and activity of monoderm bacteria, which lack an outer cell membrane and contain thick cell walls. Our data suggest that altered plant metabolism and increased activity of bacterial ATP-binding cassette (ABC) transporter genes are correlated with these shifts in community composition. Finally, inoculation experiments with monoderm isolates indicate that increased colonization of the root during drought can positively impact plant growth. Collectively, these results demonstrate the role that drought plays in restructuring the root microbiome and highlight the importance of temporal sampling when studying plant-associated microbiomes.

Reduced Arogenate Dehydratase Expression: Ramifications for Photosynthesis and Metabolism.

Arogenate dehydratase (ADT) catalyzes the final step of phenylalanine (Phe) biosynthesis. Previous work showed that ADT-deficient Arabidopsis (Arabidopsis thaliana) mutants had significantly reduced lignin contents, with stronger reductions in lines that had deficiencies in more ADT isoforms. Here, by analyzing Arabidopsis ADT mutants using our phenomics facility and ultra-performance liquid chromatography-mass spectrometry-based metabolomics, we describe the effects of the modulation of ADT on photosynthetic parameters and secondary metabolism. Our data indicate that a reduced carbon flux into Phe biosynthesis in ADT mutants impairs the consumption of photosynthetically produced ATP, leading to an increased ATP/ADP ratio, the overaccumulation of transitory starch, and lower electron transport rates. The effect on electron transport rates is caused by an increase in proton motive force across the thylakoid membrane that down-regulates photosystem II activity by the high-energy quenching mechanism. Furthermore, quantitation of secondary metabolites in ADT mutants revealed reduced flavonoid, phenylpropanoid, lignan, and glucosinolate contents, including glucosinolates that are not derived from aromatic amino acids, and significantly increased contents of putative galactolipids and apocarotenoids. Additionally, we used real-time atmospheric monitoring mass spectrometry to compare respiration and carbon fixation rates between the wild type and adt3/4/5/6, our most extreme ADT knockout mutant, which revealed no significant difference in both night- and day-adapted plants. Overall, these data reveal the profound effects of altered ADT activity and Phe metabolism on secondary metabolites and photosynthesis with implications for plant improvement.

Spatially Resolved Proteome Profiling of <200 Cells from Tomato Fruit Pericarp by Integrating Laser-Capture Microdissection with Nanodroplet Sample Preparation.

Due to sensitivity limitations, global proteome measurements generally require large amounts of biological starting material, which masks heterogeneity within the samples and differential protein expression among constituent cell types. Methods for spatially resolved proteomics are being developed to resolve protein expression for distinct cell types among highly heterogeneous tissues, but have primarily been applied to mammalian systems. Here we evaluate the performance of cell-type-specific proteome analysis of tomato fruit pericarp tissues by a platform integrating laser-capture microdissection (LCM) and a recently developed automated sample preparation system (nanoPOTS, nanodroplet processing in one pot for trace samples). Tomato fruits were cryosectioned prior to LCM and tissues were dissected and captured directly into nanoPOTS chips for processing. Following processing, samples were analyzed by nanoLC-MS/MS. Approximately 1900 unique peptides and 422 proteins were identified on average from ∼0.04 mm2 tissues comprising ∼8-15 parenchyma cells. Spatially resolved proteome analyses were performed using cells of outer epidermis, collenchyma, and parenchyma. Using ≤200 cells, a total of 1,870 protein groups were identified and the various tissues were easily resolved. The results provide spatial and tissue-specific insights into key enzymes and pathways involved in carbohydrate transport and source-sink relationships in tomato fruit. Of note, at the time of fruit ripening studied here, we identified differentially abundant proteins throughout the pericarp related to chlorophyll biogenesis, photosynthesis, and especially transport.

Multi-omic data integration links deleted in breast cancer 1 (DBC1) degradation to chromatin remodeling in inflammatory response.

This study investigated the dynamics of ubiquitinated proteins after the inflammatory stimulation of RAW 264.7 macrophage-like cells with bacterial lipopolysaccharide. Ubiquitination is a common protein post-translational modification that regulates many key cellular functions. We demonstrated that levels of global ubiquitination and K48 and K63 polyubiquitin chains change after lipopolysaccharide stimulation. Quantitative proteomic analysis identified 1199 ubiquitinated proteins, 78 of which exhibited significant changes in ubiquitination levels following stimulation. Integrating the ubiquitinome data with global proteomic and transcriptomic results allowed us to identify a subset of 88 proteins that were targeted for degradation after lipopolysaccharide stimulation. Using cellular assays and Western blot analyses, we biochemically validated DBC1 (a histone deacetylase inhibitor) as a degradation substrate that is targeted via an orchestrated mechanism utilizing caspases and the proteasome. The degradation of DBC1 releases histone deacetylase activity, linking lipopolysaccharide activation to chromatin remodeling in caspase- and proteasome-mediated signaling.

Proteome of Geobacter sulfurreducens in the presence of U(VI).

Geobacter species often play an important role in the in situ bioremediation of uranium-contaminated groundwater, but little is known about how these microbes avoid uranium toxicity. To evaluate this further, the proteome of Geobacter sulfurreducens exposed to 100 µM U(VI) acetate was compared to control cells not exposed to U(VI). Of the 1363 proteins detected from these cultures, 203 proteins had higher abundance during exposure to U(VI) compared with the control cells and 148 proteins had lower abundance. U(VI)-exposed cultures expressed lower levels of proteins involved in growth, protein and amino acid biosynthesis, as well as key central metabolism enzymes as a result of the deleterious effect of U(VI) on the growth of G. sulfurreducens. In contrast, proteins involved in detoxification, such as several efflux pumps belonging to the RND (resistance-nodulation-cell division) family, and membrane protection, and other proteins, such as chaperones and proteins involved in secretion systems, were found in higher abundance in cells exposed to U(VI). Exposing G. sulfurreducens to U(VI) resulted in a higher abundance of many proteins associated with the oxidative stress response, such as superoxide dismutase and superoxide reductase. A strain in which the gene for superoxide dismutase was deleted grew more slowly than the WT strain in the presence of U(VI), but not in its absence. The results suggested that there is no specific mechanism for uranium detoxification. Rather, multiple general stress responses are induced, which presumably enable Geobacter species to tolerate high uranium concentrations.

Quantitative phosphoproteomic analysis of soybean root hairs inoculated with Bradyrhizobium japonicum.

Root hairs are single hair-forming cells on roots that function to increase root surface area, enhancing water and nutrient uptake. In leguminous plants, root hairs also play a critical role as the site of infection by symbiotic nitrogen fixing rhizobia, leading to the formation of a novel organ, the nodule. The initial steps in the rhizobia-root hair infection process are known to involve specific receptor kinases and subsequent kinase cascades. Here, we characterize the phosphoproteome of the root hairs and the corresponding stripped roots (i.e. roots from which root hairs were removed) during rhizobial colonization and infection to gain insight into the molecular mechanism of root hair cell biology. We chose soybean (Glycine max L.), one of the most important crop plants in the legume family, for this study because of its larger root size, which permits isolation of sufficient root hair material for phosphoproteomic analysis. Phosphopeptides derived from root hairs and stripped roots, mock inoculated or inoculated with the soybean-specific rhizobium Bradyrhizobium japonicum, were labeled with the isobaric tag eight-plex iTRAQ, enriched using Ni-NTA magnetic beads and subjected to nanoRPLC-MS/MS1 analysis using HCD and decision tree guided CID/ETD strategy. A total of 1625 unique phosphopeptides, spanning 1659 nonredundant phosphorylation sites, were detected from 1126 soybean phosphoproteins. Among them, 273 phosphopeptides corresponding to 240 phosphoproteins were found to be significantly regulated (>1.5-fold abundance change) in response to inoculation with B. japonicum. The data reveal unique features of the soybean root hair phosphoproteome, including root hair and stripped root-specific phosphorylation suggesting a complex network of kinase-substrate and phosphatase-substrate interactions in response to rhizobial inoculation.

Pressurized pepsin digestion in proteomics: an automatable alternative to trypsin for integrated top-down bottom-up proteomics.

Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome.

Tripogon loliiformis tolerates rapid desiccation after metabolic and transcriptional priming during initial drying.

Crop plants and undomesticated resilient species employ different strategies to regulate their energy resources and growth. Most crop species are sensitive to stress and prioritise rapid growth to maximise yield or biomass production. In contrast, resilient plants grow slowly, are small, and allocate their resources for survival in challenging environments. One small group of plants, termed resurrection plants, survive desiccation of their vegetative tissue and regain full metabolic activity upon watering. However, the precise molecular mechanisms underlying this extreme tolerance remain unknown. In this study, we employed a transcriptomics and metabolomics approach, to investigate the mechanisms of desiccation tolerance in Tripogon loliiformis, a modified desiccation-tolerant plant, that survives gradual but not rapid drying. We show that T. loliiformis can survive rapid desiccation if it is gradually dried to 60% relative water content (RWC). Furthermore, the gene expression data showed that T. loliiformis is genetically predisposed for desiccation in the hydrated state, as evidenced by the accumulation of MYB, NAC, bZIP, WRKY transcription factors along with the phytohormones, abscisic acid, salicylic acid, amino acids (e.g., proline) and TCA cycle sugars during initial drying. Through network analysis of co-expressed genes, we observed differential responses to desiccation between T. loliiformis shoots and roots. Dehydrating shoots displayed global transcriptional changes across broad functional categories, although no enrichment was observed during drying. In contrast, dehydrating roots showed distinct network changes with the most significant differences occurring at 40% RWC. The cumulative effects of the early stress responses may indicate the minimum requirements of desiccation tolerance and enable T. loliiformis to survive rapid drying. These findings potentially hold promise for identifying biotechnological solutions aimed at developing drought-tolerant crops without growth and yield penalties.

Annotated genome sequence of a fast-growing diploid clone of red alder (Alnus rubra Bong.).

Red alder (Alnus rubra Bong.) is an ecologically significant and important fast-growing commercial tree species native to western coastal and riparian regions of North America, having highly desirable wood, pigment, and medicinal properties. We have sequenced the genome of a rapidly growing clone. The assembly is nearly complete, containing the full complement of expected genes. This supports our objectives of identifying and studying genes and pathways involved in nitrogen-fixing symbiosis and those related to secondary metabolites that underlie red alder's many interesting defense, pigmentation, and wood quality traits. We established that this clone is most likely diploid and identified a set of SNPs that will have utility in future breeding and selection endeavors, as well as in ongoing population studies. We have added a well-characterized genome to others from the order Fagales. In particular, it improves significantly upon the only other published alder genome sequence, that of Alnus glutinosa. Our work initiated a detailed comparative analysis of members of the order Fagales and established some similarities with previous reports in this clade, suggesting a biased retention of certain gene functions in the vestiges of an ancient genome duplication when compared with more recent tandem duplications.

Metabolomic, photoprotective, and photosynthetic acclimatory responses to post-flowering drought in sorghum.

Climate change is globally affecting rainfall patterns, necessitating the improvement of drought tolerance in crops. Sorghum bicolor is a relatively drought-tolerant cereal. Functional stay-green sorghum genotypes can maintain green leaf area and efficient grain filling during terminal post-flowering water deprivation, a period of ~10 weeks. To obtain molecular insights into these characteristics, two drought-tolerant genotypes, BTx642 and RTx430, were grown in replicated control and terminal post-flowering drought field plots in California's Central Valley. Photosynthetic, photoprotective, and water dynamics traits were quantified and correlated with metabolomic data collected from leaves, stems, and roots at multiple timepoints during control and drought conditions. Physiological and metabolomic data were then compared to longitudinal RNA sequencing data collected from these two genotypes. The unique metabolic and transcriptomic response to post-flowering drought in sorghum supports a role for the metabolite galactinol in controlling photosynthetic activity through regulating stomatal closure in post-flowering drought. Additionally, in the functional stay-green genotype BTx642, photoprotective responses were specifically induced in post-flowering drought, supporting a role for photoprotection in the molecular response associated with the functional stay-green trait. From these insights, new pathways are identified that can be targeted to maximize yields under growth conditions with limited water.

Zng1 is a GTP-dependent zinc transferase needed for activation of methionine aminopeptidase.

The evolution of zinc (Zn) as a protein cofactor altered the functional landscape of biology, but dependency on Zn also created an Achilles' heel, necessitating adaptive mechanisms to ensure Zn availability to proteins. A debated strategy is whether metallochaperones exist to prioritize essential Zn-dependent proteins. Here, we present evidence for a conserved family of putative metal transferases in human and fungi, which interact with Zn-dependent methionine aminopeptidase type I (MetAP1/Map1p/Fma1). Deletion of the putative metal transferase in Saccharomyces cerevisiae (ZNG1; formerly YNR029c) leads to defective Map1p function and a Zn-deficiency growth defect. In vitro, Zng1p can transfer Zn2+ or Co2+ to apo-Map1p, but unlike characterized copper chaperones, transfer is dependent on GTP hydrolysis. Proteomics reveal mis-regulation of the Zap1p transcription factor regulon because of loss of ZNG1 and Map1p activity, suggesting that Zng1p is required to avoid a compounding effect of Map1p dysfunction on survival during Zn limitation.

Omic data from evolved E. coli are consistent with computed optimal growth from genome-scale models.

After hundreds of generations of adaptive evolution at exponential growth, Escherichia coli grows as predicted using flux balance analysis (FBA) on genome-scale metabolic models (GEMs). However, it is not known whether the predicted pathway usage in FBA solutions is consistent with gene and protein expression in the wild-type and evolved strains. Here, we report that >98% of active reactions from FBA optimal growth solutions are supported by transcriptomic and proteomic data. Moreover, when E. coli adapts to growth rate selective pressure, the evolved strains upregulate genes within the optimal growth predictions, and downregulate genes outside of the optimal growth solutions. In addition, bottlenecks from dosage limitations of computationally predicted essential genes are overcome in the evolved strains. We also identify regulatory processes that may contribute to the development of the optimal growth phenotype in the evolved strains, such as the downregulation of known regulons and stringent response suppression. Thus, differential gene and protein expression from wild-type and adaptively evolved strains supports observed growth phenotype changes, and is consistent with GEM-computed optimal growth states.

Real-Time Mass Spectrometry Measurements of Respiration Rates in Biological Systems.

Many organisms process carbon and other nutrients to generate energy through aerobic respiration where organic carbon compounds are broken down and oxygen is consumed, producing carbon dioxide and water. Respiration is indicative of active metabolism, and respiration rates are proportional to the amount of living biomass in an ecosystem. Although there are many methods for measuring respiration rates in the laboratory, current systems, such as infrared gas analyzers, are limited in their ability to independently resolve isotopomer fluxes across a range of relevant gases including both CO2 and O2 in real-time. Therefore, monitoring of biological respiration in real time under controlled laboratory conditions would enable better understanding of cellular physiology. To address this challenge, we developed a real time mass spectrometry (RTMS) manifold that simultaneously measures production and consumption of multiple gases and their isotopologues in seconds with the speed and sensitivity necessary to characterize rapidly changing respiration events as they occur. This universal manifold can be fitted to a variety of instruments and affords the same analytical precision and accuracy of the instrument while allowing for the real time measurements. Here, we paired the manifold to a single quad MS with an electron impact (EI) source operated in scan mode to detect extracted target gases by their respective masses (e.g., 12CO2 at mass 44, 13CO2 at 45). We demonstrated applicability of the RTMS instrument to different biological ecosystems (bacterial cultures, plants, and soil), and in all cases, we were able to detect simultaneous and rapid measurements of multiple gases in real time, providing novel insights into complex respiratory metabolism and the influence of biological and environmental factors.

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers.

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

New Insights Into Lignification via Network and Multi-Omics Analyses of Arogenate Dehydratase Knock-Out Mutants in Arabidopsis thaliana.

Multiple Arabidopsis arogenate dehydratase (ADT) knock-out (KO) mutants, with phenotypes having variable lignin levels (up to circa 70% reduction), were studied to investigate how differential reductions in ADTs perturb its overall plant systems biology. Integrated "omics" analyses (metabolome, transcriptome, and proteome) of wild type (WT), single and multiple ADT KO lines were conducted. Transcriptome and proteome data were collapsed into gene ortholog (GO) data, with this allowing for enzymatic reaction and metabolome cross-comparisons to uncover dominant or likely metabolic biosynthesis reactions affected. Network analysis of enzymes-highly correlated to stem lignin levels-deduced the involvement of novel putative lignin related proteins or processes. These included those associated with ribosomes, the spliceosome, mRNA transport, aminoacyl tRNA biosynthesis, and phosphorylation. While prior work helped explain lignin biosynthesis regulation at the transcriptional level, our data here provide support for a new hypothesis that there are additional post-transcriptional and translational level processes that need to be considered. These findings are anticipated to lead to development of more accurate depictions of lignin/phenylpropanoid biosynthesis models in situ, with new protein targets identified for further biochemical analysis and/or plant bioengineering. Additionally, using KEGG defined functional categorization of proteomics and transcriptomics analyses, we detected significant changes to glucosinolate, α-linolenic acid, nitrogen, carotenoid, aromatic amino acid, phenylpropanoid, and photosynthesis-related metabolic pathways in ADT KO mutants. Metabolomics results also revealed that putative carotenoid and galactolipid levels were generally increased in amount, whereas many glucosinolates and phenylpropanoids (including flavonoids and lignans) were decreased in the KO mutants.

Deciphering the microbial and molecular responses of geographically diverse Setaria accessions grown in a nutrient-poor soil.

The microbial and molecular characterization of the ectorhizosphere is an important step towards developing a more complete understanding of how the cultivation of biofuel crops can be undertaken in nutrient poor environments. The ectorhizosphere of Setaria is of particular interest because the plant component of this plant-microbe system is an important agricultural grain crop and a model for biofuel grasses. Importantly, Setaria lends itself to high throughput molecular studies. As such, we have identified important intra- and interspecific microbial and molecular differences in the ectorhizospheres of three geographically distant Setaria italica accessions and their wild ancestor S. viridis. All were grown in a nutrient-poor soil with and without nutrient addition. To assess the contrasting impact of nutrient deficiency observed for two S. italica accessions, we quantitatively evaluated differences in soil organic matter, microbial community, and metabolite profiles. Together, these measurements suggest that rhizosphere priming differs with Setaria accession, which comes from alterations in microbial community abundances, specifically Actinobacteria and Proteobacteria populations. When globally comparing the metabolomic response of Setaria to nutrient addition, plants produced distinctly different metabolic profiles in the leaves and roots. With nutrient addition, increases of nitrogen containing metabolites were significantly higher in plant leaves and roots along with significant increases in tyrosine derived alkaloids, serotonin, and synephrine. Glycerol was also found to be significantly increased in the leaves as well as the ectorhizosphere. These differences provide insight into how C4 grasses adapt to changing nutrient availability in soils or with contrasting fertilization schemas. Gained knowledge could then be utilized in plant enhancement and bioengineering efforts to produce plants with superior traits when grown in nutrient poor soils.

Analyzing protease specificity and detecting in vivo proteolytic events using tandem mass spectrometry.

Although trypsin remains the most commonly used protease in MS, other proteases may be employed for increasing peptide coverage or generating overlapping peptides. Knowledge of the accurate specificity rules of these proteases is helpful for database search tools to detect peptides, and becomes crucial when label-free MS is used to discover in vivo proteolytic cleavages. Since in vivo cleavages are inferred by subtracting digestion-induced cleavages from all observed cleavages, it is important to ensure that the specificity rule used to identify digestion-induced cleavages are broad enough to capture even minor cleavages produced in digestion, to avoid erroneously identifying them as in vivo cleavages. In this study, we describe MS-Proteolysis, a software tool for identifying putative sites of in vivo proteolytic cleavage using label-free MS. The tool is used in conjunction with digestion by trypsin and three other proteases, whose specificity rules are revised and extended before inferring proteolytic cleavages. Finally, we show that comparative analysis of multiple proteases can be used to detect putative in vivo proteolytic sites on a proteome-wide scale.

The Importance of Protein Phosphorylation for Signaling and Metabolism in Response to Diel Light Cycling and Nutrient Availability in a Marine Diatom.

Diatoms are major contributors to global primary production and their populations in the modern oceans are affected by availability of iron, nitrogen, phosphate, silica, and other trace metals, vitamins, and infochemicals. However, little is known about the role of phosphorylation in diatoms and its role in regulation and signaling. We report a total of 2759 phosphorylation sites on 1502 proteins detected in Phaeodactylum tricornutum. Conditionally phosphorylated peptides were detected at low iron (n = 108), during the diel cycle (n = 149), and due to nitrogen availability (n = 137). Through a multi-omic comparison of transcript, protein, phosphorylation, and protein homology, we identify numerous proteins and key cellular processes that are likely under control of phospho-regulation. We show that phosphorylation regulates: (1) carbon retrenchment and reallocation during growth under low iron, (2) carbon flux towards lipid biosynthesis after the lights turn on, (3) coordination of transcription and translation over the diel cycle and (4) in response to nitrogen depletion. We also uncover phosphorylation sites for proteins that play major roles in diatom Fe sensing and utilization, including flavodoxin and phytotransferrin (ISIP2A), as well as identify phospho-regulated stress proteins and kinases. These findings provide much needed insight into the roles of protein phosphorylation in diel cycling and nutrient sensing in diatoms.