Integrating Discovery-driven Proteomics and Selected Reaction Monitoring To Develop a Noninvasive Assay for Geoduck Reproductive Maturation

Geoduck clams (<i>Panopea generosa</i>) are an increasingly important fishery and aquaculture product along the eastern Pacific coast from Baja California, Mexico, to Alaska. These long-lived clams are highly fecund, although sustainable hatchery production of genetically diverse larvae is hindered by the lack of sexual dimorphism, resulting in asynchronous spawning of broodstock, unequal sex ratios, and low numbers of breeders. The development of assays of gonad physiology could indicate sex and maturation stage as well as be used to assess the status of natural populations. Proteomic profiles were determined for three reproductive maturation stages in both male and female clams using data-dependent acquisition (DDA) of gonad proteins. Gonad proteomes became increasingly divergent between males and females as maturation progressed. The DDA data were used to develop targets analyzed with selected reaction monitoring (SRM) in gonad tissue as well as hemolymph. The SRM assay yielded a suite of indicator peptides that can be used as an efficient assay to determine geoduck gonad maturation status. Application of SRM in hemolymph samples demonstrates that this procedure could effectively be used to assess reproductive status in marine mollusks in a nonlethal manner

Venn diagram of number of proteins identified in Fe-replete and Fe-limited <i>T. pseudonana</i>.

<p>Proteins results presented were confidently identified from triplicate PAcIFIC analyses on the LTQ-VELOS. Fe-replete (blue) and Fe-limited (red) conditions were harvested at mid-exponential growth phase after acclimation. Numbers parenthetically annotated indicate homologous protein identifications.</p

Total peptide spectral counts from photosystem complex subunits.

<p>Spectral counts result from quadruplicate analyses on Fe-replete (blue) and Fe-limited (red) cultures. Photosystem II requires 2–3 atoms of Fe per complex, Cytochrome (Cyt) <i>b₆f</i> complex requires 6 Fe atoms per complex, and photosystem I requires 12 Fe atoms per complex. “*” indicates that the protein was determined to be significantly up- or down-regulated by QSpec (i.e. Bayes Factor >10 and log₂ fold change >0.5).</p

Diatom Proteomics Reveals Unique Acclimation Strategies to Mitigate Fe Limitation

<div><p>Phytoplankton growth rates are limited by the supply of iron (Fe) in approximately one third of the open ocean, with major implications for carbon dioxide sequestration and carbon (C) biogeochemistry. To date, understanding how alteration of Fe supply changes phytoplankton physiology has focused on traditional metrics such as growth rate, elemental composition, and biophysical measurements such as photosynthetic competence (F_v/F_m). Researchers have subsequently employed transcriptomics to probe relationships between changes in Fe supply and phytoplankton physiology. Recently, studies have investigated longer-term (i.e. following acclimation) responses of phytoplankton to various Fe conditions. In the present study, the coastal diatom, <i>Thalassiosira pseudonana</i>, was acclimated (10 generations) to either low or high Fe conditions, i.e. Fe-limiting and Fe-replete. Quantitative proteomics and a newly developed proteomic profiling technique that identifies low abundance proteins were employed to examine the full complement of expressed proteins and consequently the metabolic pathways utilized by the diatom under the two Fe conditions. A total of 1850 proteins were confidently identified, nearly tripling previous identifications made from differential expression in diatoms. Given sufficient time to acclimate to Fe limitation, <i>T. pseudonana</i> up-regulates proteins involved in pathways associated with intracellular protein recycling, thereby decreasing dependence on extracellular nitrogen (N), C and Fe. The relative increase in the abundance of photorespiration and pentose phosphate pathway proteins reveal novel metabolic shifts, which create substrates that could support other well-established physiological responses, such as heavily silicified frustules observed for Fe-limited diatoms. Here, we discovered that proteins and hence pathways observed to be down-regulated in short-term Fe starvation studies are constitutively expressed when <i>T. pseudonana</i> is acclimated (i.e., nitrate and nitrite transporters, Photosystem II and Photosystem I complexes). Acclimation of the diatom to the desired Fe conditions and the comprehensive proteomic approach provides a more robust interpretation of this dynamic proteome than previous studies.</p></div

Spectral count data and QSpec statistical analyses for key nitrogen metabolism, urea cycle, and spermine synthesis proteins identified in all experiments.

<p>Each replicate analysis (i.e. 1, 2, 3, 4) is indicative of the summation of spectral counts for that particular protein identified in 4 gas phase fractions (GPFs). Spectral counts resulting from triplicate analytical cycles of PAcIFIC were added together to provide a final count for the PAcIFIC analysis. In order to be considered significantly up or down regulated two criteria were met by QSpec: Bayes factor >10, and log₂(−Fe/+Fe)>0.5. Proteins that do not have QSpec information were only identified using the data-independent PAcIFIC method and could not be statistically evaluated. This list does not include all proteins involved in nitrogen metabolism (e.g. amino acids biosynthesis and degradation).</p

Biological processes up-regulated in Fe-replete <i>T. pseudonana</i> as reported by DAVID Biological Process Term level 4 analysis of gene ontology categories.

<p>Count: total proteins up-regulated in Fe-replete cultures that correlated with the Biological Process; %: Percent of total proteins associated with that term; P-value: probability that the number of proteins identified to be up-regulated from that biological process is significant with respect to the total number of proteins from the <i>T. pseudonana</i> proteome associated with that process (reported p-value threshold <0.05).</p

Cartoon representation of diatom cell biochemistry when acclimated to Fe-limitation.

<p>Not all metabolic pathways are shown. Black and grey pathways and proteins indicate presence during Fe-limitation, white proteins indicate significantly down-regulated proteins, colored pathways were significantly up-regulated in Fe-limited cells compared to Fe-replete cells. A) Pentose Phosphate Pathway, B) The rejoining of the pentose phosphate pathway with glycolysis and generation of pyruvate. C) Polyamine synthesis using spermine synthase. D) Proposed reduction of Fe⁺³ and eventual transport of Fe into cells. Purple: photosynthesis and glycolysis-gluconeogenesis, red: nucleotide metabolism, teal: lipid metabolism, orange: amino acid metabolism.</p

Methodological details from some recent illustrative studies examining “omics” of Fe limitation on diatoms revealing the wide range of methodological approaches that have been employed.

<p>qRT-PCR = quantitative reverse transcription polymerase chain reaction. 2D SDS PAGE LC/MS/MS = 2 dimensional SDS PAGE gel electrophoresis followed by tandem mass spectrometry protein identifications on individual gel spots.</p

Metabolic biochemistry map of proteins expressed and identified in Fe-limited <i>T. pseudonana</i>.

<p>Map includes data from triplicate PAcIFIC analyses on a tandem mass spectrometer from <i>Thalassiosira pseudonana</i> acclimated to Fe-limitation. Each node (or corner) represents a metabolite and the lines connecting the nodes represent an enzyme (i.e. protein). Metabolites were not measured in this study. Proteins that were identified in both Fe-replete and Fe-limited cultures are highlighted in grey. Proteins that were identified to be unique to Fe-limited cultures are indicated in color. From top left – light blue: sugar and glycan biosynthesis, light purple: starch and sucrose metabolism (including photosynthesis, oxidative phosphorylation, carbon fixation), dark purple: glycolysis-gluconeogenesis (including TCA cycle), red: nucleotide metabolism, teal: lipid metabolism, orange: amino acid metabolism (including urea cycle).</p

Metabolic biochemistry map of proteins expressed and identified in Fe-replete <i>T. pseudonana</i>.

<p>Map includes data from triplicate PAcIFIC analyses on a tandem mass spectrometer from <i>Thalassiosira pseudonana</i> acclimated to Fe-replete conditions. Each node (or corner) represents a metabolite and the lines connecting the nodes represent an enzyme (i.e. protein). Metabolites were not measured in this study. Proteins that were identified in both Fe-replete and Fe-limited cultures are highlighted in grey. Proteins that were identified to be unique to the Fe-replete cultures are indicated in color. From top left – light blue: sugar and glycan biosynthesis, light purple: starch and sucrose metabolism (including photosynthesis, oxidative phosphorylation, carbon fixation), dark purple: glycolysis-gluconeogenesis (including TCA cycle), red: nucleotide metabolism, teal: lipid metabolism, orange: amino acid metabolism (including urea cycle).</p