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. 2011 Sep;10(9):M110.004739.
doi: 10.1074/mcp.M110.004739. Epub 2011 May 24.

Quantitative shotgun proteomics using a uniform ¹⁵N-labeled standard to monitor proteome dynamics in time course experiments reveals new insights into the heat stress response of Chlamydomonas reinhardtii

Timo Mühlhaus  1 , Julia Weiss, Dorothea Hemme, Frederik Sommer, Michael Schroda
Affiliations

Quantitative shotgun proteomics using a uniform ¹⁵N-labeled standard to monitor proteome dynamics in time course experiments reveals new insights into the heat stress response of Chlamydomonas reinhardtii

Timo Mühlhaus et al. Mol Cell Proteomics. 2011 Sep.

Abstract

Crop-plant-yield safety is jeopardized by temperature stress caused by the global climate change. To take countermeasures by breeding and/or transgenic approaches it is essential to understand the mechanisms underlying plant acclimation to heat stress. To this end proteomics approaches are most promising, as acclimation is largely mediated by proteins. Accordingly, several proteomics studies, mainly based on two-dimensional gel-tandem MS approaches, were conducted in the past. However, results often were inconsistent, presumably attributable to artifacts inherent to the display of complex proteomes via two-dimensional-gels. We describe here a new approach to monitor proteome dynamics in time course experiments. This approach involves full ¹⁵N metabolic labeling and mass spectrometry based quantitative shotgun proteomics using a uniform ¹⁵N standard over all time points. It comprises a software framework, IOMIQS, that features batch job mediated automated peptide identification by four parallelized search engines, peptide quantification and data assembly for the processing of large numbers of samples. We have applied this approach to monitor proteome dynamics in a heat stress time course using the unicellular green alga Chlamydomonas reinhardtii as model system. We were able to identify 3433 Chlamydomonas proteins, of which 1116 were quantified in at least three of five time points of the time course. Statistical analyses revealed that levels of 38 proteins significantly increased, whereas levels of 206 proteins significantly decreased during heat stress. The increasing proteins comprise 25 (co-)chaperones and 13 proteins involved in chromatin remodeling, signal transduction, apoptosis, photosynthetic light reactions, and yet unknown functions. Proteins decreasing during heat stress were significantly enriched in functional categories that mediate carbon flux from CO₂ and external acetate into protein biosynthesis, which also correlated with a rapid, but fully reversible cell cycle arrest after onset of stress. Our approach opens up new perspectives for plant systems biology and provides novel insights into plant stress acclimation.

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Figures

Fig. 1.
Fig. 1.
Schemes for the preparation of 15N-labeled reference cells, heat stress sampling, sample processing, and data evaluation. A, To prepare a 15N-labeled reference, a Chlamydomonas cell culture was grown for at least 10 generations in medium containing 15NH4Cl as nitrogen source and incubated in a 42 °C water bath. One thirds of the culture were harvested just before and 60 min and 180 min after shifting to 42 °C and pooled. This procedure ensured that proteins absent before or after heat stress were present in the final reference (red spike). Heat stress kinetics were performed by incubating cells grown in medium containing 14NH4Cl as nitrogen source in a 42 °C water bath and sampling shortly before and 30, 60, 120, and 180 min after shifting to 42 °C. B, 15N-labeled reference cells (indicated by the red protein) were mixed with unlabeled samples (indicated by the blue protein) before protein extraction. Soluble proteins prepared by freeze/thaw cycles were acetone-precipitated, tryptically digested and analyzed by nanoLC-ESI-MS/MS. Depending on which protein was more abundant in the sample (heavy or light) peptide identification by MS2 was preferentially based on heavy (red) or light (blue) peptides. Peptide identification by four search engines in parallel, quantification of heavy and light peptides by XPRESS, integration of quantification values at the protein level and export to R! for visualization was managed by the IOMIQS framework.
Fig. 2.
Fig. 2.
Overview of peptides and proteins identified and quantified in this study. A, Venn diagram of spectra-to-peptide mappings by search engines Sequest, X!Tandem, OMSSA and Mascot. MS2 spectra were taken from 90 raw files. B, Stacked Venn diagram showing the fractions of identified and quantified proteins from the total protein-coding gene models present in V3.1 of the Chlamydomonas genome.
Fig. 3.
Fig. 3.
Comparison of chaperone expression kinetics by quantitative immunoblotting and quantitative shotgun proteomics. Accumulation of cytosolic HSP70A (A), chloroplast HSP70B (B), cytosolic HSP90A (C), and chloroplast HSP90C (D) during heat shock as measured by immunoblot analysis (green curves) or quantitative shotgun proteomics (red curves). Values from immunoblot analyses were derived from ECL signals from the chaperones normalized to the signals from CF1β. Mean values and standard deviations derive from 3 biological and 2–4 technical replicates. Shown are Z-score transformed values to facilitate comparison of protein kinetics.
Fig. 4.
Fig. 4.
Kinetics of proteins exhibiting significant changes during heat stress. Kinetics of all 244 proteins significantly changing during heat stress are plotted bin-wise as percent of the maximal value in the time course. Values and standard deviations derive from three biological and two technical replicates (listed in supplemental Table S3). A, Small heat shock proteins. B, HSP100s. C, HSP90 and co-chaperones. D, HSP70 and co-chaperones. E, HSP60s. F, FKBPs/Cyclophilins. G, Protein folding and abiotic stress. H, Proteases/Peptidases. I, Protein biosynthesis (LSU cytosol). J, Protein biosynthesis (SSU cytosol). K, Protein biosynthesis (LSU plastid). L, Protein biosynthesis (not ribosome). M, N-metabolism. N, Amino acid metabolism. (O) Proteins associated with PS I. P, Proteins associated with PS II. Q, Other proteins associated with light reactions. R, Porphyrin biogenesis. S, Vitamin biosynthesis. T, Proteins of photosynthetic and photorespiratory carbon metabolism. U, Starch synthesis and degradation. V, Glycolysis and gluconeogenesis. W, TCA cycle. X, Oxidative pentose phosphate pathway. Y, Acetate metabolism/glyoxylate cycle. Z, Fermentation. AA, Glycerolipid metabolism. AB, Mitochondrial electron transfer chain and ATP synthase. AC, Nucleotide metabolism. AD, Sugar nucleotide metabolism. AE, Metal handling. AF, S-assimilation. AG, C1-metabolism. AH, Redox regulation/oxidative stress response. AI, Signal transduction. AJ, DNA binding/transcription/chromatin remodeling. AK, RNA processing/binding. AL, Vesicle transport. AM, Pumps and transporters. AN, Protein sorting. AO, Flagellar and basal body proteins. AP, Miscellaneous. AQ, Proteins without functional annotation.
Fig. 4.
Fig. 4.
Kinetics of proteins exhibiting significant changes during heat stress. Kinetics of all 244 proteins significantly changing during heat stress are plotted bin-wise as percent of the maximal value in the time course. Values and standard deviations derive from three biological and two technical replicates (listed in supplemental Table S3). A, Small heat shock proteins. B, HSP100s. C, HSP90 and co-chaperones. D, HSP70 and co-chaperones. E, HSP60s. F, FKBPs/Cyclophilins. G, Protein folding and abiotic stress. H, Proteases/Peptidases. I, Protein biosynthesis (LSU cytosol). J, Protein biosynthesis (SSU cytosol). K, Protein biosynthesis (LSU plastid). L, Protein biosynthesis (not ribosome). M, N-metabolism. N, Amino acid metabolism. (O) Proteins associated with PS I. P, Proteins associated with PS II. Q, Other proteins associated with light reactions. R, Porphyrin biogenesis. S, Vitamin biosynthesis. T, Proteins of photosynthetic and photorespiratory carbon metabolism. U, Starch synthesis and degradation. V, Glycolysis and gluconeogenesis. W, TCA cycle. X, Oxidative pentose phosphate pathway. Y, Acetate metabolism/glyoxylate cycle. Z, Fermentation. AA, Glycerolipid metabolism. AB, Mitochondrial electron transfer chain and ATP synthase. AC, Nucleotide metabolism. AD, Sugar nucleotide metabolism. AE, Metal handling. AF, S-assimilation. AG, C1-metabolism. AH, Redox regulation/oxidative stress response. AI, Signal transduction. AJ, DNA binding/transcription/chromatin remodeling. AK, RNA processing/binding. AL, Vesicle transport. AM, Pumps and transporters. AN, Protein sorting. AO, Flagellar and basal body proteins. AP, Miscellaneous. AQ, Proteins without functional annotation.
Fig. 4.
Fig. 4.
Kinetics of proteins exhibiting significant changes during heat stress. Kinetics of all 244 proteins significantly changing during heat stress are plotted bin-wise as percent of the maximal value in the time course. Values and standard deviations derive from three biological and two technical replicates (listed in supplemental Table S3). A, Small heat shock proteins. B, HSP100s. C, HSP90 and co-chaperones. D, HSP70 and co-chaperones. E, HSP60s. F, FKBPs/Cyclophilins. G, Protein folding and abiotic stress. H, Proteases/Peptidases. I, Protein biosynthesis (LSU cytosol). J, Protein biosynthesis (SSU cytosol). K, Protein biosynthesis (LSU plastid). L, Protein biosynthesis (not ribosome). M, N-metabolism. N, Amino acid metabolism. (O) Proteins associated with PS I. P, Proteins associated with PS II. Q, Other proteins associated with light reactions. R, Porphyrin biogenesis. S, Vitamin biosynthesis. T, Proteins of photosynthetic and photorespiratory carbon metabolism. U, Starch synthesis and degradation. V, Glycolysis and gluconeogenesis. W, TCA cycle. X, Oxidative pentose phosphate pathway. Y, Acetate metabolism/glyoxylate cycle. Z, Fermentation. AA, Glycerolipid metabolism. AB, Mitochondrial electron transfer chain and ATP synthase. AC, Nucleotide metabolism. AD, Sugar nucleotide metabolism. AE, Metal handling. AF, S-assimilation. AG, C1-metabolism. AH, Redox regulation/oxidative stress response. AI, Signal transduction. AJ, DNA binding/transcription/chromatin remodeling. AK, RNA processing/binding. AL, Vesicle transport. AM, Pumps and transporters. AN, Protein sorting. AO, Flagellar and basal body proteins. AP, Miscellaneous. AQ, Proteins without functional annotation.
Fig. 5.
Fig. 5.
Clustering of the kinetic behavior of 230 Chlamydomonas proteins found to be differentially regulated by heat stress. A, UP fast (11 proteins). B, UP delayed (24 proteins). C, DOWN fast (51 proteins). D, DOWN delayed (85 proteins). E, DOWN delayed, into steady state (59 proteins). Information on which protein belongs to which cluster is compiled in Table I. The medians of all proteins within a cluster are shown in bold.
Fig. 6.
Fig. 6.
Heat map representation of protein and transcript abundance during heat stress. Relative changes in the abundance of transcript levels of 24 selected target genes in Chlamydomonas cells exposed to heat stress for 30 and 180 min were determined by qRT-PCR using the relative 2−ΔΔCt method. Transcripts of the CBLP2 house-keeping gene were used as internal standard. Values are the average of two biological replicates, each measured in triplicate (all values are compiled in supplemental Table S6). For each of the 24 targets the relative changes in protein levels at the two time points, as determined by quantitative shotgun proteomics, are also shown (values are from supplemental Table 3). Fold changes are log2 transformed.
Fig. 7.
Fig. 7.
Growth curves of unstressed and heat stressed Chlamydomonas cells. Chlamydomonas cells were grown at 25 °C to a cell density of ∼5 × 106 cells/ml and diluted 20-fold in two flasks with fresh TAP medium. Both cultures were incubated at 25 °C and cell densities were measured in triplicate during 130 h after dilution. One culture was kept continuously at 25 °C (black line), the other was incubated in a 42 °C water bath after t = 24 h (gray line) for 24 h and shifted back to 25 °C at t = 48 h. Error bars represent standard deviations from three independent growth experiments.
Fig. 8.
Fig. 8.
Schematic overview of the quantitative shotgun proteomics results. Shown is a crude outline of the biochemical pathways and cellular processes in which most of the 1116 quantified proteins participate. Boxes next to each pathway or process represent the 47 functional bins, bin numbers as listed in Table I are in gray letters. Bins with underlined numbers and names are significantly enriched in proteins changing in response to heat stress with respect to the total protein population. White boxes are proteins that do not change during heat stress or whose changes are not significant. Red boxes represent proteins increasing, blue boxes proteins decreasing during heat stress. The color intensity indicates the magnitude of change.
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