Data S8: Methods
Free amino acid analysis via HPLC. Free amino acids were extracted from lyophilized cw15
cells as described previously (Hacham et al. 2002). Briefly, ~20 mg of lyophilized cells was
homogenized by a TissueLyser (Qiagen Valencia, CA, USA) for 5 min and extracted twice
with 600 L of water: chloroform: methanol (3:5:12, v/v/v). The upper water-methanol phase
was collected, dried, and dissolved in 20 L of 20mMHCl for derivatization with AccQ-Tag
reagents and analysis using an ACQUITY UPLC system with an AccQ-Tag ULTRA C18 (1.7
µm, 2.1 x 100 mm) column according to the manufacturer’s instructions (Waters, Milford,
MA, USA). A standard concentration curve was prepared and analyzed alongside samples,
and the concentration of each amino acid (pmol/mg) was calculated according to the standard
curve for each respective amino acid.
Total Amino Acid Analysis via HPLC. Approximately three milligrams of lyophilized cell
samples were placed into glass tubes with 400 µL performic acid, and incubated at room
temperature for 30 minutes. Following oxidation, the samples were dried down, mixed with 1
mL of 0.5% (w/v) phenol in 6N HCl, and incubated at 116C for 24h. Samples were then
dried down, and re-suspended in 200 µL of 20mMHCl. Samples and standards were
derivatized and analyzed as for free amino acid as described above.
C/N ratio measurement. Samples for C/N analysis were harvested as described earlier; freeze
dried, weighed and sent for analysis of the carbon and nitrogen percentage at the Duke
Environmental Stable Isotope Laboratory at Duke University by dry combustion using a CE
Instruments NC2100 elemental analyzer (ThermoQuest Italia).
Proteomics. Three biological replicates were performed for proteomic study. To
accommodate to our 4plex iTRAQ experiment design (Supplemental Figure 1), 0.5h sample
were omitted due to slow protein change. Proteins extraction, in-solution trypsin digestion
and iTRAQ labeling were performed as described previously with some modification (Wang
et al. 2012). Briefly, after digestion, 100 µg of each sample was used for labeling using
4-plex iTRAQ reagents according to the manufacturer’s instructions (AB Sciex, Foster City,
USA). To accommodate the seven time points (0h, 1h, 2h, 4h, 6h, 12h and 24h) in this study,
two 4-plex iTRAQ experiments were performed for each biological replicate. The samples
from 0h (N-free) were labeled with reagent with 114 as reporter ion in both experiments as
control. The samples from 1h, 2h and 4h were labeled with reagents with 115,116 and 117 as
reporter ions in the first iTRAQ experiment, respectively. For the second iTRAQ experiment,
the samples from 6h, 12h, and 24h were labeled with reagents 115,116, and 117 as reporter
ions, too. After labeling, samples from each separate experiment containing iTRAQ labels
114-117 were mixed and fractionated via SCX as described previously (Alvarez et al. 2011).
Fractions from 5 to 24 min were analyzed by nano-LC-MS/MS using an LTQ-Orbitrap Velos
mass spectrometer (ThermoFisher Scientific,Rockford, USA) coupled with a nanoLC Ultra
(Eksigent, Dublin, USA). Each sample (5 µL) was loaded onto a trap column (C18
PepMap100, 300µm 1mm, 5µm, 100 Å, Dionex, Sunnyvale, USA) at a flow rate of 4
µL/min for 5 min. Peptide separation was carried out on a C18 column (Acclaim PepMap C18,
15 cm × 75 μm × 3 μm, 100 Å, Dionex) at a flow rate of 0.26 L/min. Peptides were
separated using a 145 min linear gradient ranging from 5% to 50% B (mobile phase A, 0.1%
FA; mobile phase B, 0.1% FA in ACN). The mass spectrometer was operated in positive
ionization mode. The MS survey scan was performed in the FT cell from a mass range of 300
to 1,700 m/z. The resolution was set to 60,000 @ 400 m/z and the automatic gain control
(AGC) was set to 500,000 ions. HCD fragmentation was used for MS/MS and the 10 most
intense signals in the survey scan were fragmented. A resolution of 7,500 was used in the
Orbitrap with an isolation window of 2 m/z, a target value of 100,000 ions, and with a
maximum accumulation time of 1 s. Fragmentation was performed with normalized collision
energies of 45% and activation times of 0.1 ms. Dynamic exclusion was performed with a
repeat count of 1 and exclusion duration of 75 s, and a minimum MS signal for triggering
MS/MS was set to 5,000 counts. Data was processed using Mascot Distiller v2.4 and
searched using Mascot Daemon (Matrix Science, London, UK). For data processing, the
MS/MS settings include single peak window selection from m/z 113.5 to m/z 117.5. All
searches were performed against the Chlamydomonas protein database from Phytozome 9
combined with Chlamydomonas chloroplast and mitochondria database from NCBInr
(19,603 sequences) and the common contaminants database (120 sequences, including trypsin,
keratins, bovine serum albumin etc.) using the following settings: trypsin as cleavage enzyme;
two missed cleavages; carbamidomethyl (C), iTRAQ (N terminal), iTRAQ (K) were fixed
modifications; and oxidation (M), acetyl (protein N-term), deamidated (N, Q), and iTRAQ (Y)
were selected as variable modifications. The mass error tolerance for precursor ions was set
to 10 ppm and 0.02 Da for fragment ions. Automatic isotope correction was carried out using
the values supplied with the AB Sciex reagents. The following settings were used to process
the quantification results: the protein ratio type was the ‘weighted’ geometric mean,
normalization was ‘summed intensity’, outlier removal was ‘automatic’ (Dixon's method up
to 25 data points, Rosner's method above 25 data points), the peptide threshold was ‘at least
homology’ (i.e., peptide score does not exceed absolute threshold but is an outlier from the
quasi-normal distribution of random scores), the minimum number of peptides was two and
the Mascot score is 24, and the peptides were required to be the top ranking peptide matches
(‘red’). Protein ratio was calculated by Mascot using at least 2 peptide ratios (based on
reporter ion intensity) and only those with significant ratio were considered. For each protein
in each time point, only significant ratios were observed in more than two replicates were
shown in average and the ratio of 0.8 or 1.2 were used as cutoffs. An automatic decoy
database search was also performed to assess the false positive rate (FDR) of protein
identification and the results were filtered by significance threshold to FDR1%. Identified
proteins were grouped based on 98% homology with Scaffold v3.1 (Proteome Software,
Portland, USA).
For mapping Chlamydomonas genes onto the Kyoto Encyclopedia of Genes and
Genomes (KEGG) pathway annotation, we used an annotation database (DB) for gene
number with KEGG orthology from the Phytozome website. The Phytozome peptide DB was
used for Blast2GO V2.5.0 analysis (Conesa et al. 2005). BLAST was performed using the
following settings: blast DB: nr; number of blast hits: 20; blast E cutoff: 1.0×e-3; blast
program: BLASTP; blast mode: QBlast-NCBI; high-scoring segment pair (HSP) length
cutoff: 33; low complexity filter: on. A total of 19,603 sequences were imported into
Blast2GO for BLAST analysis. Annotation was performed with an E-value hit filter of 1×e-6,
annotation cut-off of 55, GO weight of 5 and HSP hit coverage of 30.