Proteome Analysis
Extraction and digestion - Samples were randomized before
processing to avoid batch effects. Frozen rosettes were ground under
liquid N2. Proteins were extracted from 100 mg of frozen
powder per sample by adding 150 µl of extraction buffer (30 mM Tris-HCl
pH 8.0, 4% SDS). Tubes were incubated in a shaker (Eppendorf) at 4ºC at
1400 rpm for 30 min. Samples were centrifuged at 16000 g and 4ºC
for 30 min and the supernatant was transferred to a new tube. Protein
concentration was estimated based on Bradford (Bradford, 1976) using the
Bio-Rad Protein Assay reagent. Subsequently, DTT was added to a final
concentration of 50 mM and proteins were reduced for 30 min on ice. For
digestion, 140 µg of proteins were processed following the FASP method
(Wisniewski, Zougman, Nagaraj, & Mann, 2009). Peptides were desalted
using SPE C18 columns (Finisterre) and dried down in a SpeedVac
concentrator.
Peptide fractionation - To increase proteome coverage, peptide
samples were fractionated by hydrophilic interaction chromatography
(HILIC) on an Agilent 1200 series HPLC system with a YMC‑Pack Polyamine
II 250 x 3.0 mm size column with 5 μm particle size and 120 Å pore size.
Samples were dissolved in 100 μl Buffer A (75% ACN, 8 mM
KH2PO4, pH 4.5) and separated with
Buffer B (5% ACN, 100 mM KH2PO4,
pH 4.5) at a flow rate of 500 µl/min with the following gradient:
0‑7.5 min, 0% B; 7.5‑37.5 min, 0‑50% B; 37.5‑42.5 min, 50‑100% B;
42.5‑47.5 min, 100% B. Following the separation the column was washed
with 100% buffer A and re-equilibrated for 60 min. For each sample, the
27 automatically collected fractions were pooled into five fractions
that were subsequently dried down in a SpeedVac concentrator. Each
sample was then dissolved in 200 µl of 3% ACN, 0.1% TFA, desalted on
SPE C18 columns (Finisterre) and again dried in a SpeedVac concentrator.
LC-MS analysis - Mass spectrometry queues were arranged to
process comparable fractions in the same batch, with sample order
randomized within each batch. Peptide samples were dissolved in 20 µl
3% ACN, 0.1% FA and spiked with internal retention time (iRT)
standards (Biognosys) for chromatography quality control. LC-MS/MS
shotgun analyses were performed on a Thermo Orbitrap Fusion instrument
coupled to an Eksigent NanoLC Ultra (Sciex). Samples were separated on a
self‑packed reverse‑phase column (75 µm x 150 mm) with C18 material
(ReproSil-Pur, C18, 120 Å, AQ, 1.9 µm, Dr. Maisch GmbH). The column was
equilibrated with 100% solvent A (0.1% FA in water). Peptides were
eluted using the following gradient of solvent B (0.1% FA in ACN) at a
flow rate of 0.3 µl/min: 0‑50 min: 3‑25% B, 50‑60 min: 25‑35% B,
60‑70 min: 35-97% B, 70‑80 min: 97% B, 80‑85 min: 2% B. Mass spectra
were acquired in a data-dependent manner. All precursor signals were
recorded in the Orbitrap using quadrupole transmission in the mass range
of 300‑1500 m/z. Spectra were recorded with a resolution of 120000
(FWHM) at 200 m/z, a target value of 4e5 and the maximum cycle time set
to 3 s. Data dependent MS/MS were recorded in the linear ion trap using
quadrupole isolation with a window of 1.6 Da and higher-energy
collisional dissociation (HCD) fragmentation with 30% fragmentation
energy. The ion trap was operated in rapid scan mode with a target value
of 1E4 and a maximum injection time of 250 ms. Precursor signals were
selected for fragmentation with a charge state from + 2 to + 7 and a
signal intensity of at least 5e3. A dynamic exclusion list was used for
30 s and maximum parallelizing ion injections was activated. The mass
spectrometry proteomics data were handled using the local laboratory
information management system (LIMS) (Türker et al., 2010)
Phosphoproteome
Analysis
Extraction – Whole rosette tissue from each time point was
harvested and ground under liquid N2. From each
biological replicate 200 mg of ground leaf material was weighed out
under liquid N2. In addition to each biological
replicate, 200 mg of samples containing equal weighted parts of each
biological replicate and time-point were created as a reference sample
(gold-standard) for downstream dimethyl labeling. All proteins were
extracted in a 250 µl solution of 50 mM HEPES pH 8.0, 6 M urea, 2 M
thiourea, 100 mM NaCl, 10 mM EDTA, 2 mM NaOV, 5 mM NaF, 50 µg/mL
PhosSTOP (Roche). Samples were shaken at room temperature for 30 min at
1000 g with vortexing every 10 min. Extracts were then brought to
pH 8.0 using triethylammonium bicarbonate (TEAB). Protein extracts were
then reduced for 30 min with 10 mM DTT, followed by alkylation with
30 mM iodoacetamide for 1 h. Extracts were clarified to separate soluble
and insoluble fractions. The insoluble fraction was re-suspended in
300 µL 60:40 buffer containing 60% MeOH: 40% 50 mM TEAB pH 8.0
followed by shaking at 1000 rpm (Eppendorf tabletop) for 2.5 h. The
protein concentration of the soluble fraction was then measured using
the Bradford protein assay (Bradford, 1976). An amount of 1 mg of
soluble protein from each sample was then diluted with 1 vol. of 50 mM
TEAB and then water was added to a total volume of 1.2 ml and a final
urea/thiourea concentration of 1.2 M. The soluble fraction was then
digested for 20 h at 37ºC using a 1:50 ratio of trypsin (Promega) to
extracted protein while gently shaking. Each insoluble fraction was
digested by 0.5 µg chymotrypsin and 1 µg trypsin at 37ºC for 20 h
shaking at 600 rpm (Eppendorf tabletop). Digestion reactions were
stopped using TFA to a final concentration of 0.5%. The insoluble
fractions were centrifuged for 10 min at 20000 g at room
temperature and the supernatant removed. The supernatant was then dried
and re-suspended in desalting buffer comprised of 3% ACN / 0.1% TFA.
The soluble fraction and the supernatant from the insoluble fraction
were desalted using SPE C18 columns (Finisterre) and dried in a SpeedVac
concentrator.
Dimethyl labeling and phosphopeptide enrichment - Total peptide
fractions from each experimental (light label) and gold-standard (heavy
label) sample were labeled according to Boersema et al.,(Boersema, Raijmakers, Lemeer, Mohammed, & Heck, 2009). Heavy and light
samples were then mixed 1:1 and desalted prior to phosphopeptide
enrichment using TiO2. Phosphopeptide enrichment was
performed using TiO2 heavy and light dimethyl-labelled
phosphopeptides as previously described (Zhou et al., 2011).
LC-MS - Phosphorylated peptide samples were analyzed using a Q
Exactive Orbitrap mass spectrometer (Thermo Scientific). Dissolved
samples were injected using an Easy-nLC 1000 system (Thermo Scientific)
and separated on a self-made reverse-phase column (75 µm x 150 mm)
packed with C18 material (ReproSil-Pur, C18, 120 Å, AQ, 1.9 µm, Dr.
Maisch GmbH). The column was equilibrated with 100% solvent A (0.1%
formic acid (FA) in water). Peptides were eluted using the following
gradient of solvent B (0.1% FA in ACN): 0-120 min, 0-35% B,
120-122 min, 35-95% B at a flow rate of 0.3 µl/min. High accuracy mass
spectra were acquired in data-depended acquisition mode. All precursor
signals were recorded in a mass range of 300-1700 m/z and a resolution
of 70000 at 200 m/z. The maximum accumulation time for a target value of
3e6 was set to 120 ms. Up to 12 data dependent MS/MS were recorded using
quadrupole isolation with a window of 2 Da and HCD fragmentation with
28% fragmentation energy. A target value of 1e6 was set for MS/MS using
a maximum injection time of 250 ms and a resolution of 70000 at 200 m/z.
Precursor signals were selected for fragmentation with charge states
from +2 to +7 and a signal intensity of at least 1e5. All precursor
signals selected for MS/MS were dynamically excluded for 30 s.