Supplementary Materials and Methods
For protein extraction, larvae were solubilised into 50 μL of fresh 1% SDS buffer
using a Qiagen TissueLyser such that the larvae disintegrated without thawing.
Samples were then incubated for 10 min at 70°C to complete the extraction and
centrifuged for 10 min at 13,200 rpm. The supernatant was then transferred to a fresh
Eppendorf LoBind tube, precipitated for 4 h at -20°C using 300 μL of acetone,
centrifuged for 20 min at 13,200 rpm, re-dissolved in 100 μL of 1% SDS and
sonicated for 30 min. Protein concentration was then determined with a NanoDrop®
ND-1000 spectrophotometer.
For in-solution digestion, 100 μg of protein was dissolved in 50 μL 7 M urea/2 M
thiourea. Disulphide bridges were reduced with 1 mM DTT for 1 h at room
temperature (RT), after which free SH-groups were alkylated with 5 mM IAA for 1.5
h at RT. Proteins were first digested with LysC (1:50 enzyme:protein ratio; Wako,
129-02541) for 4 h at RT, the urea concentration was then reduced to <2 M with 100
mM ammonium bicarbonate, and proteins were re-digested with trypsin (1:50
enzyme:protein ratio; Promega, V2580) overnight at RT. The reaction was quenched
with 1% (final concentration) TFA, and 30 μg of peptides was purified on C18StageTips (Rappsilber et al., 2007), eluted with 80% acetonitrile and fractionated into
six fractions (pH 3, 4, 5, 6, 8, and 11) using a SAX-C18 StageTip–based protocol
(Wiśniewski et al., 2009). Peptides were finally eluted from the C18-StageTips, dried
in a SpeedVac, reconstituted in 0.1% TFA and analysed using nano-LC–coupled
mass-spectrometry; each fraction was analysed twice.
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For Nano-LC-MS/MS, peptides were separated by reversed-phase chromatography
using an Agilent 1200 series nanoflow system (Agilent Technologies) connected to a
LTQ Orbitrap classic mass spectrometer (Thermo Electron, Bremen, Germany)
equipped with a nanoelectrospray ion source (Proxeon, Odense, Denmark). Peptides
were loaded onto a precolumn + analytical column setup (precolumn: Proxeon EASYColumn SC001, 100 µm × 20 mm, packed with Reprosil-Pur C18-AQ of 120 Å
diameter packed with 5 µm particles; analytical column: Proxeon EASY-Column
SC200, 75 µm × 100 mm, packed with Reprosil-Pur C18-AQ 120 Å 3 µm particles)
using a flow rate of 700 nl/min for 20 min. Peptides were separated at a flow rate of
200 nl/min with 90 min gradients as follows: pH 3–5 fractions: 8–36% solution B; pH
6 fraction: 8–35% solution B; pH 8 fraction: 5–33% solution B; pH 11 fraction: 2–
30% solution B (A: 0.5% acetic acid; B: 0.5% acetic acid/80% acetonitrile). Eluted
peptides were sprayed directly into an LTQ Orbitrap mass spectrometer with a fused
silica spray emitter (FS360-20-10-N-20, New Objective). The ionisation voltage was
applied using a liquid junction. The Orbitrap was operated at a 180°C capillary
temperature and 2.0 kV spray voltage. Data were acquired in the data-dependent
mode with up to five MS/MS scans being recorded for each precursor ion scan.
Precursor ion spectra were recorded in profile mode in the Orbitrap (m/z 300–1900, R
= 60,000, max injection time 500 ms, max 1,000,000 charges); data-dependent
MS/MS spectra were acquired in centroid in the LTQ (max injection time 150 ms,
max 5 000 charges, normalised CE 35%, isolation width 2, wideband activation
enabled). Mono-isotopic precursor selection was enabled, singly charged ions and
ions with an unassigned charge state were rejected, and each fragmented ion was
dynamically excluded for 90 s. All measurements in the Orbitrap mass analyser were
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performed with the lock-mass option enabled (lock-masses were m/z 445.12003 and
519.13882).
References
Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification,
enrichment, pre-fractionation and storage of peptides for proteomics using
StageTips. Nature Protocols, 2, 1896-1906.
Wiśniewski JR, Zougman A, Mann M (2009) Combination of FASP and StageTipbased fractionation allows in-depth analysis of the hippocampal membrane
proteome. Journal of Proteome Research, 8, 5674-5678.
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