151125_Quantitative human cell culture

Protein extracts were reduced with 10ul of 200mM TCEP then
alkylated with 10ul or 375mM iodoacetamide in the dark for 30 minutes and
TCA/Acetone precipitated (100 ug). Protein pellets were resolubilized in 100ul
of 100mM triethyl ammonium bicarbonate (TEAB) and were digested overnight at
37C by adding 10 ug of Trypsin/LysC mixture (V5071, Promega) in 100 mM
TEAB. Individual samples (100 ug) were
labeled with a unique isobaric mass tag reagent (TMT 10-plex, Thermo Scientific)
according to the manufacturer instructions. Both pairing and labeling order of
TMT reagent and peptide sample were randomized.
Briefly, TMT-6 plex reagents (0.8ug vials) were allowed to come to room
temperature before adding 41 ul of anhydrous acetonitrile, then briefly
vortexed and centrifuged. The entire TMT
reagent vial was added to the 100 ug peptide sample and reacted at room
temperature for 1 hr. 5% hydroxylamine
(8 ul) was then added to quench the reaction.
All TMT labeled samples were combined and vacuum centrifuged to
dryness. The combined samples of TMT
labeled peptides was resuspended in 2ml of 10mM TEAB and separated into 84
fractions at 250ul/min using a 0- 90% acetonitrile gradient in 10 mM TEAB on a
150mm x 2.1mm ID Waters XBridge 5um C18 using an Agilent 1200 capillary HPLC in
normal flow mode and Agilent 1260 micro-fraction collector. The 84 fractions are concatenated into 24
fractions by combining all odd rows of each column 1 through 12 into 12
fractions and all even rows of each column into another 12 fractions.

Peptide fractions were resuspended in 20ul 2% acetonitrile
in 0.1% formic acid and analyzed by reverse phase liquid chromatography coupled
to tandem mass spectrometry. Peptides
were separated on a 75 um x 150 mm ProntoSIL-120-5-C18 H column (3µm, 120Å
(BISCHOFF), www.bischoff-chrom.com)
using 2-90% acetonitrile gradient at 300 nl/min over 90 min on a EasyLC nanoLC
1000 (Thermo Scentific). Eluting
peptides were sprayed through 1 µm emitter tip (New Objective,
www.newobjective.com) at 2.0 kV directly into a Q-Exactive Plus (QE Plus,
Thermo Scientific) mass spectrometer. Survey scans (full ms) were acquired from
350-1700
m/z with data dependent monitoring of up to 15 peptide masses (precursor ions),
each individually isolated in a 1.2 Da window and fragmented using HCD
activation collision energy 32 and 30 s dynamic exclusion. Precursor and the
fragment ions were analyzed at resolutions 70,000 and 35,000, respectively,
with automatic gain control (AGC) target values at 3e6 with 50 ms maximum
injection time (IT) and 1e5 with 200 ms maximum IT, respectively. Isotopically resolved masses in precursor
(MS) and fragmentation (MS/MS) spectra were processor in Proteome Discoverer
(PD) software (v1.4, Thermo Scientific).
All data were searched using Mascot (2.5.1; www.matrixscience.com) against
the Refseq
mouse 2012 database. The following criteria were set for all database
searches: sample’s species; trypsin as the enzyme, allowing one missed
cleavage; cysteine carbamidomethylation and N-terminal TMT label as fixed
modifications; TMT label on lysine, methionine oxidation, asparagine and
glutamine deamidation as variable modifications. Peptide identifications from
Mascot searches were filtered at 1% False Discovery Rate (FDR) confidence
threshold, based on a concatenated decoy database search, using the Proteome
Discoverer. Proteome Discoverer uses only the peptide identifications with the
highest Mascot score for the same peptide matched spectrum from the different
extraction methods.