Tmtpro 16plex label reagent set

Whole-cell protein lysates were collected from DIPG007 cells from the following conditions: (a) untreated (n=3); (b) stably knocked down for H3-3A/H3F3A (H3-3AKD sh1) (n=3); (c) treated with AU-15330 PROTAC (1 μM for 24 h) (n=4); and (d) vehicle (Veh = DMSO for 24 h) (n=3). Samples were then sent to the Proteomics Resource Facility at the University of Michigan for analysis. Briefly, 100 μg of the lysate/sample/replicate was prepared and labeled following the directions of the TMTpro™ 16plex Label Reagent Set (Thermo Fisher Scientific #A44521). Twelve fractions were used for LC-MS/MS analysis. Generated data were aligned to a human protein database (20291 entries; reviewed; downloaded on 12/13/2021) and sorted for high (≤1%) or medium-low (≤5%) FDR confidence. A heatmap of protein abundance was generated using GraphPad Prism 8.4.3 (GraphPad software).

To compare the proteomes of the U251N shCTRL and shCAPN2 cells, 10⁶ cells were collected and washed with DPBS. Proteins were extracted and desalted with S-Trap™ mini columns according to the manufacturer’s instructions (ProtiFi™). Proteins were digested with trypsin (V5111, Promega). Peptide concentrations were determined using Pierce™ BCA Protein Assay Kit. 25 µg of each sample were labelled with TMTpro™ 16plex Label Reagent Set (A44520, Thermo Scientific™) and pooled. 100 µg of the pooled sample were fractionated on a high pH liquid chromatography (HPLC, Agilent 1100 Series). 54 fractions were collected and concatenated to 18 samples which were submitted for LC–MS/MS analysis on a Velos Pro Orbitrap Elite™ (Thermo Scientific™) mass spectrometer coupled to an Easy nanoLC 1000 (Thermo Scientific™) with a 200 cm µPAC™ column (PharmaFluidics). Data were analyzed using MSFragger in Fragpipe 16.0 [51 (link)] and the UniProt human reference proteome database downloaded on the 14^th of June 2021 (20,845 protein entries including 245 potential contaminants). We allowed for two missed cleavages, a fragment and a precursor mass tolerance of 20 ppm each. A mass shift of 304.207146 Da was set for the N-term and lysine (K) representing the tandem mass tags (TMT), a mass shift of 57.0214560 Da for cysteine (C) representing carbamidomethylations and of 40.010600 Da for N-term acetylation.

Reduction and alkylation were carried out using dithiothreitol and indole-3-acetic acid. Proteins were precipitated with the chloroform/water/methanol method (57 (link)) to remove remnant lipids. Pellets were digested with lysyl endopeptidase (Wako) overnight at room temperature. The samples were further digested for 6 hours at room temperature, using sequencing grade modified trypsin (Promega). Peptides were then tagged with tandem mass tags from the TMTpro 16-plex Label Reagent Set (Thermo Fisher Scientific) following the manufacturer’s instructions.

Isobaric labeling of
peptides was performed using TMTpro 16plex Label Reagent Set (Fisher
Scientific, UK) according to the manufacturer’s recommended
protocol (lot number: WC320807). After confirming labeling efficiency
of >97% by short 1 h liquid chromatography (LC)–MS runs,
labeled
peptides from each temperature point were combined to a single sample
per biological replicate and desalted with C18 Macro Spin Columns
(Harvard Apparatus, USA). The pooled sample was subject to fractionation
using basic-pH reversed-phase LC on a Gemini C18 column (250 mm ×
3 mm, 3 μm, 110 Å; Phenomenex) on a Dionex Ultimate 3000
off-line LC system, generating 12 fractions which were dried. All
solvents used were high-performance LC grade (Fisher Scientific).
Mobile phase A consisted of 20 mM ammonium formate pH 8.0 and mobile
phase B of 100% acetonitrile (MeCN). Peptides were separated over
a 49 min linear gradient of 1–49% B at 250 nL/min. Peptide
elution was monitored by UV detection at 214 nm. Each of the 12 fractions
was acidified to a final concentration of 1% trifluoroacetic acid
(TFA) and dried using a speed-vac.