Titansphere

Dimethyl labeled (1:1 mixed conditions) from three biological replicates were fractionated using Ultimate 3000 HPLC (Thermo Scientific) equipped with a 4.6 × 250- mm TSKgel Amide-80 5-μm particle column (Tosoh Biosciences). The buffers used for the separation were 0.1% TFA (HILIC buffer A) and 99.9% acetonitrile, 0.1% TFA (HILIC buffer B). The peptide samples were resuspended in 80% HILIC buffer B and injected onto the HILIC column. The chromatography was performed using the following elution gradient: 80% B held for 20 min followed by 80% B to 60% B in 40 min and finally 0% B for 10 min at a flow rate of 0.4 mL/min. In total, 16 fractions were collected per biological replicate and subjected to phosphopeptide enrichment using titanium dioxide (TiO₂, Titansphere, GL Science) as previously described [20 (link),23 (link)]. Briefly, TiO₂ spheres were prepared at 50 mg/mL, and 25 μL (1.25 mg TiO2) were added to each sample fraction, incubated for 10 min/RT to bind phosphopeptides. Samples were washed and eluted with 200 μL 0.4 M NH₄OH followed with 200 μL 0.2 M NH₄OH/50% acetonitrile and then dried using a speedvac (Genevac).

Coomassie Blue stained SDS-PAGE gel bands corresponding to ROR1, ROR2 and CAM-1 ICDs were excised, reduced, cysteine-alkylated, and digested with trypsin in situ[33] (link). Aliquots of the resulting peptides were enriched for phosphopeptides using TiO₂ affinity chromatography (Titansphere; GL Sciences Inc., Torrance, CA). Peptides before and after phospho-enrichment were analyzed by capillary reverse phase liquid chromatography tandem mass spectrometry on an orbitrap mass spectrometer (Orbitrap XL; Thermo Fisher, San Jose, CA). Mass spectra were recorded in data-dependent experiments whereby eight collision-induced dissociation product ion spectra were collected per full MS scan [34] (link). Mass spectra were searched against a database of human proteins using the Mascot program (Matrix Science) and putative phosphorylation sites manually verified.

For phosphopeptide analysis, desalted peptide mixtures underwent further purification using TiO₂ beads (5 μm, Titansphere; GL Sciences, Torrance, CA, USA). The bound fraction containing enriched phosphopeptides was eluted with 5% ammonium hydroxide solution containing 40% acetonitrile. Enriched phosphopeptides were vacuum-dried and re-suspended in 0.1% formic acid solution for subsequent nano-liquid chromatography/tandem mass spectrometry (nano LC-MS/MS).

The TiO₂-based phosphopeptide enrichment of all three biological replicates (36 fractions) was carried out as described by Larsen et al. [23 (link)]. The TiO₂ beads (Titansphere; GL Sciences, Inc., Tokyo, Japan) were suspended in DHB solution (80% ACN, 1% TFA, and 5% 2,5-Dihydroxybenzoic acid) at room temperature for 1 h. Each fraction was then resuspended in 5% DHB solution and incubated with TiO₂ beads for 30 min at room temperature with gentle rotation. TiO₂ beads enriched with phosphopeptides were washed thrice with DHB solution and then with 40% ACN twice. The peptide mixture was acidified using 1% Trifluoroacetic acid (TFA) and desalted using a C18 Sep-Pak cartridge (WAT051910, Waters Corporation, Milford, MA, USA). The peptides were concentrated by vacuum centrifugation and subjected to a C18 StageTip cleanup before mass spectrometry analysis.

Titanium dioxide (TiO₂) based phosphopeptide enrichment was carried out as described previously (28 (link)). Briefly, TiO₂ beads (Titansphere; GL Sciences, Inc.) were suspended in DHB solution (80% ACN, 1% TFA, and 5% 2,5-Dihydroxybenzoic acid) at room temperature for 1 h. Each of the fractions were then resuspended in 5% DHB solution and incubated with TiO₂ beads for 30 min at room temperature with gentle rotation. TiO₂ beads enriched with phosphopeptides were washed thrice with DHB solution and then with 40% ACN twice. The enriched phosphopeptides were eluted thrice into tubes containing 20% TFA on ice using 2% ammonia solution. The peptides were concentrated by vacuum centrifugation and subjected to C18 cleanup before mass spectrometry analysis.