DOI: 10.25345/c5w221
title: MassIVE MSV000087222 - In vivo phosphoproteomics uncovers novel requirements for proteins in epithelial growth and division
title: Materials. Aqua 5um C18, 125A pore diameter, and Luna 5 um strong cation exchange, 100A pore diameter, were purchased in bulk from Phenomenex (Torrance, CA). Deactivated fused silica tubing (0.100mm internal diameter x 0.360mm outer diameter, 0.050 mmi.d. x 0.360mm o.d., and 0.250mm i.d. x 0.360mm o.d. were obtained from Polymicro (Lisle, IL). HPLC grade water and acetonitrile were from EMD Chemicals Inc. (Gibbstown, NJ). HPLC grade formic acid was purchased from Mallinckrodt Baker, Inc. (Phillipsburg, NJ). Protein Digestions. The precipitated proteins in 100mM Tris-HCl, pH 8.5, 8M urea, reduced in 5mM TCEP, incubated at room temperature for 30min, and carboxymethylated by adding CAM to 10mM and incubated at room temperature for 30min in dark. Proteins were then digested with endoproteinase Lys-C followed by trypsin, at 37 oC overnight. Digestion reactions were quenched by adding formic acid to 5%. The peptide mixtures were aliquoted and stored at -80 oC prior to use. 1/10 of digested peptides were sent for MS analysis directly without further process. 4/10 of digested peptides were used for phosphopeptide enrichment through IMAC, and last 5/10 peptides were used for phosphopeptides enrichment through TiO2. Phosphopeptide Enrichment. Enriched phosphopeptides by IMAC: 6 times of IMAC loading buffer (30% acetonitrile, 250mM acetic acid) was added into digested peptides and mixed with 30 ul equilibrated PHOS-Select gel (Sigma-Aldrich) in a blocked spin column(Sigma-Aldrich). This peptide solution was incubated for 2hrs with end-over-end rotation. Then the resin was washed two times with IMAC loading buffer, and once with pure water. Phosphopeptides were eluted with 0.3 MNH4OH, after elution, pH was adjusted to 2.7 by adding formic acid. Enriched phosphopeptides by TiO2: 6 times of TiO2 loading buffer (80% acetonitrile, 2.5% TFA and was saturated with phthalic acid) added into peptide solution, loaded onto equilibrated TiO2 spin tip (GL Science, Saitama, Japan) by centrifugating at 1000g for 15 min. The TiO2 spin tip was washed twice with loading buffer, twice with washing buffer I (80% acetonitrile, 0.1% TFA) and once with washing buffer II (0.1% TFA). All washing steps were done by centrifugating at 1000g for 5min. Phosphopeptides were eluted with 0.3 MNH4OH, after elution, pH was adjusted to 2.7 by adding formic acid. Multidimensional Protein Identification Technology. MudPIT was carried out as described previously with the following modifications. Peptide mixtures were loaded onto a 250um i.d. capillary packed first with 3.5cm of 5um Luna SCX, followed by 2.5cm of 5um Aqua C18. The biphasic column was washed with buffer A for more than 20 column volumes. After desalting, the biphasic column was connected via a 2-um filtered union (UpChurch Scientific) to a 100um i.d. column, which had been pulled to a 5um tip, then packed with 10 cm of 5um C18 RP particles (Aqua). The split three-phase column was placed in line with an Agilent 1200 quaternary HPLC pump (Palo Alto, CA) and a Velos Orbitrap Elite (VOE) mass spectrometer (Thermo Fisher Scientific). The direct analysis samples and enriched samples were analyzed using 16- and 12-step MudPIT, on VOE, respectively. The solvent solutions used were described above with the additional buffer C for salt bumps consisting of buffer A with 500u mM ammonium acetate. Tandem Mass Spectrometry (MS/MS). Instrument settings common to the three systems were as follows: MS spray voltage set at 2.5 kV; MS transfer tube temperature set at 275 oC; 50 ms MS1 injection time; 1 MS1 microscan; MS1 data acquired in profile mode; 15 MS2 dependent scans; 1 MS2 microscan; and MS2 data acquired in centroid mode. Instrument settings: MS1 scans acquired in Orbitrap (OT) at 60000 resolution; full MS1 range acquired from 400-1500 m/z; MS1 AGC targets set to 1.00E+06; MS1 charge states between 2-5; ddMS2 acquired in and IT; MS2 collision energy and fragmentation 35% CID; MS2 AGC targets of 1.00E+05; MS2 max injection times of 150 ms. Data Analysis. Collected MS/MS spectra were searched with the ProLuCID algorithm against a database of 44464 protein sequences combining 22007 non-redundant Drosophila melanogaster proteins (NCBI, 2018-06-03 release), 225 common contaminants, and their corresponding 22232 randomized amino acid sequences. All cysteines were considered as fully carboxamidomethylated (+57.0215 Da statically added), while methionine oxidation (+15.9949 Da), serine, threonine, and tryptophan phosphorylation (+79.9663 Da) were searched as a differential modification. DTASelect v1.9 and swallow, an in-house developed software, were used to filter ProLuCID search results at given FDRs at the spectrum, peptide, and protein levels. Here, all controlled FDRs were less than 1%. Our in-house developed software, NSAF7 v0.0.1, was used to generate spectral count-based label free quantitation results.