Pierce tio2 phosphopeptide enrichment and clean up kit

Generally, the phosphorylated part of a protein is about 0.01- 0.1%, so it’s almost impossible to use oocytes for identification of phosphorylation sites. Therefore, we used NIH3T3 cells instead, then generated phospho-specific antibody and verify the antibody in oocytes. We used 5 IP reactions, each of which employed 1x10⁶ NIH3T3 cells, 30 μl protein A/G beads and 5 μg Pnma5 antibody. Then the immuno-complexed beads were eluted by 0.2 M glycine (pH 2.7), and the phosphorylated portion of the immuno-complex was enriched by Pierce™ TiO₂ Phosphopeptide Enrichment and Clean-up Kit (Thermo Scientific, Rockford, IL) and sent to Testing & Analysis Center, Nanjing Medical University for LC-MS (liquid chromatograph-mass spectrometer). We pick Thr553 (phosphorylation possibility 93.4%) and the whole process of antibody production & purification was performed by Zhong Ding Biotechnique, Ltd (Nanjing, Jiangsu, China). A short phospho peptide MPTGT(ph)EAAQGVE (“C” at the C-term is an extra residue for conjugation) was synthesized and injected into rabbit for the serum production. The phospho-specific antibody was purified from the serum through column filled with phospho peptide-conjugated resin and then absorbed through column filled with non-phospho peptide (MPTGTEAAQGVE)-conjugated resin to remove residual non-phospho-specific antibody.

Aliquots containing 450 μg of each individual sample were digested with trypsin using the filter-aided sample preparation method. The phosphopeptides were enriched using Pierce TiO₂ Phosphopeptide Enrichment and Clean Up Kit (Thermo Fisher Scientific). The purified phosphopeptide samples were evaporated to dryness, reconstituted in 50 mM TEAB, and labeled using TMT 10-plex isobaric mass tagging reagents (Thermo Fisher Scientific). The TMT-labeled phosphopeptide samples were mixed into corresponding sets and purified using Pierce C-18 Spin Columns (Thermo Fisher Scientific). Purified samples were dried on Speedvac and reconstituted in 15 μl of 3% acetonitrile, 0.1% formic acid for analysis.

Proteins were digested using the filter-aided sample preparation (FASP)^{28 (link)}. 500 µg protein amount per sample were used for the phospho-enrichment. The Orbitrap Fusion Tribrid mass spectrometer interfaced to an Easy-nLC1000 was used to analyze the TMT labeled samples. Briefly: phosphopeptides were enriched using Pierce™ TiO₂ Phosphopeptide Enrichment and Clean-Up Kit (Thermo Fisher Scientific), the resulting eluates evaporated in a vacuum centrifuge and subjected to isobaric mass tagging reagent 6-plex TMT® (Thermo Fisher Scientific), and finally desalted using Pierce™ C18 spin columns (Thermo Fisher Scientific). Further separation of the TMT labeled peptides for total protein analysis was performed with Strong Cation Exchange Chromatography (SCX). MS raw data files for each TMT set were matched for identification and relative protein quantification using Proteome Discoverer version 1.4. The networks analyses were generated through the use of IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) using previous described algorithms for the use of IPA^{29 (link)}. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE^{30 (link)} partner repository with the dataset identifier PXD009198.