Reversible tyrosine phosphorylation is a key biochemical process that regulates many responses and is often deregulated in cancer and immunological disorders. In this study, we report a large-scale enrichment and immunoprecipitation of phosphotyrosine-containing peptides from three mouse primary tissues, brain, lung and liver with two commercial available anti-phosphotyrosine antibodies, pY100 and 4G10, to determine complementary enrichment patterns of these antibodies.

In order to obtain more complete phosphoproteome information of each tissue, phosphorylated serine, threonine (pST) and pohsphorylated tyrosine (pY) were enriched via titanium dioxide (TiO₂). We analysed a small portion of these fractions and identified 2256 pST phosphoproteins and 5168 unique phosphopeptides across the three tissues. After pY enrichment, we identified 715, 529 and 274 phosphotyrosine containing peptides and 491, 338 and 203 phosphoproteins from brain, lung and liver tissue samples, using pY 100 and 4G10 antibodies with 1% FDR. Brain tissue had the largest number of unique phosphotyrosine peptides (43.8%) and phosphoproteins (42.7%) of the three tissues examined. Without the pY-enrichment, the distribution of phosphorylated serine, threonine and tyrosine was calculated to be 86.1%, 11.4% and 2.3% for brain tissue, 89.8%, 9% and 1.0% for lung and 84.7%, 12.1% and 2.5% for liver tissue. Combining the identifications from pST and enriched pY-containing peptides, a total of 1683, 861 and 1379 phosphoproteins were identified from brain, lung and liver tissue, respectively. The overlap of pY proteins and pST proteins is around 9% for brain and lung samples and 4% for liver tissue. Each antibody enriched different sets of pY-containing peptides and a recognition specificity analysis shows the 4G10 seems to prefer the sequence window containing negatively charged amino acids. An Ingenuity Pathway Analysis of the pY-containing proteins shows that leukocyte extravasation signaling and integrin signaling are enriched the most in the lung tissue while the Fcy receptor-mediated phagocytosis in macrophages and monocytes signaling enriched in similar level across the three tissues.