Phosphoproteome Analysis of HeLa Cells Using Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)

Abstract

Identification of phosphorylated proteins remains a difficult task despite technological advances in protein purification methods and mass spectrometry. Here, we report identification of tyrosine-phosphorylated proteins by coupling stable isotope labeling with amino acids in cell culture (SILAC) to mass spectrometry. We labeled HeLa cells with stable isotopes of tyrosine, or, a combination of arginine and lysine to identify tyrosine phosphorylated proteins. This allowed identification of 118 proteins, of which only 45 proteins were previously described as tyrosine-phosphorylated proteins. A total of 42 in vivo tyrosine phosphorylation sites were mapped, including 34 novel ones. We validated the phosphorylation status of a subset of novel proteins including cytoskeleton associated protein 1, breast cancer anti-estrogen resistance 3, chromosome 3 open reading frame 6, WW binding protein 2, Nice-4 and RNA binding motif protein 4. Our strategy can be used to identify potential kinase substrates without prior knowledge of the signaling pathways and can also be applied to profiling to specific kinases in cells. Because of its sensitivity and general applicability, our approach will be useful for investigating signaling pathways in a global fashion and for using phosphoproteomics for functional annotation of genomes.

Time Series Proteome Profiling To Study Endoplasmic Reticulum Stress Response

“…Additionally, SILAC can allow accurate temporal proteome profiling 15 and monitoring of protein phosphorylation to study signaling pathways. [16][17][18][19][20] In this study, we implemented the SILAC strategy in combination with SDS-PAGE and LC-MS/MS to monitor temporal changes in the expression and dynamics of proteins within the endoplasmic reticulum (ER) compartment of human primary fibroblast cells exposed to ER stress inducers tunicamycin (Tun) and thapsigargin (Thp). To demonstrate the feasibility of temporal profiling using this method, we have chosen a model system using Tun and Thp, as these ER stress agents have been documented to induce temporal changes in a number of ER resident proteins within minutes to hours.…”

Finishing touches: Post-translational modification of protein factors involved in mammalian pre-mRNA 3′ end formation

“…Pre-treatment agents include pervanadate, a tyrosine phosphatase inhibitor that leads to accumulation of phosphotyrosines (Amanchy, Kalume, Iwahori, Zhong, & Pandey, 2005;Rush et al, 2005), ionizing radiation (Matsuoka et al, 2007), nocodazole (Beausoleil et al, 2006), tumor necrosis factor-alpha (TNF-α) (Cantin et al, 2006) and epidermal growth factor (EGF) (Olsen et al, 2006). In order to compare treated with untreated cells, SILAC, stable isotope labeling with amino acids in cell culture, is frequently used (Ong et al, 2002).…”

Phosphoproteomic Analysis of Pancreatic Ductal Adenocarcinoma Cells Reveals Differential Phosphorylation of Cell Adhesion, Cell Junction and Structural Proteins

“…It is essential to stabilize microtubules during the elongation of dendrites and neuritis, and it can also interact with other cellular components, including filamentous actin and signaling proteins [44,45]. The functions of MAP-7 and MAP-1B are modulated by phosphorylation, and multiple phosphorylation sites have been previously identified in human cervix epithelial adenocarcinoma (Hela) cells by MS-based phosphoproteomic study [46][47][48][49]. In addition, differential phosphorylation was observed in stathmin 1 isoform a and transforming acidic coiled-coil containing protein 2 isoform d (TACC2).…”

“…In the past decade, researchers successfully profiled the phosphoproteomes of various mammalian cells and tissues and were able to identify thousands of phosphopeptides from over a thousand phosphoproteins by MS [46][47][48][49]. However, most of the reports have been focused on analysis of a single sample, and information of comparative phosphoproteomic studies of cancer and normal samples was limited.…”