Phosphoprotein Profiling by PA-GeLC−MS/MS

Abstract

A significant consequence of protein phosphorylation is to alter protein-protein interactions, leading to dynamic regulation of the components of protein complexes that direct many core biological processes. Recent proteomic studies have populated databases with extensive compilations of cellular phosphoproteins and phosphorylation sites and a similarly deep coverage of the subunit compositions and interactions in multiprotein complexes. However, considerably less data are available on the dynamics of phosphorylation, composition of multiprotein complexes or that define their interdependence. We describe a method to identify candidate phosphoprotein complexes by combining phosphoprotein affinity chromatography, separation by size, denaturing gel electrophoresis, protein identification by tandem mass spectrometry, and informatics analysis. Toward developing phosphoproteome profiling, we have isolated native phosphoproteins using a phosphoprotein affinity matrix, Pro-Q Diamond resin (Molecular Probes-Invitrogen). This resin quantitatively retains phosphoproteins and associated proteins from cell extracts. Pro-Q Diamond purification of a yeast whole cell extract followed by 1-D PAGE separation, proteolysis and ESI LC-MS/MS, a method we term PA-GeLC-MS/MS, yielded 108 proteins, a majority of which were known phosphoproteins. To identify proteins that were purified as parts of phosphoprotein complexes, the Pro-Q eluate was separated into two fractions by size, <100 kDa and >100 kDa, before analysis by PAGE and ESI LC-MS/MS and the component proteins queried against databases to identify protein-protein interactions. The <100 kDa fraction was enriched in phosphoproteins indicating the presence of monomeric phosphoproteins. The >100 kDa fraction contained 171 proteins of 20-80 kDa, nearly all of which participate in known protein-protein interactions. Of these 171, few are known phosphoproteins, consistent with their purification by participation in protein complexes. By comparing the results of our phosphoprotein profiling with the informational databases on phosphoproteomics, protein-protein interactions and protein complexes, we have developed an approach to examining the correlation between protein interactions and protein phosphorylation.

Alternative splicing of FBP-interacting repressor coordinates c-Myc, P27Kip1/cyclinE and Ku86/XRCC5 expression as a molecular sensor for bleomycin-induced DNA damage pathway

“…Eight human colorectal cancer cell lines and a human cervical cancer cell line (HeLa) were characterized for CIN, MIN status according to previous publications [29-34]. The expression of Ku86/Ku70, FIR, and PARP-1 was significantly decreased in CIN (HeLa, HT29, CaCO2, SW480, and SW837) cells compared with MIN (RKO, SW48, HCT116, and DLD1) cells (Figure 9).…”

SAP155-Mediated Splicing of FUSE-Binding Protein-Interacting Repressor Serves as a Molecular Switch for c-myc Gene Expression

“…For immunoprecipitation by anti-FLAG antibody-conjugated beads, the nuclear fraction (NF) was reacted with magnetic Magnosphere MS300/carboxyl beads (Como Bio) precoated with anti-mouse IgG to reduce nonspecific protein binding and then reacted with anti-FLAG antibody for 1 hour at 4 C. After immunoprecipitation, the IgG and anti-FLAG antibody-conjugated beads were washed 5 times with 50 mmol/L phosphate buffer and the bound proteins were eluted with extraction buffer [40 mmol/L Tris-HCl (pH 6.8), 1% SDS, 1 mmol/L DTT] for 1 hour at 60 C. The immunoprecipitates were then analyzed by gel-based liquid chromatography-mass spectrometry (GeLC-MS) and protein identification (17). For immunoprecipitation by anti-SAP155 antibody-conjugated beads, Dynabeads ProteinG (Invitrogen) was prepared by same procedures as anti-FLAG antibody.…”

Recent Developments in Mass Spectrometry Analysis of Phosphoproteomes

“…An effective protein-level enrichment strategy is to use metal oxide affinity chromatography (MOAC) (Wolschin et al, 2005a). Commercial kits (e.g., Pro-Q Diamond resin from Invitrogen) have become available for purification of phosphoproteins (Kristjansdottir et al, 2008). The isolated phosphoproteins are purified by polyacrylamide gel electrophoresis either in the 1D-or 2D-format (i.e., in combination with IEF); visualization of separated phosphoproteins is accomplished by staining the gel with Pro-Q diamond fluorescent dye (Agrawal and Thelen, 2005;Steinberg et al, 2003;Schulenberg et al, 2004).…”