Selective Detection Of Phosphopeptides Research Articles

An ICP-MS, ESI-MS and molecular modelling investigation of homogeneous gallium affinity tagging (HMAT) of phosphopeptides

Protein phosphorylation and de-phosphorylation, provide one of the most common signalling pathways within cells, being involved in regulating cellular processes, mediating enzyme inhibition, protein–protein recognition and protein degradation. Compared with normal proteomics, phosphoproteomics poses some additional challenges requiring more initial separation and additional sensitivity to detect and quantify potentially ultra-low abundance species. In this work, the selective detection of phosphopeptides is described based on the incorporation of a metal tag, gallium–N,N-biscarboxymethyl lysine (Ga-LysNTA), in solution before separation and detection by liquid chromatography coupled to inductively coupled plasma mass spectrometry (LC–ICP-MS). Experimental and theoretical characterisation of the resulting Ga–phosphopeptide complex is presented based on linear ion trap electrospray ionisation mass spectrometry (ESI-MS), Fourier transform mass spectrometry (FT-MS) and molecular modelling data. Linear ion trap electrospray ionisation mass spectrometry (ESI-MS) was employed to study the interaction of the gallium tag with platelet derived growth factor beta receptor (ß-PDGF), a small phosphopeptide. In addition high resolution Fourier transform mass spectrometry (FT-MS) was used for accurate mass determination and multistage tandem mass spectrometry of the gallium–ß-PDGF complex identified the fragmentation pathway. Finally, molecular modelling was used to investigate the energetically favoured structures of both the Ga-LysNTA material and the ß-PDGF–Ga-LysNTA complex.

Fe 3O 4@Al 2O 3 magnetic core–shell microspheres for rapid and highly specific capture of phosphopeptides with mass spectrometry analysis

Selective detection of phosphopeptides from complex biological samples is a challenging and highly relevant task in many proteomics applications. In this study, a novel phosphopeptide enrichment approach based on the strong interaction of Fe 3O 4@Al 2O 3 magnetic core–shell microspheres with phosphopeptides has been developed. With a well-defined core–shell structure, the Fe 3O 4@Al 2O 3 magnetic core–shell microspheres not only have a shell of aluminum oxide, giving them a high-trapping capacity for the phosphopeptides, but also have magnetic property that enables easy isolation by positioning an external magnetic field. The prepared Fe 3O 4@Al 2O 3 magnetic core–shell microspheres have been successfully applied to the enrichment of phosphopeptides from the tryptic digest of standard phosphoproteins β-casein and ovalbumin. The excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of β-casein and bovine serum albumin with molar ratio of 1:50 as well as tryptic digest product of casein and five protein mixtures. The results also proved a stronger selective ability of Fe 3O 4@Al 2O 3 magnetic core–shell microspheres over Fe 3+-immobilized magnetic silica microspheres, commercial Fe 3+–IMAC (immobilized metal affinity chromatography) resin, and TiO 2 beads. Finally, the Al 2O 3 coated Fe 3O 4 microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. These results show that Fe 3O 4@Al 2O 3 magnetic core–shell microspheres are very good materials for rapid and selective separation and enrichment of phosphopeptides.

Selective detection and identification of phosphopeptides by dansyl MS/MS/MS fragmentation

Protein phosphorylation regulates many cellular processes and pathways, such as cell cycle progression, signal transduction cascades and gene expression. Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics applications. Often phosphopeptides are present in small amounts and need selective isolation or enrichment before identification. Here we report a novel approach to label selectively phospho-Ser/-Thr residues by exploiting the features of a novel linear ion trap mass spectrometer. Using dansyl labelling and MS3 fragmentation, we developed a method useful for the large-scale proteomic profiling of phosphorylation sites. The new residues in the sequence were stable and easily identifiable under general conditions for tandem mass spectrometric sequencing.

Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns.

Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics applications. Often phosphopeptides are present in small amounts and need selective isolation or enrichment before identification. Here we report a novel automated method for the enrichment of phosphopeptides from complex mixtures. The method employs a two-dimensional column setup, with titanium oxide-based solid-phase material (Titansphere) as the first dimension and reversed-phase material as the second dimension. Phosphopeptides are separated from nonphosphorylated peptides by trapping them under acidic conditions on a TiO(2) precolumn. Nonphosphorylated peptides break through and are trapped on a reversed-phase precolumn after which they are analyzed by nanoflow LC-ESI-MS/MS. Subsequently, phosphopeptides are desorbed from the TiO(2) column under alkaline conditions, reconcentrated onto the reversed-phase precolumn, and analyzed by nanoflow LC-ESI-MS/MS. The selectivity and practicality of using TiO(2) precolumns for trapping phosphopeptides are demonstrated via the analysis of a model peptide RKISASEF, in a 1:1 mixture of a non- and a monophosphorylated form. A sample of 125 fmol of the phosphorylated peptide could easily be isolated from the nonphosphorylated peptide with a recovery above 90%. In addition, proteolytic digests of three different autophosphorylation forms of the 153-kDa homodimeric cGMP-dependent protein kinase are analyzed. From proteolytic digests of the fully autophosphorylated protein at least eight phosphorylation sites are identified, including two previously uncharacterized sites, namely, Ser-26 and Ser-44. Ser-26 is characterized as a minor phosphorylation site in purified PKG samples, while Ser-44 is identified as a novel in vitro autophosphorylation target. These results clearly show that TiO(2) has strong affinity for phosphorylated peptides, and thus, we conclude that this material has a high potential in the field of phosphoproteomics.