Ole Nørregaard Jensen

Highly Selective Enrichment of Phosphorylated Peptides from Peptide Mixtures Using Titanium Dioxide Microcolumns

Reversible phosphorylation of proteins regulates the majority of all cellular processes, e.g. proliferation, differentiation, and apoptosis. A fundamental understanding of these biological processes at the molecular level requires characterization of the phosphorylated proteins. Phosphorylation is often substoichiometric, and an enrichment procedure of phosphorylated peptides derived from phosphorylated proteins is a necessary prerequisite for the characterization of such peptides by modern mass spectrometric methods. We report a highly selective enrichment procedure for phosphorylated peptides based on TiO2microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased the selectivity of the enrichment of phosphorylated peptides by TiO2. We demonstrated that this new procedure was more selective for binding phosphorylated peptides than IMAC using MALDI mass spectrometry. In addition, we showed that LC-ESI-MSMS was biased toward monophosphorylated peptides, whereas MALDI MS was not. Other substituted aromatic carboxylic acids were also capable of specifically reducing binding of nonphosphorylated peptides, whereas phosphoric acid reduced binding of both phosphorylated and nonphosphorylated peptides. A putative mechanism for this intriguing effect is presented.

Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome

In signal transduction in eukaryotes, protein phosphorylation is a key event. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling networks also requires quantitation of these phosphorylation events. In this article, we outline several methods for enrichment of phosphorylated proteins and peptides and discuss various options for their identification and quantitation with special emphasis on mass spectrometry-based techniques.

Quantitative Phosphoproteomics Applied to the Yeast Pheromone Signaling Pathway

Cellular processes such as proliferation, differentiation, and adaptation to environmental changes are regulated by protein phosphorylation. Development of sensitive and comprehensive analytical methods for determination of protein phosphorylation is therefore a necessity in the pursuit of a detailed molecular view of complex biological processes. We present a quantitative modification-specific proteomic approach that combines stable isotope labeling by amino acids in cell culture (SILAC) for quantitation with IMAC for phosphopeptide enrichment and three stages of mass spectrometry (MS/MS/MS) for identification. This integrated phosphoproteomic technology identified and quantified phosphorylation in key regulator and effector proteins of a prototypical G-protein-coupled receptor signaling pathway, the yeast pheromone response. SILAC encoding of yeast proteomes was achieved by incorporation of [13C6]arginine and [13C6]lysine in a double auxotroph yeast strain. Pheromone-treated yeast cells were mixed with SILAC-encoded cells as the control and lysed, and extracted proteins were digested with trypsin. Phosphopeptides were enriched by a combination of strong cation exchange chromatography and IMAC. Phosphopeptide fractions were analyzed by LC-MS using a linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. MS/MS and neutral loss-directed MS/MS/MS analysis allowed detection and sequencing of phosphopeptides with exceptional accuracy and specificity. Of more than 700 identified phosphopeptides, 139 were differentially regulated at least 2-fold in response to mating pheromone. Among these regulated proteins were components belonging to the mitogen-activated protein kinase signaling pathway and to downstream processes including transcriptional regulation, the establishment of polarized growth, and the regulation of the cell cycle.

Large-scale Analysis of in Vivo Phosphorylated Membrane Proteins by Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry

Global analyses of protein phosphorylation require specific enrichment methods because of the typically low abundance of phosphoproteins. To date, immobilized metal ion affinity chromatography (IMAC) for phosphopeptides has shown great promise for large-scale studies, but has a reputation for poor specificity. We investigated the potential of IMAC in combination with capillary liquid chromatography coupled to tandem mass spectrometry for the identification of plasma membrane phosphoproteins of Arabidopsis. Without chemical modification of peptides, over 75% pure phosphopeptides were isolated from plasma membrane digests and detected and sequenced by mass spectrometry. We present a scheme for two-dimensional peptide separation using strong anion exchange chromatography prior to IMAC that both decreases the complexity of IMAC-purified phosphopeptides and yields a far greater coverage of monophosphorylated peptides. Among the identified sequences, six originated from different isoforms of the plasma membrane H+-ATPase and defined two previously unknown phosphorylation sites at the regulatory C terminus. The potential for large-scale identification of phosphorylation sites on plasma membrane proteins will have wide-ranging implications for research in signal transduction, cell-cell communication, and membrane transport processes.

Phosphoproteomics of the Arabidopsis Plasma Membrane and a New Phosphorylation Site Database

Functional genomic technologies are generating vast amounts of data describing the presence of transcripts or proteins in plant cells. Together with classical genetics, these approaches broaden our understanding of the gene products required for specific responses. Looking to the future, the focus of research must shift to the dynamic aspects of biology: molecular mechanisms of function and regulation. Phosphorylation is a key regulatory factor in all aspects of plant biology; but it is difficult, if not impossible, for most researchers to identify in vivo phosphorylation sites within their proteins of interest. We have developed a large-scale strategy for the isolation of phosphopeptides and identification by mass spectrometry (Nühse et al., 2003b). Here, we describe the identification of more than 300 phosphorylation sites from Arabidopsis thaliana plasma membrane proteins. These data will be a valuable resource for many fields of plant biology and overcome a major impediment to the elucidation of signal transduction pathways. We present an analysis of the characteristics of phosphorylation sites, their conservation among orthologs and paralogs, and the existence of putative motifs surrounding the sites. These analyses yield general principles for predicting other phosphorylation sites in plants and provide indications of specificity determinants for responsible kinases. In addition, more than 50 sites were mapped on receptor-like kinases and revealed an unexpected complexity of regulation. Finally, the data also provide empirical evidence on the topology of transmembrane proteins. This information indicates that prediction programs incorrectly identified the cytosolic portion of the protein in 25% of the transmembrane proteins found in this study. All data are deposited in a new searchable database for plant phosphorylation sites maintained by PlantsP (http://plantsp.sdsc.edu) that will be updated as the project expands to encompass additional tissues and organelles.

Highly selective enrichment of phosphorylated peptides using titanium dioxide

The characterization of phosphorylated proteins is a challenging analytical task since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric. Highly efficient enrichment procedures are therefore required. Here we describe a protocol for selective phosphopeptide enrichment using titanium dioxide (TiO2) chromatography. The selectivity toward phosphopeptides is obtained by loading the sample in a 2,5-dihydroxybenzoic acid (DHB) or phthalic acid solution containing acetonitrile and trifluoroacetic acid (TFA) onto a TiO2 micro-column. Although phosphopeptide enrichment can be achieved by using TFA and acetonitrile alone, the selectivity is dramatically enhanced by adding DHB or phthalic acid since these compounds, in conjunction with the low pH caused by TFA, prevent binding of nonphosphorylated peptides to TiO2. Using an alkaline solution (pH ≥ 10.5) both monophosphorylated and multiphosphorylated peptides are eluted from the TiO2 beads. This highly efficient method for purification of phosphopeptides is well suited for the characterization of phosphoproteins from both in vitro and in vivo studies in combination with mass spectrometry (MS). It is a very easy and fast method. The entire protocol requires less than 15 min per sample if the buffers have been prepared in advance (not including lyophilization).

Cross-frequency coupling supports multi-item working memory in the human hippocampus

Recent findings indicate that the hippocampus supports not only long-term memory encoding but also plays a role in working memory (WM) maintenance of multiple items; however, the neural mechanism underlying multi-item maintenance is still unclear. Theoretical work suggests that multiple items are being maintained by neural assemblies synchronized in the gamma frequency range (25–100 Hz) that are locked to consecutive phase ranges of oscillatory activity in the theta frequency range (4–8 Hz). Indeed, cross-frequency coupling of the amplitude of high-frequency activity to the phase of slower oscillations has been described both in animals and in humans, but has never been linked to a theoretical model of a cognitive process. Here we used intracranial EEG recordings in human epilepsy patients to test pivotal predictions from theoretical work. First, we show that simultaneous maintenance of multiple items in WM is accompanied by cross-frequency coupling of oscillatory activity in the hippocampus, which is recruited during multi-item WM. Second, maintenance of an increasing number of items is associated with modulation of beta/gamma amplitude with theta band activity of lower frequency, consistent with the idea that longer cycles are required for an increased number of representations by gamma cycles. This effect cannot be explained by a difference in theta or beta/gamma power. Third, we describe how the precision of cross-frequency coupling predicts individual WM performance. These data support the idea that working memory in humans depends on a neural code using phase information.

Biomarker Discovery from Pancreatic Cancer Secretome Using a Differential Proteomic Approach

Quantitative proteomics can be used as a screening tool for identification of differentially expressed proteins as potential biomarkers for cancers. Candidate biomarkers from such studies can subsequently be tested using other techniques for use in early detection of cancers. Here we demonstrate the use of stable isotope labeling with amino acids in cell culture (SILAC) method to compare the secreted proteins (secretome) from pancreatic cancer-derived cells with that from non-neoplastic pancreatic ductal cells. We identified 145 differentially secreted proteins (>1.5-fold change), several of which were previously reported as either up-regulated (e.g. cathepsin D, macrophage colony stimulation factor, and fibronectin receptor) or down-regulated (e.g. profilin 1 and IGFBP-7) proteins in pancreatic cancer, confirming the validity of our approach. In addition, we identified several proteins that have not been correlated previously with pancreatic cancer including perlecan (HSPG2), CD9 antigen, fibronectin receptor (integrin β1), and a novel cytokine designated as predicted osteoblast protein (FAM3C). The differential expression of a subset of these novel proteins was validated by Western blot analysis. In addition, overexpression of several proteins not described previously to be elevated in human pancreatic cancer (CD9, perlecan, SDF4, apoE, and fibronectin receptor) was confirmed by immunohistochemical labeling using pancreatic cancer tissue microarrays suggesting that these could be further pursued as potential biomarkers. Lastly the protein expression data from SILAC were compared with mRNA expression data obtained using gene expression microarrays for the two cell lines (Panc1 and human pancreatic duct epithelial), and a correlation coefficient (r) of 0.28 was obtained, confirming previously reported poor associations between RNA and protein expression studies.

Analytical strategies for phosphoproteomics

Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events in which the complex interplay between protein kinases and protein phosphatases strictly controls biological processes such as proliferation, differentiation, and apoptosis. Defective or altered signaling pathways often result in abnormalities leading to various diseases, emphasizing the importance of understanding protein phosphorylation. Phosphorylation is a transient modification, and phosphoproteins are often very low abundant. Consequently, phosphoproteome analysis requires highly sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phospho‐specific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis will be on the affinity methods utilized specifically for phosphoprotein and phosphopeptide enrichment prior to MS analysis, and on recent applications of these methods in cell biological applications.

Interpreting the protein language using proteomics.

Post-translational modifications define the functional and structural plasticity of proteins in archaea, prokaryotes and eukaryotes. Multi-site protein modification modulates protein activity and macromolecular interactions and is involved in a range of fundamental molecular processes. Combining state-of-the-art technologies in molecular cell biology, protein mass spectrometry and bioinformatics, it is now feasible to discover and study the structural and functional roles of distinct protein post-translational modifications.