Quantitative phosphoproteomics dissection of seven-transmembrane receptor signaling using full and biased agonists.

Seven-transmembrane receptors (7TMRs) signal through the well described heterotrimeric G proteins but can also activate G protein-independent signaling pathways of which the impact and complexity are less understood. The angiotensin II type 1 receptor (AT(1)R) is a prototypical 7TMR and an important drug target in cardiovascular diseases. "Biased agonists" with intrinsic "functional selectivity" that simultaneously blocks Galpha(q) protein activity and activates G protein-independent pathways of the AT(1)R confer important perspectives in treatment of cardiovascular diseases. In this study, we performed a global quantitative phosphoproteomics analysis of the AT(1)R signaling network. We analyzed ligand-stimulated SILAC (stable isotope labeling by amino acids in cell culture) cells by high resolution (LTQ-Orbitrap) MS and compared the phosphoproteomes of the AT(1)R agonist angiotensin II and the biased agonist [Sar(1),Ile(4),Ile(8)]angiotensin II (SII angiotensin II), which only activates the Galpha(q) protein-independent signaling. We quantified more than 10,000 phosphorylation sites of which 1183 were regulated by angiotensin II or its analogue SII angiotensin II. 36% of the AT(1)R-regulated phosphorylations were regulated by SII angiotensin II. Analysis of phosphorylation site patterns showed a striking distinction between protein kinases activated by Galpha(q) protein-dependent and -independent mechanisms, and we now place protein kinase D as a key protein involved in both Galpha(q)-dependent and -independent AT(1)R signaling. This study provides substantial novel insight into angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity and quantity of Galpha(q) protein-independent signaling and uncovers novel signaling pathways. We foresee that the amount and diversity of G protein-independent signaling may be more pronounced than previously recognized for other 7TMRs as well. Quantitative mass spectrometry is a promising tool for evaluation of the signaling properties of biased agonists to other receptors in the future.

Upregulated expression of human neutrophil peptides 1, 2 and 3 (HNP 1-3) in colon cancer serum and tumours: a biomarker study.

BACKGROUND: Molecular markers for localized colon tumours and for prognosis following therapy are needed. Proteomics research is currently producing numerous biomarker studies with clinical potential. We investigate the protein composition of plasma and of tumour extracts with the aim of identifying biomarkers for colon cancer. METHODS: By Surface Enhanced Laser Desorption/Ionisation--Time Of Flight/Mass spectrometry (SELDI-TOF/MS) we compare the protein profiles of colon cancer serum with serum from healthy individuals and the protein profiles of colon tumours with normal colon tissue. By size exclusion chromatography, we investigate the binding of HNP 1-3 to high mass plasma proteins. By microflow we investigate the effect of HNP 1-3 on mammalian cells. RESULTS: Human Neutrophil Peptides -1, -2 and -3 (HNP 1-3), also known as alfa-defensin-1, -2 and -3, are present in elevated concentrations in serum from colon cancer patients and in protein extracts from colon tumours. A fraction of HNP 1-3 in serum is bound to unidentified high mass plasma proteins. HNP 1-3 purified from colon tumours are lethal to mammalian cells. CONCLUSIONS: HNP 1-3 may serve as blood markers for colon cancer in combination with other diagnostic tools. We propose that HNP 1-3 are carried into the bloodstream by attaching to high mass plasma proteins in the tumour microenvironment. We discuss the effect of HNP 1-3 on tumour progression.

Predicting kinase activity in angiotensin receptor phosphoproteomes based on sequence-motifs and interactions.

Recent progress in the understanding of seven-transmembrane receptor (7TMR) signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT1aR) is one of the most studied 7TMRs with respect to selective activation of the ß-arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data. The method builds upon NetworKIN, which applies sophisticated linear motif analysis in combination with contextual network modelling to predict kinase-substrate associations with high accuracy and sensitivity. These predictions form the basis for subsequently nonparametric statistical analysis to identify likely activated kinases. This suggested that AT1aR-dependent signalling activates 48 of the 285 kinases detected in HEK293 cells. Of these, Aurora B, CLK3 and PKG1 have not previously been described in the pathway whereas others, such as PKA, PKB and PKC, are well known. In summary, we have developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets.

Candidate biomarker verification: Critical examination of a serum protein pattern for human colorectal cancer.

PURPOSE: We critically examine a candidate serum protein pattern for human colorectal cancer (CRC) with respect to reproducibility, sample handling, and disease specificity. EXPERIMENTAL DESIGN: Serum samples from CRC patients, patients with benign colon tumors and healthy individuals, were obtained at two collection sites and analyzed by SELDI-TOF MS on 8 days, over a period of 5 weeks. The spectra were subjected to multivariate analysis. Tissues from normal colon and CRC were analyzed by SELDI-TOF MS. Selected mass peaks were identified. RESULTS: Using an elaborate experimental design we developed a multivariate classifier that correctly classified CRC and control serum measured on an independent day. The classifier did not discriminate between samples from CRC patients and patients with benign colon tumors, and, secondly, did not correctly classify serum from an independent collection site. All discriminatory mass peaks were identified as high abundant plasma proteins. Tissue profiling provided support of increased proteolytic activity in CRC tissue. CONCLUSION AND CLINICAL RELEVANCE: Critical verification did not justify advancing the identified CRC serum protein pattern into clinical validation without improvement. We believe that proteomics biomarker research could benefit if the presented, or a similar, verification scheme was more commonly employed in explorative biomarker studies.

Preanalytical and analytical variation of surface-enhanced laser desorption-ionization time-of-flight mass spectrometry of human serum.

BACKGROUND: Surface-enhanced laser desorption-ionization time-of-flight (SELDI-TOF) mass spectrometry of human serum is a potential diagnostic tool in human diseases. In the present study, the preanalytical and analytical variation of SELDI-TOF mass spectrometry of serum was assessed in healthy individuals. METHODS: Serum and plasma were obtained from healthy human individuals. Protein peaks in human serum and plasma were determined by SELDI-TOF mass spectrometry. RESULTS: The protein peaks in serum in healthy individuals showed no variation with gender, age, fasting, or diurnal rhythm. The intensity of protein peaks changed significantly during clotting of serum from 30 to 60 min, followed by smaller changes from 60 to 120 min. The intensity of protein peaks changed after 60-min storage of serum at room temperature, whereas no changes were observed for storage on ice. The number of reproducible protein peaks in serum was determined on WCX2, SAX2, and IMAC30 arrays as 29, 34, and 36, respectively. The average coefficient of variation (CV) of the mass value of protein peaks was 0.03%. The intra-assay CV of peak intensity on IMAC30 arrays was 16% (10%-36%, n=8) for 36 peaks, inter-assay CV was 18% (6%-34%, n=4) for 16 peaks, and inter-individual CV was 38% (16%-56%, n=16) for 20 peaks. CONCLUSIONS: The pre-analytical and analytical conditions of SELDI-TOF mass spectrometry of serum have a significant impact on the protein peaks, with the number of peaks low and the assay variation high. Consequently, SELDI-TOF mass spectrometry of serum needs further evaluation before application in clinical diagnostics.