SimPhospho: a software tool enabling confident phosphosite assignment

Motivation: Mass spectrometry combined with enrichment strategies for phosphorylated peptides has been successfully employed for two decades to identify sites of phosphorylation. However, unambiguous phosphosite assignment is considered challenging. Given that site-specific phosphorylation events function as different molecular switches, validation of phosphorylation sites is of utmost importance. In our earlier study we developed a method based on simulated phospho-peptide spectral libraries, which enables highly sensitive and accurate phosphosite assignments. To promote more widespread use of this method, we here introduce a software implementation with improved usability and performance.Results: We present SimPhospho, a fast and user-friendly tool for accurate simulation of phospho-peptide tandem mass spectra. Simulated phosphopeptide spectral libraries are used to validate and supplement database search results, with a goal to improve reliable phosphoproteome identification and reporting. The presented program can be easily used together with the TransProteomic Pipeline and integrated in a phosphoproteomics data analysis workflow.Availability and implementation: SimPhospho is open source and it is available for Windows, Linux and Mac operating systems. The software and its user's manual with detailed description of data analysis as well as test data can be found at https://sourceforge.net/projects/simphospho/.Contact: [email protected] or G.L. [email protected] information: Supplementary data are available at Bioinformatics online

Phosphorylation of NFATC1 at PIM1 target sites is essential for its ability to promote prostate cancer cell migration and invasion

Background Progression of prostate cancer from benign local tumors to metastatic carcinomas is a multistep process. Here we have investigated the signaling pathways that support migration and invasion of prostate cancer cells, focusing on the role of the NFATC1 transcription factor and its post-translational modifications. We have previously identified NFATC1 as a substrate for the PIM1 kinase and shown that PIM1-dependent phosphorylation increases NFATC1 activity without affecting its subcellular localization. Both PIM kinases and NFATC1 have been reported to promote cancer cell migration, invasion and angiogenesis, but it has remained unclear whether the effects of NFATC1 are phosphorylation-dependent and which downstream targets are involved. Methods We used mass spectrometry to identify PIM1 phosphorylation target sites in NFATC1, and analysed their functional roles in three prostate cancer cell lines by comparing phosphodeficient mutants to wild-type NFATC1. We used luciferase assays to determine effects of phosphorylation on NFAT-dependent transcriptional activity, and migration and invasion assays to evaluate effects on cell motility. We also performed a microarray analysis to identify novel PIM1/NFATC1 targets, and validated one of them with both cellular expression analyses and in silico in clinical prostate cancer data sets. Results Here we have identified ten PIM1 target sites in NFATC1 and found that prevention of their phosphorylation significantly decreases the transcriptional activity as well as the pro-migratory and pro-invasive effects of NFATC1 in prostate cancer cells. We observed that also PIM2 and PIM3 can phosphorylate NFATC1, and identified several novel putative PIM1/NFATC1 target genes. These include the ITGA5 integrin, which is differentially expressed in the presence of wild-type versus phosphorylation-deficient NFATC1, and which is coexpressed with PIM1 and NFATC1 in clinical prostate cancer specimens. Conclusions Based on our data, phosphorylation of PIM1 target sites stimulates NFATC1 activity and enhances its ability to promote prostate cancer cell migration and invasion. Therefore, inhibition of the interplay between PIM kinases and NFATC1 may have therapeutic implications for patients with metastatic forms of cancer.Peer reviewe

Faecal Metaproteomic Analysis Reveals a Personalized and Stable Functional Microbiome and Limited Effects of a Probiotic Intervention in Adults

Recent metagenomic studies have demonstrated that the overall functional potential of the intestinal microbiome is rather conserved between healthy individuals. Here we assessed the biological processes undertaken in-vivo by microbes and the host in the intestinal tract by conducting a metaproteome analysis from a total of 48 faecal samples of 16 healthy adults participating in a placebo-controlled probiotic intervention trial. Half of the subjects received placebo and the other half consumed Lactobacillus rhamnosus GG for three weeks (10(10) cfu per day). Faecal samples were collected just before and at the end of the consumption phase as well as after a three-week follow-up period, and were processed for microbial composition and metaproteome analysis. A common core of shared microbial protein functions could be identified in all subjects. Furthermore, we observed marked differences in expressed proteins between subjects that resulted in the definition of a stable and personalized microbiome both at the mass-spectrometry-based proteome level and the functional level based on the KEGG pathway analysis. No significant changes in the metaproteome were attributable to the probiotic intervention. A detailed taxonomic assignment of peptides and comparison to phylogenetic microarray data made it possible to evaluate the activity of the main phyla as well as key species, including Faecalibacterium prausnitzii. Several correlations were identified between human and bacterial proteins. Proteins of the human host accounted for approximately 14% of the identified metaproteome and displayed variations both between and within individuals. The individually different human intestinal proteomes point to personalized host-microbiota interactions. Our findings indicate that analysis of the intestinal metaproteome can complement gene-based analysis and contributes to a thorough understanding of the activities of the microbiome and the relevant pathways in health and disease.Peer reviewe

Phosphorylation of NFATC1 at PIM1 target sites is essential for its ability to promote prostate cancer cell migration and invasion

Background Progression of prostate cancer from benign local tumors to metastatic carcinomas is a multistep process. Here we have investigated the signaling pathways that support migration and invasion of prostate cancer cells, focusing on the role of the NFATC1 transcription factor and its post-translational modifications. We have previously identified NFATC1 as a substrate for the PIM1 kinase and shown that PIM1-dependent phosphorylation increases NFATC1 activity without affecting its subcellular localization. Both PIM kinases and NFATC1 have been reported to promote cancer cell migration, invasion and angiogenesis, but it has remained unclear whether the effects of NFATC1 are phosphorylation-dependent and which downstream targets are involved. Methods We used mass spectrometry to identify PIM1 phosphorylation target sites in NFATC1, and analysed their functional roles in three prostate cancer cell lines by comparing phosphodeficient mutants to wild-type NFATC1. We used luciferase assays to determine effects of phosphorylation on NFAT-dependent transcriptional activity, and migration and invasion assays to evaluate effects on cell motility. We also performed a microarray analysis to identify novel PIM1/NFATC1 targets, and validated one of them with both cellular expression analyses and in silico in clinical prostate cancer data sets. Results Here we have identified ten PIM1 target sites in NFATC1 and found that prevention of their phosphorylation significantly decreases the transcriptional activity as well as the pro-migratory and pro-invasive effects of NFATC1 in prostate cancer cells. We observed that also PIM2 and PIM3 can phosphorylate NFATC1, and identified several novel putative PIM1/NFATC1 target genes. These include the ITGA5 integrin, which is differentially expressed in the presence of wild-type versus phosphorylation-deficient NFATC1, and which is coexpressed with PIM1 and NFATC1 in clinical prostate cancer specimens. Conclusions Based on our data, phosphorylation of PIM1 target sites stimulates NFATC1 activity and enhances its ability to promote prostate cancer cell migration and invasion. Therefore, inhibition of the interplay between PIM kinases and NFATC1 may have therapeutic implications for patients with metastatic forms of cancer

Ataxin-3 Plays a Role in Mouse Myogenic Differentiation through Regulation of Integrin Subunit Levels

BACKGROUND: During myogenesis several transcription factors and regulators of protein synthesis and assembly are rapidly degraded by the ubiquitin-proteasome system (UPS). Given the potential role of the deubiquitinating enzyme (DUB) ataxin-3 in the UPS, and the high expression of the murine ataxin-3 homolog in muscle during embryogenesis, we sought to define its role in muscle differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Using immunofluorescence analysis, we found murine ataxin-3 (mATX3) to be highly expressed in the differentiated myotome of E9.5 mouse embryos. C2C12 myoblasts depleted of mATX3 by RNA interference exhibited a round morphology, cell misalignment, and a delay in differentiation following myogenesis induction. Interestingly, these cells showed a down-regulation of alpha5 and alpha7 integrin subunit levels both by immunoblotting and immunofluorescence. Mouse ATX3 was found to interact with alpha5 integrin subunit and to stabilize this protein by repressing its degradation through the UPS. Proteomic analysis of mATX3-depleted C2C12 cells revealed alteration of the levels of several proteins related to integrin signaling. CONCLUSIONS: Ataxin-3 is important for myogenesis through regulation of integrin subunit levels.This work was financed by the Fundacao para a Ciencia e a Tecnologia (FCT) (POCI/SAU-MMO/60412/2002) and by National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) grant RO1 NS038712 to HLP. MCC, FB, AJR, and RJT were supported by the FCT fellowships (SFRH/BD/9759/2003 and SFRH/BPD/28560/2006), (SFRH/BPD/17368/2004), (SFRH/BD/17066/2004), (SFRH/BD/29947/2006), respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Enrichment and sequencing of phosphopeptides on indium tin oxide coated glass slides

Unambiguous identification of phosphorylation sites is of premier importance to biologists, who seek to understand the role of phosphorylation from the perspective of site-specific control of biological phenomena. Despite this widely asked and highly specific information, many methods developed are aimed at analysis of complete proteomes, indeed even phospho-proteomes, surpassing the basic requests of many biologists. We have therefore further developed a simple method that specifically deals with the analysis of multiple phosphorylation sites on singular proteins or small collections of proteins. With this method, the whole purification process, from sample application to MALDI-MS analysis, can be performed on commercially available indium tin oxide (ITO) coated glass slides. We show that fifteen (15) samples can be purified within one hour, and that low femtomole sensitivity can be achieved. This limit of identification is demonstrated by the successful MS/MS-based identification of 6 fmol of monophosphopeptide from β-casein. We demonstrate that the method can be applied for identifying phosphorylation sites from recombinant and cell-derived biological protein samples. Since ITO-coated glass slides are inexpensive and available from several suppliers the method is readily and inexpensively available to other researchers. Taken together, the presented protocols and materials render this method as an extremely fast and sensitive phosphopeptide identification protocol that should aid biologists in discovery and validation of phosphorylation sites

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry for quantitation and molecular stability assessment of insulin entrapped within PLGA nanoparticles

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) was evaluated for both qualitative and quantitative analysis of insulin entrapped within poly(D,L‐lactic‐co‐glycolic acid) nanoparticles. Quantitation was performed by adding an internal standard (arg‐insulin) to defined and unknown sample solutions, in order to reduce point‐to‐point and sample‐to‐sample variability. The ratio of the peak height of insulin to the peak height of arg‐insulin was plotted against the insulin concentration. In this way, an excellent linear relationship was found (R2 > 0.99). This method of quantitation was compared with classical UV spectroscopy and reverse‐phase high‐performance liquid chromatography measurements. All methods provided close final drug loading values for the insulin‐loaded nanoparticle batches tested. Additionally, with respect to molecular stability, covalent insulin dimers were found only at trace levels in those nanoparticles. Compared with other methods, MALDI‐TOF MS is a valuable tool for the characterization of proteins from nanoparticles, because no extensive extraction and complex sampling procedures are required