Nutrition factors associated with rib stress injury history in elite rowers.

OBJECTIVES: To investigate associations between nutrition factors (diet restriction, menstrual status, calcium intake, vitamin D and K status), bone mineral density (BMD) and rib stress injury (RSI) history. DESIGN: Cross-sectional. METHODS: 133 elite rowers completed a self-report questionnaire to collect information regarding training and injury history, menstrual status and diet restriction, and a calcium intake questionnaire (SCQ2002). BMD and body composition were assessed by dual-energy X-ray absorptiometry. A sub-group (n = 68) had vitamin D and K status assessed from fasted morning blood. History of RSI was self-reported and verified against medical records. Characteristics of injured and uninjured rowers were compared (one-way ANOVA), while relationships with BMD (multiple linear regression) and RSI (multiple logistic regression) were modelled. RESULTS: Diet restriction was inversely related to spine BMD and rib BMD. Within sex, vitamin D and K status, and calcium intake were not associated with injury. Among rowers with RSI history, lightweight males had lower total bone mass, femur BMD and rib BMD, whereas heavyweight females had lower rib BMD. In relation to RSI history, the best models included rib, spine or femur BMD with age, body fat and sex. A female-specific model included rib BMD, current menstrual dysfunction, age and body fat levels. CONCLUSIONS: BMD, including that of the rib, diet restriction, menstrual function and weight category were associated with rib injury history and should be considered in the management of elite rowers.

Early DNA methylation changes in children developing beta cell autoimmunity at a young age.

AIMS/HYPOTHESIS: Type 1 diabetes is a chronic autoimmune disease of complex aetiology, including a potential role for epigenetic regulation. Previous epigenomic studies focused mainly on clinically diagnosed individuals. The aim of the study was to assess early DNA methylation changes associated with type 1 diabetes already before the diagnosis or even before the appearance of autoantibodies. METHODS: Reduced representation bisulphite sequencing (RRBS) was applied to study DNA methylation in purified CD4+ T cell, CD8+ T cell and CD4-CD8- cell fractions of 226 peripheral blood mononuclear cell samples longitudinally collected from seven type 1 diabetes-specific autoantibody-positive individuals and control individuals matched for age, sex, HLA risk and place of birth. We also explored correlations between DNA methylation and gene expression using RNA sequencing data from the same samples. Technical validation of RRBS results was performed using pyrosequencing. RESULTS: We identified 79, 56 and 45 differentially methylated regions in CD4+ T cells, CD8+ T cells and CD4-CD8- cell fractions, respectively, between type 1 diabetes-specific autoantibody-positive individuals and control participants. The analysis of pre-seroconversion samples identified DNA methylation signatures at the very early stage of disease, including differential methylation at the promoter of IRF5 in CD4+ T cells. Further, we validated RRBS results using pyrosequencing at the following CpG sites: chr19:18118304 in the promoter of ARRDC2; chr21:47307815 in the intron of PCBP3; and chr14:81128398 in the intergenic region near TRAF3 in CD4+ T cells. CONCLUSIONS/INTERPRETATION: These preliminary results provide novel insights into cell type-specific differential epigenetic regulation of genes, which may contribute to type 1 diabetes pathogenesis at the very early stage of disease development. Should these findings be validated, they may serve as a potential signature useful for disease prediction and management.

Reproducibility-optimized detection of differential DNA methylation.

Aim: DNA methylation is a key epigenetic mechanism regulating gene expression. Identifying differentially methylated regions is integral to DNA methylation analysis and there is a need for robust tools reliably detecting regions with significant differences in their methylation status. Materials & methods: We present here a reproducibility-optimized test statistic (ROTS) for detection of differential DNA methylation from high-throughput sequencing or array-based data. Results: Using both simulated and real data, we demonstrate the ability of ROTS to identify differential methylation between sample groups. Conclusion: Compared with state-of-the-art methods, ROTS shows competitive sensitivity and specificity in detecting consistently differentially methylated regions.

Phosphoproteome and drug-response effects mediated by the three protein phosphatase 2A inhibitor proteins CIP2A, SET, and PME-1.

Protein phosphatase 2A (PP2A) critically regulates cell signaling and is a human tumor suppressor. PP2A complexes are modulated by proteins such as cancerous inhibitor of protein phosphatase 2A (CIP2A), protein phosphatase methylesterase 1 (PME-1), and SET nuclear proto-oncogene (SET) that often are deregulated in cancers. However, how they impact cellular phosphorylation and how redundant they are in cellular regulation is poorly understood. Here, we conducted a systematic phosphoproteomics screen for phosphotargets modulated by siRNA-mediated depletion of CIP2A, PME-1, and SET (to reactivate PP2A) or the scaffolding A-subunit of PP2A (PPP2R1A) (to inhibit PP2A) in HeLa cells. We identified PP2A-modulated targets in diverse cellular pathways, including kinase signaling, cytoskeleton, RNA splicing, DNA repair, and nuclear lamina. The results indicate nonredundancy among CIP2A, PME-1, and SET in phosphotarget regulation. Notably, PP2A inhibition or reactivation affected largely distinct phosphopeptides, introducing a concept of nonoverlapping phosphatase inhibition- and activation-responsive sites (PIRS and PARS, respectively). This phenomenon is explained by the PPP2R1A inhibition impacting primarily dephosphorylated threonines, whereas PP2A reactivation results in dephosphorylation of clustered and acidophilic sites. Using comprehensive drug-sensitivity screening in PP2A-modulated cells to evaluate the functional impact of PP2A across diverse cellular pathways targeted by these drugs, we found that consistent with global phosphoproteome effects, PP2A modulations broadly affect responses to more than 200 drugs inhibiting a broad spectrum of cancer-relevant targets. These findings advance our understanding of the phosphoproteins, pharmacological responses, and cellular processes regulated by PP2A modulation and may enable the development of combination therapies.

Optimization of TripleTOF spectral simulation and library searching for confident localization of phosphorylation sites.

Tandem mass spectrometry (MS/MS) has been used in analysis of proteins and their post-translational modifications. A recently developed data analysis method, which simulates MS/MS spectra of phosphopeptides and performs spectral library searching using SpectraST, facilitates confident localization of phosphorylation sites. However, its performance has been evaluated only on MS/MS spectra acquired using Orbitrap HCD mass spectrometers so far. In this study, we have investigated whether this approach would be applicable to another type of mass spectrometers, and optimized the simulation and search conditions to achieve sensitive and confident site localization. Synthetic phosphopeptides and enriched K562 cell phosphopeptides were analyzed using a TripleTOF 6600 mass spectrometer before and after enzymatic dephosphorylation. Dephosphorylated peptides identified by X!Tandem database searching were subjected to spectral simulation of all possible single phosphorylations using SimPhospho software. Phosphopeptides were identified and localized by SpectraST searching against a library of the simulated spectra. Although no synthetic phosphopeptide was localized at 1% false localization rate under the previous conditions, optimization of the spectral simulation and search conditions for the TripleTOF datasets achieved the localization and improved the sensitivity. Furthermore, the optimized conditions enabled sensitive localization of K562 phosphopeptides at 1% false discovery and localization rates. These results suggest that accurate phosphopeptide simulation of TripleTOF MS/MS spectra is possible and the simulated spectral libraries can be used in SpectraST searching for confident localization of phosphorylation sites.

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 phosphopeptide 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 phosphopeptide 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 Trans-Proteomic 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/. Supplementary information: Supplementary data are available at Bioinformatics online.

Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling.

Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantification. To allow data normalization in situations where depletion of RAS or PP2A inhibitor CIP2A causes a large uni-directional change in the phosphopeptide abundance, we developed a novel normalization strategy, named pairwise normalization. This normalization is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of the pairwise normalization was verified by various independent methods. Additionally, we demonstrate how the selected normalization method influences the downstream analyses and interpretation of pathway activities. Consequently, bioinformatics analysis of RAS and CIP2A regulated phosphoproteomes revealed a significant overlap in their functional pathways. This is most likely biologically meaningful as we observed a synergistic survival effect between CIP2A and RAS expression as well as KRAS activating mutations in TCGA pan-cancer data set, and synergistic relationship between CIP2A and KRAS depletion in colony growth assays.

Confident site localization using a simulated phosphopeptide spectral library.

We have investigated if phosphopeptide identification and simultaneous site localization can be achieved by spectral library searching. This allows taking advantage of comparison of specific spectral features, which would lead to improved discrimination of differential localizations. For building a library, we propose a spectral simulation strategy where all possible single phosphorylations can be simply and accurately (re)constructed on enzymatically dephosphorylated peptides, by predicting the diagnostic fragmentation events produced in beam-type CID. To demonstrate the performance of our approach, enriched HeLa phosphopeptides were dephosphorylated with alkaline phosphatase and analyzed with higher energy collisional dissociation (HCD), which were then used for creating a spectral library of simulated phosphopeptides. Spectral library searching using SpectraST was performed on data sets of synthetic phosphopeptides and the HeLa phosphopeptides, and subsequently compared to Mascot and Sequest database searching followed by phosphoRS and Ascore afforded localization, respectively. Our approach successfully led to accurate localization, and it outperformed other methods, when phosphopeptides were covered by the library. These results suggest that the searching with simulated spectral libraries serves as a crucial approach for both supplementing and validating the phosphorylation sites obtained by database searching and localization tools. For future development, simulation of multiply phosphorylated peptides remains to be implemented.

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 ß-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.