Automated Enrichment of Phosphotyrosine Peptides for High-Throughput Proteomics.

Phosphotyrosine (pY) enrichment is critical for expanding the fundamental and clinical understanding of cellular signaling by mass spectrometry-based proteomics. However, current pY enrichment methods exhibit a high cost per sample and limited reproducibility due to expensive affinity reagents and manual processing. We present rapid-robotic phosphotyrosine proteomics (R2-pY), which uses a magnetic particle processor and pY superbinders or antibodies. R2-pY can handle up to 96 samples in parallel, requires 2 days to go from cell lysate to mass spectrometry injections, and results in global proteomic, phosphoproteomic, and tyrosine-specific phosphoproteomic samples. We benchmark the method on HeLa cells stimulated with pervanadate and serum and report over 4000 unique pY sites from 1 mg of peptide input, strong reproducibility between replicates, and phosphopeptide enrichment efficiencies above 99%. R2-pY extends our previously reported R2-P2 proteomic and global phosphoproteomic sample preparation framework, opening the door to large-scale studies of pY signaling in concert with global proteome and phosphoproteome profiling.

Anticodon sequence determines the impact of mistranslating tRNAAla variants.

Transfer RNAs (tRNAs) maintain translation fidelity through accurate charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA at the ribosome. Mistranslation occurs when an amino acid not specified by the genetic message is incorporated into proteins and has applications in biotechnology, therapeutics and is relevant to disease. Since the alanyl-tRNA synthetase uniquely recognizes a G3:U70 base pair in tRNAAla and the anticodon plays no role in charging, tRNAAla variants with anticodon mutations have the potential to mis-incorporate alanine. Here, we characterize the impact of the 60 non-alanine tRNAAla anticodon variants on the growth of Saccharomyces cerevisiae. Overall, 36 tRNAAla anticodon variants decreased growth in single- or multi-copy. Mass spectrometry analysis of the cellular proteome revealed that 52 of 57 anticodon variants, not decoding alanine or stop codons, induced mistranslation when on single-copy plasmids. Variants with G/C-rich anticodons resulted in larger growth deficits than A/U-rich variants. In most instances, synonymous anticodon variants impact growth differently, with anticodons containing U at base 34 being the least impactful. For anticodons generating the same amino acid substitution, reduced growth generally correlated with the abundance of detected mistranslation events. Differences in decoding specificity, even between synonymous anticodons, resulted in each tRNAAla variant mistranslating unique sets of peptides and proteins. We suggest that these differences in decoding specificity are also important in determining the impact of tRNAAla anticodon variants.

IsobaricQuant enables cross-platform quantification, visualization, and filtering of isobarically-labeled peptides.

In mass spectrometry (MS)-based quantitative proteomics, labeling with isobaric mass tags such as iTRAQ and TMT can substantially improve sample throughput and reduce peptide missing values. Nonetheless, the quantification of labeled peptides tends to suffer from reduced accuracy due to the co-isolation of co-eluting precursors of similar mass-to-charge. Acquisition approaches such as multistage MS3 or ion mobility separation address this problem, yet are difficult to audit and limited to expensive instrumentation. Here we introduce IsobaricQuant, an open-source software tool for quantification, visualization, and filtering of peptides labeled with isobaric mass tags, with specific focus on precursor interference. IsobaricQuant is compatible with MS2 and MS3 acquisition strategies, has a viewer that allows assessing interference, and provides several scores to aid the filtering of scans with compression. We demonstrate that IsobaricQuant quantifications are accurate by comparing it with commonly used software. We further show that its QC scores can successfully filter out scans with reduced quantitative accuracy at MS2 and MS3 levels, removing inaccurate peptide quantifications and decreasing protein CVs. Finally, we apply IsobaricQuant to a PISA dataset and show that QC scores improve the sensitivity of the identification of protein targets of a kinase inhibitor. IsobaricQuant is available at https://github.com/Villen-Lab/isobaricquant.

Coisolation of Peptide Pairs for Peptide Identification and MS/MS-Based Quantification.

Stable-isotope labeling with amino acids in cell culture (SILAC)-based metabolic labeling is a widely adopted proteomics approach that enables quantitative comparisons among a variety of experimental conditions. Despite its quantitative capacity, SILAC experiments analyzed with data-dependent acquisition (DDA) do not fully leverage peptide pair information for identification and suffer from undersampling compared to label-free proteomic experiments. Herein, we developed a DDA strategy that coisolates and fragments SILAC peptide pairs and uses y-ions for their relative quantification. To facilitate the analysis of this type of data, we adapted the Comet sequence database search engine to make use of SILAC peptide paired fragments and developed a tool to annotate and quantify MS/MS spectra of coisolated SILAC pairs. This peptide pair coisolation approach generally improved expectation scores compared to the traditional DDA approach. Fragment ion quantification performed similarly well to precursor quantification in the MS1 and achieved more quantifications. Lastly, our method enables reliable MS/MS quantification of SILAC proteome mixtures with overlapping isotopic distributions. This study shows the feasibility of the coisolation approach. Coupling this approach with intelligent acquisition strategies has the potential to improve SILAC peptide sampling and quantification.

DIAmeter: matching peptides to data-independent acquisition mass spectrometry data.

MOTIVATION: Tandem mass spectrometry data acquired using data independent acquisition (DIA) is challenging to interpret because the data exhibits complex structure along both the mass-to-charge (m/z) and time axes. The most common approach to analyzing this type of data makes use of a library of previously observed DIA data patterns (a 'spectral library'), but this approach is expensive because the libraries do not typically generalize well across laboratories. RESULTS: Here, we propose DIAmeter, a search engine that detects peptides in DIA data using only a peptide sequence database. Although some existing library-free DIA analysis methods (i) support data generated using both wide and narrow isolation windows, (ii) detect peptides containing post-translational modifications, (iii) analyze data from a variety of instrument platforms and (iv) are capable of detecting peptides even in the absence of detectable signal in the survey (MS1) scan, DIAmeter is the only method that offers all four capabilities in a single tool. AVAILABILITY AND IMPLEMENTATION: The open source, Apache licensed source code is available as part of the Crux mass spectrometry analysis toolkit (http://crux.ms). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

mRNA destabilization is the dominant effect of mammalian microRNAs by the time substantial repression ensues.

MicroRNAs (miRNAs) regulate target mRNAs through a combination of translational repression and mRNA destabilization, with mRNA destabilization dominating at steady state in the few contexts examined globally. Here, we extend the global steady-state measurements to additional mammalian contexts and find that regardless of the miRNA, cell type, growth condition, or translational state, mRNA destabilization explains most (66%->90%) miRNA-mediated repression. We also determine the relative dynamics of translational repression and mRNA destabilization for endogenous mRNAs as a miRNA is induced. Although translational repression occurs rapidly, its effect is relatively weak, such that by the time consequential repression ensues, the effect of mRNA destabilization dominates. These results imply that consequential miRNA-mediated repression is largely irreversible and provide other insights into the nature of miRNA-mediated regulation. They also simplify future studies, dramatically extending the known contexts and time points for which monitoring mRNA changes captures most of the direct miRNA effects.

Clinical features associated with the invasive component in lentigo maligna of the head and neck: A retrospective study of 175 cases.

BACKGROUND: Lentigo maligna (LM) can develop into lentigo maligna melanoma (LMM) with risk of metastatic dissemination. LMM may be underestimated on the basis of the initial biopsy. The invasion may affect both the therapeutic options and the prognosis. OBJECTIVES: To identify the clinical features associated with invasive forms of LM and factors associated with its recurrence. METHODS: A retrospective, single-centre study of consecutive LM and LMM histologically confirmed and treated by surgery between 2009 and 2014. RESULTS: In total, 175 patients with LM/LMM were surgically treated in our establishment. In men, lesions were more likely to be in the "peripheral zone" (41.8%), while in women they were seen more often in the "central zone" (P=0.001). In multivariate analysis, only the peripheral zone was found to be associated with a risk of invasion (P=0.008). The rate of recurrence was 9% and lesions were more likely to be primary LMM (P=0.0006) excised with clear margins. CONCLUSION: The treatment of choice in LM with non-clear margins must be re-excision, especially for lesions situated in the peripheral zone. Close follow-up is recommended due to risk of recurrence, even in the case of clear margins.

R2-P2 rapid-robotic phosphoproteomics enables multidimensional cell signaling studies.

Recent developments in proteomics have enabled signaling studies where > 10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited in throughput, reproducibility, and robustness, hampering experiments that involve multiple perturbations, such as those needed to map kinase-substrate relationships, capture pathway crosstalks, and network inference analysis. To address these challenges, we introduce rapid-robotic phosphoproteomics (R2-P2), an end-to-end automated method that uses magnetic particles to process protein extracts to deliver mass spectrometry-ready phosphopeptides. R2-P2 is rapid, robust, versatile, and high-throughput. To showcase the method, we applied it, in combination with data-independent acquisition mass spectrometry, to study signaling dynamics in the mitogen-activated protein kinase (MAPK) pathway in yeast. Our results reveal broad and specific signaling events along the mating, the high-osmolarity glycerol, and the invasive growth branches of the MAPK pathway, with robust phosphorylation of downstream regulatory proteins and transcription factors. Our method facilitates large-scale signaling studies involving hundreds of perturbations opening the door to systems-level studies aiming to capture signaling complexity.

TORC1 and TORC2 converge to regulate the SAGA co-activator in response to nutrient availability.

Gene expression regulation is essential for cells to adapt to changes in their environment. Co-activator complexes have well-established roles in transcriptional regulation, but less is known about how they sense and respond to signaling cues. We have previously shown that, in fission yeast, one such co-activator, the SAGA complex, controls gene expression and the switch from proliferation to differentiation in response to nutrient availability. Here, using a combination of genetic, biochemical, and proteomic approaches, we show that SAGA responds to nutrients through the differential phosphorylation of its Taf12 component, downstream of both the TORC1 and TORC2 pathways. Taf12 phosphorylation increases early upon starvation and is controlled by the opposing activities of the PP2A phosphatase, which is activated by TORC1, and the TORC2-activated Gad8AKT kinase. Mutational analyses suggest that Taf12 phosphorylation prevents cells from committing to differentiation until starvation reaches a critical level. Overall, our work reveals that SAGA is a direct target of nutrient-sensing pathways and has uncovered a mechanism by which TORC1 and TORC2 converge to control gene expression and cell fate decisions.

Novel insights into Sabino1 and splashed white coat color patterns in horses.

Within the framework of genome-wide analyses using the novel Axiom® genotyping array, we investigated the distribution of two previously described coat color patterns, namely sabino1 (SBI), associated with the KIT gene (KI16+1037A), and splashed white, associated with the PAX3 gene (ECA6:g.11429753C>T; PAX3C70Y ), including a total of 899 horses originating from eight different breeds (Achal Theke, Purebred Arabian, Partbred Arabian, Anglo-Arabian, Shagya Arabian, Haflinger, Lipizzan and Noriker). Based on the data we collected we were able to demonstrate that, besides Quarter horses, the PAX3C70Y allele is also present in Noriker (seven out of 189) and Lipizzan (three out of 329) horses. The SB1 allele was present in three breeds (Haflinger, 14 out of 98; Noriker, four out of 189; Lipizzan one out of 329). Furthermore, we examined the phenotypes of SB1- and PAX3C70Y -carrier horses for their characteristic white spotting patterns. None of the SB1/sb1-carrier horses met the criteria defining the Sabino1 pattern according to current applied protocols. From 10 heterozygous PAX3C70Y -carrier horses, two had nearly a splashed white phenotype. The results of this large-scale experiment on the genetic association of white spotting patterns in horses underline the influence of gene interactions and population differences on complex traits such as Sabino1 and splashed white.

Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1.

The eIF4E-binding protein (4E-BP) is a phosphorylation-dependent regulator of protein synthesis. The nonphosphorylated or minimally phosphorylated form binds translation initiation factor 4E (eIF4E), preventing binding of eIF4G and the recruitment of the small ribosomal subunit. Signaling events stimulate serial phosphorylation of 4E-BP, primarily by mammalian target of rapamycin complex 1 (mTORC1) at residues T37/T46, followed by T70 and S65. Hyperphosphorylated 4E-BP dissociates from eIF4E, allowing eIF4E to interact with eIF4G and translation initiation to resume. Because overexpression of eIF4E is linked to cellular transformation, 4E-BP is a tumor suppressor, and up-regulation of its activity is a goal of interest for cancer therapy. A recently discovered small molecule, eIF4E/eIF4G interaction inhibitor 1 (4EGI-1), disrupts the eIF4E/eIF4G interaction and promotes binding of 4E-BP1 to eIF4E. Structures of 14- to 16-residue 4E-BP fragments bound to eIF4E contain the eIF4E consensus binding motif, (54)YXXXXLΦ(60) (motif 1) but lack known phosphorylation sites. We report here a 2.1-Å crystal structure of mouse eIF4E in complex with m(7)GTP and with a fragment of human 4E-BP1, extended C-terminally from the consensus-binding motif (4E-BP150-84). The extension, which includes a proline-turn-helix segment (motif 2) followed by a loop of irregular structure, reveals the location of two phosphorylation sites (S65 and T70). Our major finding is that the C-terminal extension (motif 3) is critical to 4E-BP1-mediated cell cycle arrest and that it partially overlaps with the binding site of 4EGI-1. The binding of 4E-BP1 and 4EGI-1 to eIF4E is therefore not mutually exclusive, and both ligands contribute to shift the equilibrium toward the inhibition of translation initiation.

[Dermatofibrosarcoma protuberans: Surgical margins using Slow-Mohs micrographic surgery. A clinical retrospective study about 20 cases]. / Marges d'exérèse des dermatofibrosarcomes cervico-faciaux par technique de Slow-Mohs : étude clinique rétrospective sur 20 cas.

OBJECTIVES: The main objective of this study is to determine the necessary surgical margins to obtain a complete R0 resection for head and neck dermatofibrosarcoma protuberans (DFSP) using Slow-Mohs micrographic surgery. The secondary objective is to study the recurrence rate of these tumors. PATIENTS AND METHODS: Slow-Mohs micrographic surgery was used for patients included between 2005 and 2015 at Bordeaux universitary hospital. For each patient the age, the sex and death occurrence, the initial surgical margins, the surgical margins for complete R0 resection, the occurrence of local or general recurrence during follow-up were reported. Surgery was realized under local anesthesia. The closure of the tumor site was realized secondarily using a skin graft or local flap. RESULTS: Twenty patients were included in the study. Initial surgical margins were 10mm (9 patients) or 15mm (11 patients). Complete resection was obtained from the first surgery for fifteen patients (75%). The average surgical margin for a complete R0 resection was 15,25±5,7mm (10-25). None of the patients presented recurrences during the entire follow-up (38 months) CONCLUSION: A complete R0 resection of head and neck DFSP is obtained from the first surgery in 75% of the cases, with minimum surgical margins (12,75±2,55mm) using the Slow-Mohs micrographic surgery. This allows a reduction of surgical margins and local recurrences. This technique provides a preservation of soft-tissues, which plays a key role for head and neck surgery.

Phosphorylation of ubiquitin at Ser65 affects its polymerization, targets, and proteome-wide turnover.

Ubiquitylation is an essential post-translational modification that regulates numerous cellular processes, most notably protein degradation. Ubiquitin can itself be phosphorylated at nearly every serine, threonine, and tyrosine residue. However, the effect of this modification on ubiquitin function is largely unknown. Here, we characterized the effects of phosphorylation of yeast ubiquitin at serine 65 in vivo and in vitro. We find this post-translational modification to be regulated under oxidative stress, occurring concomitantly with the restructuring of the ubiquitin landscape into a highly polymeric state. Phosphomimetic mutation of S65 recapitulates the oxidative stress phenotype, causing a dramatic accumulation of ubiquitylated proteins and a proteome-wide reduction of protein turnover rates. Importantly, this mutation impacts ubiquitin chain disassembly, chain linkage distribution, ubiquitin interactions, and substrate targeting. These results demonstrate that phosphorylation is an additional mode of ubiquitin regulation with broad implications in cellular physiology.

Human papillomavirus does not play a role in the Barrett esophagus: a French cohort.

The role of human papillomavirus (HPV) in Barrett's esophagus (BE) has been examined but remains unclear. The purpose of the study is to dispute the connection between HPV and BE in a prospective case-control study. Biopsies were performed above and inside the Barrett's segment for BE patients and in the distal third of the esophagus for control patients for histological interpretation and for virological analysis. Biopsies for virological analysis were placed in a virus transport medium and immediately frozen in liquid nitrogen. Virological analysis involved real-time PCR using the SyBr® green protocol with modified SPF10 general primers. A total of 180 patients (119 control and 61 BE, respectively) were included. In BE patients, 31, 18, and 12 patients had, respectively, no dysplasia, low-grade dysplasia, and high grade dysplasia. Overall, nine were found to be HPV positive: five were control patients and four BE patients. HPV positive status was not associated with BE. No factors were associated with HPV, in particular the degree of BE dysplasia. HPV infection appears unlikely to be significant in the etiology of BE compared with control patients. (ClinicalTrials.gov, Number NCT02549053).

Genome-wide association study for jumping performances in French sport horses.

A genome-wide association study was performed to identify single nucleotide polymorphisms (SNPs) associated with jumping performances of warmbloods in France. The 999 horses included in the study for jumping performances were sport horses [mostly Selle Français (68%), Anglo-Arabians (13%) and horses from the other European studbooks]. Horses were genotyped using the Illumina EquineSNP50 BeadChip. Of the 54,602 SNPs available on this chip, 44,424 were retained after quality testing. Phenotypes were obtained by deregressing official breeding values for jumping competitions to use all available information, that is, the performances of each horse as well as those of its relatives. Two models were used to test the effects of the genotypes on deregressed phenotypes: a single-marker mixed model and a haplotype-based mixed model (significant: P < 1E-05; suggestive: P < 1E-04). Both models included a polygenic effect to take into account familial structures. For jumping performances, one suggestive quantitative trait locus (QTL) located on chromosome 1 (BIEC2_31196 and BIEC2_31198) was detected with both models. This QTL explains 0.7% of the phenotypic variance. RYR2, a gene encoding a major calcium channel in cardiac muscle in humans and mice, is located 0.55 Mb from this potential QTL.

Is the use of formulae a reliable way to predict the accuracy of genomic selection?

We studied four formulae used to predict the accuracy of genomic selection prior to genotyping. The objectives of our study were to investigate the impact of the parameters of each formula on the values of accuracy calculated using these formulae, and to check whether the accuracies reported in the literature are in agreement with the formulae. First, we computed the marginal distribution of accuracy (by integration) for each parameter of all four formulae: heritability h(2) , reference population size T, number of markers M and number of effective segments in the genome Me . Then, we collected 145 accuracies and corresponding parameters reported in 13 publications on genomic selection (mainly in dairy cattle), and performed analysis of variance to test the differences between observed and predicted accuracy with effects of formulae and parameters. The variation of accuracy for different values of each parameter indicated that two parameters, T and Me, had a significant impact and that considerable differences existed between the formulae (mean accuracies differed by up to 0.20 point). The results of our meta-analysis showed a big formula effect on the accuracies predicted using each formula, and also a significant effect of the value obtained for Me calculated from Ne (effective population size). Each formula can therefore be demonstrated to be optimal depending on the assumption used for Me . In conclusion, no rules can be applied to predict the reliability of genomic selection using these formulae.

A case of early childhood caries from a Medieval site in Southern Portugal: a multidisciplinary approach.

Dental caries is the most prevalent chronic infectious disease during childhood both in historical and contemporary times, but research focused on the oral health of non-adults from the past is still scant. As such, this study proposes a multidisciplinary approach to the differential diagnosis of severe dental lesions in a medieval non-adult skeleton. The skeleton of a three-year-old child recovered in the medieval necropolis of Cacela Velha (Portugal) was studied through macroscopic, radiological, elemental and stable isotope analyses. This individual exhibited enamel destruction and dentine exposure in both the maxillary and mandibular teeth, with the latter also showing changes in coloration. Elemental analysis showed that his skull presented lower values of Si, Cl, and Ca and higher of Cu compared to the control, while the concentration of P and S were significantly lower in the teeth. Early childhood caries is the most probable diagnosis for the dental lesions observed, apparently stemming from a reticulate of factors that include potential malnutrition, and the consumption of sugars in complementary feeding - even though historical sources point to the scarcity of sugar in Portugal during most of the Middle Ages.

A conserved function of corepressors is to nucleate assembly of the transcriptional preinitiation complex.

The plant corepressor TPL is recruited to diverse chromatin contexts, yet its mechanism of repression remains unclear. Previously, we have leveraged the fact that TPL retains its function in a synthetic transcriptional circuit in the yeast model Saccharomyces cerevisiae to localize repressive function to two distinct domains. Here, we employed two unbiased whole genome approaches to map the physical and genetic interactions of TPL at a repressed locus. We identified SPT4, SPT5 and SPT6 as necessary for repression with the SPT4 subunit acting as a bridge connecting TPL to SPT5 and SPT6. We also discovered the association of multiple additional constituents of the transcriptional preinitiation complex at TPL-repressed promoters, specifically those involved in early transcription initiation events. These findings were validated in yeast and plants through multiple assays, including a novel method to analyze conditional loss of function of essential genes in plants. Our findings support a model where TPL nucleates preassembly of the transcription activation machinery to facilitate rapid onset of transcription once repression is relieved.

Automated Enrichment of Phosphotyrosine Peptides for High-Throughput Proteomics.

Phosphotyrosine (pY) enrichment is critical for expanding fundamental and clinical understanding of cellular signaling by mass spectrometry-based proteomics. However, current pY enrichment methods exhibit a high cost per sample and limited reproducibility due to expensive affinity reagents and manual processing. We present rapid-robotic phosphotyrosine proteomics (R2-pY), which uses a magnetic particle processor and pY superbinders or antibodies. R2-pY handles 96 samples in parallel, requires 2 days to go from cell lysate to mass spectrometry injections, and results in global proteomic, phosphoproteomic and tyrosine specific phosphoproteomic samples. We benchmark the method on HeLa cells stimulated with pervanadate and serum and report over 4000 unique pY sites from 1 mg of peptide input, strong reproducibility between replicates, and phosphopeptide enrichment efficiencies above 99%. R2-pY extends our previously reported R2-P2 proteomic and global phosphoproteomic sample preparation framework, opening the door to large-scale studies of pY signaling in concert with global proteome and phosphoproteome profiling.