Systematic errors in peptide and protein identification and quantification by modified peptides

The principle of shotgun proteomics is to use peptide mass spectra in order to identify corresponding sequences in a protein database. The quality of peptide and protein identification and quantification critically depends on the sensitivity and specificity of this assignment process. Many peptides in proteomic samples carry biochemical modifications, and a large fraction of unassigned spectra arise from modified peptides. Spectra derived from modified peptides can erroneously be assigned to wrong amino acid sequences. However, the impact of this problem on proteomic data has not yet been investigated systematically. Here we use combinations of different database searches to show that modified peptides can be responsible for 20-50 % of false positive identifications in deep proteomic datasets. These false positive hits are particularly problematic as they have significantly higher scores and higher intensities than other false positive matches. Furthermore, these wrong peptide assignments lead to hundreds of false protein identifications and systematic biases in protein quantification. We devise a "cleaned search" strategy to address this problem and show that this considerably improves the sensitivity and specificity of proteomic data. In summary, we show that modified peptides cause systematic errors in peptide and protein identification and quantification and should therefore be considered to further improve the quality of proteomic data annotation

Social Comparisons, Social Support, and Self-Perceptions in Group Exercise for People With Parkinson's Disease

People with Parkinson's disease (PD) may experience declining function and limited interaction with others with PD. Group exercise provides opportunities for physical accomplishment and social support, as well as potential social challenges. We used interpretative phenomenological analysis to examine experiences of social comparison, social support, and self-perceptions of 20 people with PD in group exercise. Participants experienced (a) reticence evolving to inspiration, (b) anxiety relief through camaraderie and breaking taboos, and (c) maintaining athletic identity through participating and helping others. Practical implications include facilitating a safe space and support to alleviate anxiety and sustain participation

Keeping the proportions of protein complex components in check

How do cells maintain relative proportions of protein complex components? Advances in quantitative, genome-wide measurements have begun to shed light onto the roles of protein synthesis and degradation in establishing the precise proportions in living cells: on the one hand, ribosome profiling studies indicate that proteins are already produced in the correct relative proportions. On the other hand, proteomic studies found that many complexes contain subunits that are made in excess and subsequently degraded. Here, we discuss these seemingly contradictory findings, emerging principles, and remaining open questions. We conclude that establishing precise protein levels involves both coordinated synthesis and post-translational fine-tuning via protein degradation

Quantitative GTPase affinity purification identifies Rho family protein interaction partners

Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, RhoD) depending on their nucleotide loading state. The method works with cell line or tissue-derived protein lysates in combination with SILAC-based or label free quantification, respectively. We demonstrate that qGAP identifies known and novel binding partners that can be validated in an independent assay. Our interaction network for six Rho GTPases contains many novel binding partners, reveals highly promiscuous interaction of several effectors and mirrors evolutionary relationships among Rho GTPases

Extensive allele-specific translational regulation in hybrid mice

Translational regulation is mediated through the interaction between diffusible trans-factors and cis-elements residing within mRNA transcripts. In contrast to extensively studied transcriptional regulation, cis-regulation on translation remains underexplored. Using deep sequencing-based transcriptome and polysome profiling, we globally profiled allele-specific translational efficiency for the first time in an F1 hybrid mouse. Out of 7,156 genes with reliable quantification of both alleles, we found 1,008 (14.1%) exhibiting significant allelic divergence in translational efficiency. Systematic analysis of sequence features of the genes with biased allelic translation revealed that local RNA secondary structure surrounding the start codon and proximal out-of-frame upstream AUGs could affect translational efficiency. Finally, we observed that the cis-effect was quantitatively comparable between transcriptional and translational regulation. Such effects in the two regulatory processes were more frequently compensatory, suggesting that the regulation at the two levels could be coordinated in maintaining robustness of protein expression

TERC polymorphisms are associated both with susceptibility to colorectal cancer and with longer telomeres.

Shorter telomeres have been associated with increased risk of malignancy, including colorectal cancer (CRC). Telomere length is heritable and may be an intermediate phenotype linked to genetic susceptibility to CRC

The association of bone, fingernail and blood manganese with cognitive and olfactory function in Chinese workers

Occupational manganese (Mn) exposure has been associated with cognitive and olfactory dysfunction; however, few studies have incorporated cumulative biomarkers of Mn exposure such as bone Mn (BnMn). Our goal was to assess the cross-sectional association between BnMn, blood Mn (BMn), and fingernail Mn (FMn) with cognitive and olfactory function among Mn-exposed workers. A transportable in vivo neutron activation analysis (IVNAA) system was designed and utilized to assess BnMn among 60 Chinese workers. BMn and FMn were measured using inductively coupled plasma mass spectrometry. Cognitive and olfactory function was assessed using Animal and Fruit Naming tests, World Health Organization/University of California-Los Angeles Auditory Verbal Learning Test (AVLT) and the University of Pennsylvania Smell Identification Test (UPSIT). Additional data were obtained via questionnaire. Regression models adjusted for age, education, factory of employment, and smoking status (UPSIT only), were used to assess the relationship between Mn biomarkers and test scores. In adjusted models, increasing BnMn was significantly associated with decreased performance on average AVLT scores [β (95% confidence interval (CI)) = -0.65 (-1.21, -0.09)] and Animal Naming scores [β (95% CI) = -1.54 (-3.00, -0.07)]. Increasing FMn was significantly associated with reduced performance measured by the average AVLT [β (95% CI) = -0.35 (-0.70, -0.006)] and the difference in AVLT scores [β (95% CI) = -0.40 (-0.77, -0.03)]. BMn was not significantly associated with any test scores; no significant associations were observed with Fruit Naming or UPSIT tests. BnMn and FMn, but not BMn, are associated with cognitive function in Mn-exposed workers. None of th

Wnt11/Fzd7 signaling compartmentalizes AKAP2/PKA to regulate L-type Ca2+ channel

Calcium influx through the voltage-gated L-type calcium channels (LTCC) mediates a wide range of physiological processes from contraction to secretion. Despite extensive research on regulation of LTCC conductance by PKA phosphorylation in response to β-adrenergic stimulation, the science remains incomplete. Here, we show that Wnt11, a non-canonical Wnt ligand, through its G protein-coupled receptor (GPCR) Fzd7 attenuates the LTCC conductance by preventing the proteolytic processing of its C terminus. This is mediated across species by protein kinase A (PKA), which is compartmentalized by A-kinase anchoring proteins (AKAP). Systematic analysis of all AKAP family members revealed AKAP2 anchoring of PKA is central to the Wnt11-dependent regulation of the channel. The identified Wnt11/AKAP2/PKA signalosome is required for heart development, controlling the intercellular electrical coupling in the developing zebrafish heart. Altogether, our data revealed Wnt11/Fzd7 signaling via AKAP2/PKA as a conserved alternative GPCR system regulating Ca(2+) homeostasis

Phosphorylation of the ribosomal protein RPL12/uL11 affects translation during mitosis

Emerging evidence indicates that heterogeneity in ribosome composition can give rise to specialized functions. Until now, research mainly focused on differences in core ribosomal proteins and associated factors. The effect of posttranslational modifications has not been studied systematically. Analyzing ribosome heterogeneity is challenging because individual proteins can be part of different subcomplexes (40S, 60S, 80S, and polysomes). Here we develop polysome proteome profiling to obtain unbiased proteomic maps across ribosomal subcomplexes. Our method combines extensive fractionation by sucrose gradient centrifugation with quantitative mass spectrometry. The high resolution of the profiles allows us to assign proteins to specific subcomplexes. Phosphoproteomics on the fractions reveals that phosphorylation of serine 38 in RPL12/uL11, a known mitotic CDK1 substrate, is strongly depleted in polysomes. Follow-up experiments confirm that RPL12/uL11 phosphorylation regulates the translation of specific subsets of mRNAs during mitosis. Together, our results show that posttranslational modification of ribosomal proteins can regulate translation