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

Untersuchungen zur Expression von Erythropoetinrezeptoren in Zellen außerhalb des hämatopoetischen Systems und deren funktioneller Relevanz

In neuronalen und nicht-hämatopoetischen Rattenzellen wurden der EPOR-WT und alternative Rezeptoren identifiziert. Neben dem WT-Rezeptor ließen sich zwei weitere alternative Rezeptoren in ST14A-Zellen nachweisen. Bei der Analyse der resultierenden Proteinstruktur der alternativen Transkripte zeigten sich nach Verbleib des Introns 5 sowie Intron 7 veränderte Rezeptoren. Im Speziesvergleich fielen Unterschiede von Ratte einerseits und Maus/Mensch andererseits auf. Es wurden stabil transfizierte Zellen hergestellt, hier konnten signifikante Proliferationsunterschiede gezeigt werden

Quantitative Proteomic Approach Identifies Vpr Binding Protein as Novel Host Factor Supporting Influenza A Virus Infections in Human Cells

Influenza A virus infections are a major cause for respiratory disease in humans, which affects all age groups and contributes substantially to global morbidity and mortality. IAV have a large natural host reservoir in avian species. However, many avian IAV strains lack adaptation to other hosts and hardly propagate in humans. While seasonal or pandemic influenza A virus (IAV) strains replicate efficiently in permissive human cells, many avian IAV cause abortive non-productive infections in these hosts despite successful cell entry. However, the precise reasons for these differential outcomes are poorly defined. We hypothesized that the distinct course of an IAV infection with a given virus strain is determined by the differential interplay between specific host and viral factors. By using Spike-in SILAC mass spectrometry-based quantitative proteomics we characterized sets of cellular factors whose abundance is specifically up- or down-regulated in the course of permissive vs. non-permissive IAV infection, respectively. This approach allowed for the definition and quantitative comparison of about 3500 proteins in human lung epithelial cells in response to seasonal or low-pathogenic avian H3N2 IAV. Many identified proteins were similarly regulated by both virus strains, but also 16 candidates with distinct changes in permissive vs. non-permissive infection were found. RNAi-mediated knockdown of these differentially regulated host factors identified Vpr binding protein (VprBP) as pro-viral host factor since its down-regulation inhibited efficient propagation of seasonal IAV while over-expression increased viral replication of both seasonal and avian IAV. These results not only show that there are similar differences in the overall changes during permissive and non-permissive imfluenza virus infections, but also provide a basis to evaluate VprBP as novel anti-IAV drug target

Engineering, decoding and systems-level characterization of chimpanzee cytomegalovirus

The chimpanzee cytomegalovirus (CCMV) is the closest relative of human CMV (HCMV). Because of the high conservation between these two species and the ability of human cells to fully support CCMV replication, CCMV holds great potential as a model system for HCMV. To make the CCMV genome available for precise and rapid gene manipulation techniques, we captured the genomic DNA of CCMV strain Heberling as a bacterial artificial chromosome (BAC). Selected BAC clones were reconstituted to infectious viruses, growing to similar high titers as parental CCMV. DNA sequencing confirmed the integrity of our clones and led to the identification of two polymorphic loci and a deletion-prone region within the CCMV genome. To re-evaluate the CCMV coding potential, we analyzed the viral transcriptome and proteome and identified several novel ORFs, splice variants, and regulatory RNAs. We further characterized the dynamics of CCMV gene expression and found that viral proteins cluster into five distinct temporal classes. In addition, our datasets revealed that the host response to CCMV infection and the de-regulation of cellular pathways are in line with known hallmarks of HCMV infection. In a first functional experiment, we investigated a proposed frameshift mutation in UL128 that was suspected to restrict CCMV's cell tropism. In fact, repair of this frameshift re-established productive CCMV infection in endothelial and epithelial cells, expanding the options of CCMV as an infection model. Thus, BAC-cloned CCMV can serve as a powerful tool for systematic approaches in comparative functional genomics, exploiting the close phylogenetic relationship between CCMV and HCMV

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

Cross-regulation of viral kinases with cyclin A secures shutoff of host DNA synthesis

Herpesviruses encode conserved protein kinases (CHPKs) to stimulate phosphorylation-sensitive processes during infection. How CHPKs bind to cellular factors and how this impacts their regulatory functions is poorly understood. Here, we use quantitative proteomics to determine cellular interaction partners of human herpesvirus (HHV) CHPKs. We find that CHPKs can target key regulators of transcription and replication. The interaction with Cyclin A and associated factors is identified as a signature of β-herpesvirus kinases. Cyclin A is recruited via RXL motifs that overlap with nuclear localization signals (NLS) in the non-catalytic N termini. This architecture is conserved in HHV6, HHV7 and rodent cytomegaloviruses. Cyclin A binding competes with NLS function, enabling dynamic changes in CHPK localization and substrate phosphorylation. The cytomegalovirus kinase M97 sequesters Cyclin A in the cytosol, which is essential for viral inhibition of cellular replication. Our data highlight a fine-tuned and physiologically important interplay between a cellular cyclin and viral kinases