Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometry.

Ubiquitination controls a plethora of cellular processes. Modifications by linear polyubiquitin have so far been linked with acquired and innate immunity, lymphocyte development and genotoxic stress response. Until now, a single E3 ligase complex (LUBAC), one specific deubiquitinase (OTULIN) and a very few linear polyubiquitinated substrates have been identified. Current methods for studying lysine-based polyubiquitination are not suitable for the detection of linear polyubiquitin-modified proteins. Here, we present an approach to discovering linear polyubiquitin-modified substrates by combining a lysine-less internally tagged ubiquitin (INT-Ub.7KR) with SILAC-based mass spectrometry. We applied our approach in TNFα-stimulated T-REx HEK293T cells and validated several newly identified linear polyubiquitin targets. We demonstrated that linear polyubiquitination of the novel LUBAC substrate TRAF6 is essential for NFκB signaling.

The fungal ligand chitin directly binds TLR2 and triggers inflammation dependent on oligomer size.

Chitin is the second most abundant polysaccharide in nature and linked to fungal infection and asthma. However, bona fide immune receptors directly binding chitin and signaling immune activation and inflammation have not been clearly identified because polymeric crude chitin with unknown purity and molecular composition has been used. By using defined chitin (N-acetyl-glucosamine) oligomers, we here identify six-subunit-long chitin chains as the smallest immunologically active motif and the innate immune receptor Toll-like receptor (TLR2) as a primary fungal chitin sensor on human and murine immune cells. Chitin oligomers directly bind TLR2 with nanomolar affinity, and this fungal TLR2 ligand shows overlapping and distinct signaling outcomes compared to known mycobacterial TLR2 ligands. Unexpectedly, chitin oligomers composed of five or less subunits are inactive, hinting to a size-dependent system of immuno-modulation that appears conserved in plants and humans. Since blocking of the chitin-TLR2 interaction effectively prevents chitin-mediated inflammation in vitro and in vivo, our study highlights the chitin-TLR2 interaction as a potential target for developing novel therapies in chitin-related pathologies and fungal disease.

Construction and assessment of individualized proteogenomic databases for large-scale analysis of nonsynonymous single nucleotide variants.

Next-generation sequencing projects focusing on genomes and transcriptomes identify millions of single nucleotide variants (SNVs), many of which result in single amino acid substitutions. These nonsynonymous (ns) SNVs are typically not incorporated into protein sequence databases used to identify MS/MS data. Here, we perform a comparative analysis of the assembly of nsSNV-containing proteogenomic databases. We use a comprehensive transcriptome and proteome dataset of HeLa cells from the literature to derive and to incorporate SNVs into databases applicable to proteomics search engines, and to assess their performance in the identification of nsSNVs. We assemble the databases by (1) translation of SNV-containing transcripts into all possible reading frames, (2) translation of predicted reading frame, (3) prediction of nsSNVs and subsequent incorporation into canonical protein sequences. We show substantial differences between generated databases in terms of represented nsSNVs and theoretical search space, affecting sensitivity and specificity of database search. We query the databases with >2.2M high-resolution MS/MS spectra using MaxQuant software and identify 451 variant peptides, containing 401 nsSNVs. We conclude that prediction of reading frame and, if applicable, SNV effect result in comprehensive yet compact databases necessary to retain sensitivity in large-scale analysis of nsSNVs called from transcriptomics data.

Putative link between Polo-like kinases (PLKs) and Toll-like receptor (TLR) signaling in transformed and primary human immune cells.

Toll-like receptors (TLRs) are important sentinels of bacterial and viral infection and thus fulfil a critical sensory role in innate immunity. Polo-like kinases (PLKs), a five membered family of Ser/Thr protein kinases, have long been studied for their role in mitosis and thus represent attractive therapeutic targets in cancer therapy. Recently, PLKs were implicated in TLR signaling in mice but the role of PLKs in TLR signaling in untransformed primary immune cells has not been addressed, even though PLK inhibitors are in clinical trials. We here identified several phospho-serine and phospho-threonine residues in the known TLR pathway kinases, Interleukin-1 receptor-associated kinase (IRAK) 2 and IRAK4. These sites lie in canonical polo-box motifs (PBM), sequence motifs known to direct recruitment of PLKs to client proteins. Interestingly, PLK1 was phosphorylated and PLK 2 and 3 mRNA induced upon TLR stimulation in primary immune cells, respectively. In whole blood, PLK inhibition disparately affected TLR mediated cytokine responses in a donor- and inhibitor-dependent fashion. Collectively, PLKs may thus potentially interface with TLR signaling in humans. We propose that temporary PLK inhibitor-mediated blockade of TLR-signaling in certain patients receiving such inhibitors during cancer treatment may cause adverse effects such as an increased risk of infections due to a then compromised ability of the TLR recognition system to sense and initiate cytokine responses to invading microbes.

Phosphopeptide Enrichment from Bacterial Samples Utilizing Titanium Oxide Affinity Chromatography.

Mass spectrometry (MS)-based proteomics detected hundreds of phosphorylation sites on serine, threonine and tyrosine in numerous bacterial proteins, firmly establishing the presence and importance of this posttranslational modification in prokaryotes. Recent biological follow up of these results revealed that vital processes in bacterial cell, such as cell division, differentiation, spore germination and persistence, are regulated by protein phosphorylation, raising the need to study this modification on a global scale under additional physiological conditions. Due to low abundance and low stoichiometric levels of protein phosphorylation, initial protocols for phosphopeptide enrichment and analysis required relatively high amounts of starting material, extensive fractionation and MS measurement time. Here we present a protocol for phosphopeptide enrichment and detection based on TiO2 chromatography and high resolution MS that enables in-depth detection and quantification of phosphorylation sites from significantly lower amounts of starting material and in a fraction of MS measurement time.

Parallel reaction monitoring on a Q Exactive mass spectrometer increases reproducibility of phosphopeptide detection in bacterial phosphoproteomics measurements.

Increasing number of studies report the relevance of protein Ser/Thr/Tyr phosphorylation in bacterial physiology, yet the analysis of this type of modification in bacteria still presents a considerable challenge. Unlike in eukaryotes, where tens of thousands of phosphorylation events likely occupy more than two thirds of the proteome, the abundance of protein phosphorylation is much lower in bacteria. Even the state-of-the-art phosphopeptide enrichment protocols fail to remove the high background of abundant unmodified peptides, leading to low signal intensity and undersampling of phosphopeptide precursor ions in consecutive data-dependent MS runs. Consequently, large-scale bacterial phosphoproteomic datasets often suffer from poor reproducibility and a high number of missing values. Here we explore the application of parallel reaction monitoring (PRM) on a Q Exactive mass spectrometer in bacterial phosphoproteome analysis, focusing especially on run-to-run sampling reproducibility. In multiple measurements of identical phosphopeptide-enriched samples, we show that PRM outperforms data-dependent acquisition (DDA) in terms of detection frequency, reaching almost complete sampling efficiency, compared to 20% in DDA. We observe a similar trend over multiple heterogeneous phosphopeptide-enriched samples and conclude that PRM shows a great promise in bacterial phosphoproteomics analyses where reproducible detection and quantification of a relatively small set of phosphopeptides is desired. SIGNIFICANCE: Bacterial phosphorylated peptides occur in low abundance compared to their unmodified counterparts, and are therefore rarely reproducibly detected in shotgun (DDA) proteomics measurements. Here we show that parallel reaction monitoring complements DDA analyses and makes detection of known, targeted phosphopeptides more reproducible. This will be of significance in replicated MS measurements that have a goal to reproducibly detect and quantify phosphopeptides of interest.