Benefit of In Silico Predicted Spectral Libraries in Data-Independent Acquisition Data Analysis Workflows.

Data-independent acquisition (DIA) has become a well-established method for MS-based proteomics. However, the list of options to analyze this type of data is quite extensive, and the use of spectral libraries has become an important factor in DIA data analysis. More specifically the use of in silico predicted libraries is gaining more interest. By working with a differential spike-in of human standard proteins (UPS2) in a constant yeast tryptic digest background, we evaluated the sensitivity, precision, and accuracy of the use of in silico predicted libraries in data DIA data analysis workflows compared to more established workflows. Three commonly used DIA software tools, DIA-NN, EncyclopeDIA, and Spectronaut, were each tested in spectral library mode and spectral library-free mode. In spectral library mode, we used independent spectral library prediction tools PROSIT and MS2PIP together with DeepLC, next to classical data-dependent acquisition (DDA)-based spectral libraries. In total, we benchmarked 12 computational workflows for DIA. Our comparison showed that DIA-NN reached the highest sensitivity while maintaining a good compromise on the reproducibility and accuracy levels in either library-free mode or using in silico predicted libraries pointing to a general benefit in using in silico predicted libraries.

High-Throughput Nanoflow Proteomics Using a Dual-Column Electrospray Source.

With current trends in proteomics, especially regarding clinical and low input (to single cell) samples, it is increasingly important to both maximize the throughput of the analysis and maintain as much sensitivity as possible. The new generation of mass spectrometers (MS) are taking a huge leap in sensitivity, allowing analysis of samples with shorter liquid chromatography (LC) methods while digging as deep in the proteome. However, the throughput can be doubled by implementing a dual column nano-LC-MS configuration. For this purpose, we used a dual-column setup with a two-outlet electrospray source and compared it to a classic dual-column setup with a single-outlet source.

Limited Evidence for Protein Products of Noncoding Transcripts in the HEK293T Cellular Cytosol.

Ribosome profiling has revealed translation outside canonical coding sequences, including translation of short upstream ORFs, long noncoding RNAs, overlapping ORFs, ORFs in UTRs, or ORFs in alternative reading frames. Studies combining mass spectrometry, ribosome profiling, and CRISPR-based screens showed that hundreds of ORFs derived from noncoding transcripts produce (micro)proteins, whereas other studies failed to find evidence for such types of noncanonical translation products. Here, we attempted to discover translation products from noncoding regions by strongly reducing the complexity of the sample prior to mass spectrometric analysis. We used an extended database as the search space and applied stringent filtering of the identified peptides to find evidence for novel translation events. We show that, theoretically our strategy facilitates the detection of translation events of transcripts from noncoding regions but experimentally only find 19 peptides that might originate from such translation events. Finally, Virotrap-based interactome analysis of two N-terminal proteoforms originating from noncoding regions showed the functional potential of these novel proteins.

N-glycosylation Site Analysis Reveals Sex-related Differences in Protein N-glycosylation in the Rice Brown Planthopper (Nilaparvata lugens).

Glycosylation is a common modification of proteins and critical for a wide range of biological processes. Differences in protein glycosylation between sexes have already been observed in humans, nematodes and trematodes, and have recently also been reported in the rice pest insect Nilaparvata lugens Although protein N-glycosylation in insects is nowadays of high interest because of its potential for exploitation in pest control strategies, the functionality of differential N-glycosylation between sexes is yet unknown. In this study, therefore, the occurrence and role of sex-related protein N-glycosylation in insects were examined. A comprehensive investigation of the N-glycosylation sites from the adult stages of N. lugens was conducted, allowing a qualitative and quantitative comparison between sexes at the glycopeptide level. N-glycopeptide enrichment via lectin capturing using the high mannose/paucimannose-binding lectin Concanavalin A, or the Rhizoctonia solani agglutinin which interacts with complex N-glycans, resulted in the identification of over 1300 N-glycosylation sites derived from over 600 glycoproteins. Comparison of these N-glycopeptides revealed striking differences in protein N-glycosylation between sexes. Male- and female-specific N-glycosylation sites were identified, and some of these sex-specific N-glycosylation sites were shown to be derived from proteins with a putative role in insect reproduction. In addition, differential glycan composition between males and females was observed for proteins shared across sexes. Both lectin blotting experiments as well as transcript expression analyses with complete insects and insect tissues confirmed the observed differences in N-glycosylation of proteins between sexes. In conclusion, this study provides further evidence for protein N-glycosylation to be sex-related in insects. Furthermore, original data on N-glycosylation sites of N. lugens adults are presented, providing novel insights into planthopper's biology and information for future biological pest control strategies.

Use of Hybrid Data-Dependent and -Independent Acquisition Spectral Libraries Empowers Dual-Proteome Profiling.

In the context of bacterial infections, it is imperative that physiological responses can be studied in an integrated manner, meaning a simultaneous analysis of both the host and the pathogen responses. To improve the sensitivity of detection, data-independent acquisition (DIA)-based proteomics was found to outperform data-dependent acquisition (DDA) workflows in identifying and quantifying low-abundant proteins. Here, by making use of representative bacterial pathogen/host proteome samples, we report an optimized hybrid library generation workflow for DIA mass spectrometry relying on the use of data-dependent and in silico-predicted spectral libraries. When compared to searching DDA experiment-specific libraries only, the use of hybrid libraries significantly improved peptide detection to an extent suggesting that infection-relevant host-pathogen conditions could be profiled in sufficient depth without the need of a priori bacterial pathogen enrichment when studying the bacterial proteome. Proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD017904 and PXD017945.

Disruption of endocytosis through chemical inhibition of clathrin heavy chain function.

Clathrin-mediated endocytosis (CME) is a highly conserved and essential cellular process in eukaryotic cells, but its dynamic and vital nature makes it challenging to study using classical genetics tools. In contrast, although small molecules can acutely and reversibly perturb CME, the few chemical CME inhibitors that have been applied to plants are either ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 (ES9) as an inhibitor of clathrin heavy chain (CHC) function in both Arabidopsis and human cells through affinity-based target isolation, in vitro binding studies and X-ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9-17, which lacks the undesirable side effects of ES9 while retaining the ability to target CHC. ES9 and ES9-17 have expanded the chemical toolbox used to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different systems.

Removing the Hidden Data Dependency of DIA with Predicted Spectral Libraries.

Data-independent acquisition (DIA) generates comprehensive yet complex mass spectrometric data, which imposes the use of data-dependent acquisition (DDA) libraries for deep peptide-centric detection. Here, it is shown that DIA can be redeemed from this dependency by combining predicted fragment intensities and retention times with narrow window DIA. This eliminates variation in library building and omits stochastic sampling, finally making the DIA workflow fully deterministic. Especially for clinical proteomics, this has the potential to facilitate inter-laboratory comparison.

Targeted Proteomics for Studying Pathogenic Bacteria.

Mass spectrometry-based proteomics has been extensively used to map bacterial proteomes, which has led to a better understanding of the molecular mechanisms underlying bacterial infection and bacteria-host interactions. Quantitative proteomics using selected or parallel reaction monitoring is considered one of the most sensitive and specific quantitative MS-based approaches and has significantly advanced proteome studies of pathogenic bacteria. Here, recent applications of targeted proteomics for bacteria identification, biomarker discovery, and the characterization of bacterial virulence and antimicrobial resistance are reviewed among others. Results of such studies are expected to further contribute to improve the fight against the most common human pathogenic bacteria.

Update on the moFF Algorithm for Label-Free Quantitative Proteomics.

moFF is a modular and operating-system-independent tool for quantitative analysis of label-free mass-spectrometry-based proteomics data. The moFF workflow, comprising matching-between-runs and apex quantification, can be applied to any upstream search engine's output, along with the corresponding Thermo or mzML raw file. We here present moFF 2.0, with improvements in speed through multithreading, the use of a new raw file access library, and a novel filtering approach in the matching-between-runs module. This filter allows moFF to correctly identify features that are present in one run but not in another, as demonstrated using spiked-in iRT peptides. Moreover, moFF 2.0 also provides a new peptide summary export that can be used in downstream statistical analysis. moFF is open source and freely available and can be downloaded from https://github.com/compomics/moFF.

GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis.

Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor.

Quality control in mass spectrometry-based proteomics.

Mass spectrometry is a highly complex analytical technique and mass spectrometry-based proteomics experiments can be subject to a large variability, which forms an obstacle to obtaining accurate and reproducible results. Therefore, a comprehensive and systematic approach to quality control is an essential requirement to inspire confidence in the generated results. A typical mass spectrometry experiment consists of multiple different phases including the sample preparation, liquid chromatography, mass spectrometry, and bioinformatics stages. We review potential sources of variability that can impact the results of a mass spectrometry experiment occurring in all of these steps, and we discuss how to monitor and remedy the negative influences on the experimental results. Furthermore, we describe how specialized quality control samples of varying sample complexity can be incorporated into the experimental workflow and how they can be used to rigorously assess detailed aspects of the instrument performance.

Chronic Kidney Disease Circulating Calciprotein Particles and Extracellular Vesicles Promote Vascular Calcification: A Role for GRP (Gla-Rich Protein).

OBJECTIVE: Inhibition of mineral crystal formation is a crucial step in ectopic calcification. Serum calciprotein particles (CPPs) have been linked to chronic kidney disease (CKD) calcification propensity, but additional knowledge is required to understand their function, assemblage, and composition. The role of other circulating nanostructures, such as extracellular vesicles (EVs) in vascular calcification is currently unknown. Here, we investigated the association of GRP (Gla-rich protein) with circulating CPP and EVs and the role of CKD CPPs and EVs in vascular calcification. APPROACH AND RESULTS: Biological CPPs and EVs were isolated from healthy and CKD patients and comparatively characterized using ultrastructural, analytic, molecular, and immuno-based techniques. Our results show that GRP is a constitutive component of circulating CPPs and EVs. CKD stage 5 serum CPPs and EVs are characterized by lower levels of fetuin-A and GRP, and CPPs CKD stage 5 have increased mineral maturation, resembling secondary CPP particles. Vascular smooth muscle cell calcification assays reveal that CPPs CKD stage 5 and EVs CKD stage 5 are taken up by vascular smooth muscle cells and induce vascular calcification by promoting cell osteochondrogenic differentiation and inflammation. These effects were rescued by incubation of CPPs CKD stage 5 with γ-carboxylated GRP. In vitro, formation and maturation of basic calcium phosphate crystals was highly reduced in the presence of γ-carboxylated GRP, fetuin-A, and MGP (matrix gla protein), and a similar antimineralization system was identified in vivo. CONCLUSIONS: Uremic CPPs and EVs are important players in the mechanisms of widespread calcification in CKD. We propose a major role for cGRP as inhibitory factor to prevent calcification at systemic and tissue levels.

The Response of the Root Proteome to the Synthetic Strigolactone GR24 in Arabidopsis.

Strigolactones are plant metabolites that act as phytohormones and rhizosphere signals. Whereas most research on unraveling the action mechanisms of strigolactones is focused on plant shoots, we investigated proteome adaptation during strigolactone signaling in the roots of Arabidopsis thaliana. Through large-scale, time-resolved, and quantitative proteomics, the impact of the strigolactone analog rac-GR24 was elucidated on the root proteome of the wild type and the signaling mutant more axillary growth 2 (max2). Our study revealed a clear MAX2-dependent rac-GR24 response: an increase in abundance of enzymes involved in flavonol biosynthesis, which was reduced in the max2-1 mutant. Mass spectrometry-driven metabolite profiling and thin-layer chromatography experiments demonstrated that these changes in protein expression lead to the accumulation of specific flavonols. Moreover, quantitative RT-PCR revealed that the flavonol-related protein expression profile was caused by rac-GR24-induced changes in transcript levels of the corresponding genes. This induction of flavonol production was shown to be activated by the two pure enantiomers that together make up rac-GR24. Finally, our data provide much needed clues concerning the multiple roles played by MAX2 in the roots and a comprehensive view of the rac-GR24-induced response in the root proteome.

Expanding the Interactome of TES by Exploiting TES Modules with Different Subcellular Localizations.

The multimodular nature of many eukaryotic proteins underlies their temporal or spatial engagement in a range of protein cocomplexes. Using the multimodule protein testin (TES), we here report a proteomics approach to increase insight in cocomplex diversity. The LIM-domain containing and tumor suppressor protein TES is present at different actin cytoskeleton adhesion structures in cells and influences cell migration, adhesion and spreading. TES module accessibility has been proposed to vary due to conformational switching and variants of TES lacking specific domains target to different subcellular locations. By applying iMixPro AP-MS ("intelligent Mixing of Proteomes"-affinity purification-mass spectrometry) to a set of tagged-TES modular variants, we identified proteins residing in module-specific cocomplexes. The obtained distinct module-specific interactomes combine to a global TES interactome that becomes more extensive and richer in information. Applying pathway analysis to the module interactomes revealed expected actin-related canonical pathways and also less expected pathways. We validated two new TES cocomplex partners: TGFB1I1 and a short form of the glucocorticoid receptor. TES and TGFB1I1 are shown to oppositely affect cell spreading providing biological validity for their copresence in complexes since they act in similar processes.

Protein aggregation as an antibiotic design strategy.

Taking advantage of the xenobiotic nature of bacterial infections, we tested whether the cytotoxicity of protein aggregation can be targeted to bacterial pathogens without affecting their mammalian hosts. In particular, we examined if peptides encoding aggregation-prone sequence segments of bacterial proteins can display antimicrobial activity by initiating toxic protein aggregation in bacteria, but not in mammalian cells. Unbiased in vitro screening of aggregating peptide sequences from bacterial genomes lead to the identification of several peptides that are strongly bactericidal against methicillin-resistant Staphylococcus aureus. Upon parenteral administration in vivo, the peptides cured mice from bacterial sepsis without apparent toxic side effects as judged from histological and hematological evaluation. We found that the peptides enter and accumulate in the bacterial cytosol where they cause aggregation of bacterial polypeptides. Although the precise chain of events that leads to cell death remains to be elucidated, the ability to tap into aggregation-prone sequences of bacterial proteomes to elicit antimicrobial activity represents a rich and unexplored chemical space to be mined in search of novel therapeutic strategies to fight infectious diseases.

The Arabidopsis Iron-Sulfur Protein GRXS17 is a Target of the Ubiquitin E3 Ligases RGLG3 and RGLG4.

The stability of signaling proteins in eukaryotes is often controlled by post-translational modifiers. For polyubiquitination, specificity is assured by E3 ubiquitin ligases. Although plant genomes encode hundreds of E3 ligases, only few targets are known, even in the model Arabidopsis thaliana. Here, we identified the monothiol glutaredoxin GRXS17 as a substrate of the Arabidopsis E3 ubiquitin ligases RING DOMAIN LIGASE 3 (RGLG3) and RGLG4 using a substrate trapping approach involving tandem affinity purification of RING-dead versions. Simultaneously, we used a ubiquitin-conjugating enzym (UBC) panel screen to pinpoint UBC30 as a cognate E2 UBC capable of interacting with RGLG3 and RGLG4 and mediating auto-ubiquitination of RGLG3 and ubiquitination of GRXS17 in vitro. Accordingly, GRXS17 is ubiquitinated and degraded in an RGLG3- and RGLG4-dependent manner in planta. The truncated hemoglobin GLB3 also interacted with RGLG3 and RGLG4 but appeared to obstruct RGLG3 ubiquitination activity rather than being its substrate. Our results suggest that the RGLG family is intimately linked to the essential element iron.

CEP5 and XIP1/CEPR1 regulate lateral root initiation in Arabidopsis.

Roots explore the soil for water and nutrients through the continuous production of lateral roots. Lateral roots are formed at regular distances in a steadily elongating organ, but how future sites for lateral root formation become established is not yet understood. Here, we identified C-TERMINALLY ENCODED PEPTIDE 5 (CEP5) as a novel, auxin-repressed and phloem pole-expressed signal assisting in the formation of lateral roots. In addition, based on genetic and expression data, we found evidence for the involvement of its proposed receptor, XYLEM INTERMIXED WITH PHLOEM 1 (XIP1)/CEP RECEPTOR 1 (CEPR1), during the process of lateral root initiation. In conclusion, we report here on the existence of a peptide ligand-receptor kinase interaction that impacts lateral root initiation. Our results represent an important step towards the understanding of the cellular communication implicated in the early phases of lateral root formation.

The Arabidopsis metacaspase9 degradome.

Metacaspases are distant relatives of the metazoan caspases, found in plants, fungi, and protists. However, in contrast with caspases, information about the physiological substrates of metacaspases is still scarce. By means of N-terminal combined fractional diagonal chromatography, the physiological substrates of metacaspase9 (MC9; AT5G04200) were identified in young seedlings of Arabidopsis thaliana on the proteome-wide level, providing additional insight into MC9 cleavage specificity and revealing a previously unknown preference for acidic residues at the substrate prime site position P1'. The functionalities of the identified MC9 substrates hinted at metacaspase functions other than those related to cell death. These results allowed us to resolve the substrate specificity of MC9 in more detail and indicated that the activity of phosphoenolpyruvate carboxykinase 1 (AT4G37870), a key enzyme in gluconeogenesis, is enhanced upon MC9-dependent proteolysis.

qcML: an exchange format for quality control metrics from mass spectrometry experiments.

Quality control is increasingly recognized as a crucial aspect of mass spectrometry based proteomics. Several recent papers discuss relevant parameters for quality control and present applications to extract these from the instrumental raw data. What has been missing, however, is a standard data exchange format for reporting these performance metrics. We therefore developed the qcML format, an XML-based standard that follows the design principles of the related mzML, mzIdentML, mzQuantML, and TraML standards from the HUPO-PSI (Proteomics Standards Initiative). In addition to the XML format, we also provide tools for the calculation of a wide range of quality metrics as well as a database format and interconversion tools, so that existing LIMS systems can easily add relational storage of the quality control data to their existing schema. We here describe the qcML specification, along with possible use cases and an illustrative example of the subsequent analysis possibilities. All information about qcML is available at http://code.google.com/p/qcml.

Open-Source, Platform-Independent Library and Online Scripting Environment for Accessing Thermo Scientific RAW Files.

Mass spectrometers typically output data in proprietary binary formats. While converter suites and standardized XML formats have been developed in response, these conversion steps come with non-negligible computational time and storage space overhead. As a result, simple, everyday data inspection tasks are often beyond the skills of the mass spectrometrist, who is unable to freely access the acquired data. We therefore here describe the unthermo library for convenient, platform-independent access to Thermo Scientific RAW files and the associated online playground to transform small and easily understandable scriptlets into executable programs for end-users. By fostering the provision of code examples and snippet exchange, the interested mass spectrometrist or researcher can use this playground to quickly assemble custom scripts for their particular purpose. In this way, the data in these RAW files can be mined much more readily and directly by the user, and fast, automated raw data extraction or analysis can finally become part of the daily routine of the mass spectrometrist.