Discovery and Mechanistic Elucidation of NQO1-Bioactivatable Small Molecules That Overcome Resistance to Degraders.

Degraders hold the promise to efficiently inactivate previously intractable disease-relevant targets. Unlike traditional inhibitors, degraders act substoichiometrically and rely on the hijacked proteolysis machinery, which can also act as an entry point for resistance. To fully harness the potential of targeted protein degradation, it is crucial to comprehend resistance mechanisms and formulate effective strategies to overcome them. We conducted a chemical screening to identify synthetic lethal vulnerabilities of cancer cells that exhibit widespread resistance to degraders. Comparative profiling followed by tailored optimization delivered the small molecule RBS-10, which shows preferential cytotoxicity against cells pan-resistant to degraders. Multiomics deconvolution of the mechanism of action revealed that RBS-10 acts as a prodrug bioactivated by the oxidoreductase enzyme NQO1, which is highly overexpressed in our resistance models. Collectively, our work informs on NQO1 as an actionable vulnerability to overcome resistance to degraders and as a biomarker to selectively exploit bioactivatable prodrugs in cancer.

Identification of a de novo mutation in TLK1 associated with a neurodevelopmental disorder and immunodeficiency.

Background: The Tousled-like kinases 1 and 2 (TLK1/TLK2) regulate DNA replication, repair and chromatin maintenance. TLK2 variants are associated with 'Intellectual Disability, Autosomal Dominant 57' (MRD57), a neurodevelopmental disorder (NDD) characterized by intellectual disability (ID), autism spectrum disorder (ASD) and microcephaly. Several TLK1 variants have been reported in NDDs but their functional significance is unknown. Methods: A male patient presenting with ID, seizures, global developmental delay, hypothyroidism, and primary immunodeficiency was determined to have a novel, heterozygous variant in TLK1 (c.1435C>G, p.Q479E) by genome sequencing (GS). Single cell gel electrophoresis, western blot, flow cytometry and RNA-seq were performed in patient-derived lymphoblast cell lines. In silico, biochemical and proteomic analysis were used to determine the functional impact of the p.Q479E variant and previously reported NDD-associated TLK1 variant, p.M566T. Results: Transcriptome sequencing in patient-derived cells confirmed expression of TLK1 transcripts carrying the p.Q479E variant and revealed alterations in genes involved in class switch recombination and cytokine signaling. Cells expressing the p.Q479E variant exhibited reduced cytokine responses and higher levels of spontaneous DNA damage but not increased sensitivity to radiation or DNA repair defects. The p.Q479E and p.M566T variants impaired kinase activity but did not strongly alter localization or proximal protein interactions. Conclusion: Our study provides the first functional characterization of TLK1 variants associated with NDDs and suggests potential involvement in central nervous system and immune system development. Our results indicate that, like TLK2 variants, TLK1 variants may impact development in multiple tissues and should be considered in the diagnosis of rare NDDs.

Characterization of p38α Signaling Networks in Cancer Cells Using Quantitative Proteomics and Phosphoproteomics.

p38α (encoded by MAPK14) is a protein kinase that regulates cellular responses to almost all types of environmental and intracellular stresses. Upon activation, p38α phosphorylates many substrates both in the cytoplasm and nucleus, allowing this pathway to regulate a wide variety of cellular processes. While the role of p38α in the stress response has been widely investigated, its implication in cell homeostasis is less understood. To investigate the signaling networks regulated by p38α in proliferating cancer cells, we performed quantitative proteomic and phosphoproteomic analyses in breast cancer cells in which this pathway had been either genetically targeted or chemically inhibited. Our study identified with high confidence 35 proteins and 82 phosphoproteins (114 phosphosites) that are modulated by p38α and highlighted the implication of various protein kinases, including MK2 and mTOR, in the p38α-regulated signaling networks. Moreover, functional analyses revealed an important contribution of p38α to the regulation of cell adhesion, DNA replication, and RNA metabolism. Indeed, we provide experimental evidence supporting that p38α facilitates cancer cell adhesion and showed that this p38α function is likely mediated by the modulation of the adaptor protein ArgBP2. Collectively, our results illustrate the complexity of the p38α-regulated signaling networks, provide valuable information on p38α-dependent phosphorylation events in cancer cells, and document a mechanism by which p38α can regulate cell adhesion.

Time-resolved transcriptomic profiling of the developing rabbit's lungs: impact of premature birth and implications for modelling bronchopulmonary dysplasia.

BACKGROUND: Premature birth, perinatal inflammation, and life-saving therapies such as postnatal oxygen and mechanical ventilation are strongly associated with the development of bronchopulmonary dysplasia (BPD); these risk factors, alone or combined, cause lung inflammation and alter programmed molecular patterns of normal lung development. The current knowledge on the molecular regulation of lung development mainly derives from mechanistic studies conducted in newborn rodents exposed to postnatal hyperoxia, which have been proven useful but have some limitations. METHODS: Here, we used the rabbit model of BPD as a cost-effective alternative model that mirrors human lung development and, in addition, enables investigating the impact of premature birth per se on the pathophysiology of BPD without further perinatal insults (e.g., hyperoxia, LPS-induced inflammation). First, we characterized the rabbit's normal lung development along the distinct stages (i.e., pseudoglandular, canalicular, saccular, and alveolar phases) using histological, transcriptomic and proteomic analyses. Then, the impact of premature birth was investigated, comparing the sequential transcriptomic profiles of preterm rabbits obtained at different time intervals during their first week of postnatal life with those from age-matched term pups. RESULTS: Histological findings showed stage-specific morphological features of the developing rabbit's lung and validated the selected time intervals for the transcriptomic profiling. Cell cycle and embryo development, oxidative phosphorylation, and WNT signaling, among others, showed high gene expression in the pseudoglandular phase. Autophagy, epithelial morphogenesis, response to transforming growth factor ß, angiogenesis, epithelium/endothelial cells development, and epithelium/endothelial cells migration pathways appeared upregulated from the 28th day of gestation (early saccular phase), which represents the starting point of the premature rabbit model. Premature birth caused a significant dysregulation of the inflammatory response. TNF-responsive, NF-κB regulated genes were significantly upregulated at premature delivery and triggered downstream inflammatory pathways such as leukocyte activation and cytokine signaling, which persisted upregulated during the first week of life. Preterm birth also dysregulated relevant pathways for normal lung development, such as blood vessel morphogenesis and epithelial-mesenchymal transition. CONCLUSION: These findings establish the 28-day gestation premature rabbit as a suitable model for mechanistic and pharmacological studies in the context of BPD.

Comparative analyses of vertebrate CPEB proteins define two subfamilies with coordinated yet distinct functions in post-transcriptional gene regulation.

BACKGROUND: Vertebrate CPEB proteins bind mRNAs at cytoplasmic polyadenylation elements (CPEs) in their 3' UTRs, leading to cytoplasmic changes in their poly(A) tail lengths; this can promote translational repression or activation of the mRNA. However, neither the regulation nor the mechanisms of action of the CPEB family per se have been systematically addressed to date. RESULTS: Based on a comparative analysis of the four vertebrate CPEBs, we determine their differential regulation by phosphorylation, the composition and properties of their supramolecular assemblies, and their target mRNAs. We show that all four CPEBs are able to recruit the CCR4-NOT deadenylation complex to repress the translation. However, their regulation, mechanism of action, and target mRNAs define two subfamilies. Thus, CPEB1 forms ribonucleoprotein complexes that are remodeled upon a single phosphorylation event and are associated with mRNAs containing canonical CPEs. CPEB2-4 are regulated by multiple proline-directed phosphorylations that control their liquid-liquid phase separation. CPEB2-4 mRNA targets include CPEB1-bound transcripts, with canonical CPEs, but also a specific subset of mRNAs with non-canonical CPEs. CONCLUSIONS: Altogether, these results show how, globally, the CPEB family of proteins is able to integrate cellular cues to generate a fine-tuned adaptive response in gene expression regulation through the coordinated actions of all four members.

Proteomic Tools for the Quantitative Analysis of Artificial Peptide Libraries: Detection and Characterization of Target-Amplified PD-1 Inhibitors.

We report a quantitative proteomics data analysis pipeline, which coupled to protein-directed dynamic combinatorial chemistry (DDC) experiments, enables the rapid discovery and direct characterization of protein-protein interaction (PPI) modulators. A low-affinity PD-1 binder was incubated with a library of >100 D-peptides under thiol-exchange favoring conditions, in the presence of the target protein PD-1, and we determined the S-linked dimeric species that resulted, amplified in the protein samples versus the controls. We chemically synthesized the target dimer candidates and validated them by thermophoresis binding and protein-protein interaction assays. The results provide a proof-of-concept for using this strategy in the high-throughput search of improved drug-like peptide binders that block therapeutically relevant protein-protein interactions.

Biomarker candidates for progression and clinical management of COVID-19 associated pneumonia at time of admission.

COVID-19 pathophysiology is currently not fully understood, reliable prognostic factors remain elusive, and few specific therapeutic strategies have been proposed. In this scenario, availability of biomarkers is a priority. MS-based Proteomics techniques were used to profile the proteome of 81 plasma samples extracted in four consecutive days from 23 hospitalized COVID-19 associated pneumonia patients. Samples from 10 subjects that reached a critical condition during their hospital stay and 10 matched non-severe controls were drawn before the administration of any COVID-19 specific treatment and used to identify potential biomarkers of COVID-19 prognosis. Additionally, we compared the proteome of five patients before and after glucocorticoids and tocilizumab treatment, to assess the changes induced by the therapy on our selected candidates. Forty-two proteins were differentially expressed between patients' evolution groups at 10% FDR. Twelve proteins showed lower levels in critical patients (fold-changes 1.20-3.58), of which OAS3 and COG5 found their expression increased after COVID-19 specific therapy. Most of the 30 proteins over-expressed in critical patients (fold-changes 1.17-4.43) were linked to inflammation, coagulation, lipids metabolism, complement or immunoglobulins, and a third of them decreased their expression after treatment. We propose a set of candidate proteins for biomarkers of COVID-19 prognosis at the time of hospital admission. The study design employed is distinctive from previous works and aimed to optimize the chances of the candidates to be validated in confirmatory studies and, eventually, to play a useful role in the clinical practice.

Functional analysis of TLK2 variants and their proximal interactomes implicates impaired kinase activity and chromatin maintenance defects in their pathogenesis.

INTRODUCTION: The Tousled-like kinases 1 and 2 (TLK1 and TLK2) are involved in many fundamental processes, including DNA replication, cell cycle checkpoint recovery and chromatin remodelling. Mutations in TLK2 were recently associated with 'Mental Retardation Autosomal Dominant 57' (MRD57, MIM# 618050), a neurodevelopmental disorder characterised by a highly variable phenotype, including mild-to-moderate intellectual disability, behavioural abnormalities, facial dysmorphisms, microcephaly, epilepsy and skeletal anomalies. METHODS: We re-evaluate whole exome sequencing and array-CGH data from a large cohort of patients affected by neurodevelopmental disorders. Using spatial proteomics (BioID) and single-cell gel electrophoresis, we investigated the proximity interaction landscape of TLK2 and analysed the effects of p.(Asp551Gly) and a previously reported missense variant (c.1850C>T; p.(Ser617Leu)) on TLK2 interactions, localisation and activity. RESULTS: We identified three new unrelated MRD57 families. Two were sporadic and caused by a missense change (c.1652A>G; p.(Asp551Gly)) or a 39 kb deletion encompassing TLK2, and one was familial with three affected siblings who inherited a nonsense change from an affected mother (c.1423G>T; p.(Glu475Ter)). The clinical phenotypes were consistent with those of previously reported cases. The tested mutations strongly impaired TLK2 kinase activity. Proximal interactions between TLK2 and other factors implicated in neurological disorders, including CHD7, CHD8, BRD4 and NACC1, were identified. Finally, we demonstrated a more relaxed chromatin state in lymphoblastoid cells harbouring the p.(Asp551Gly) variant compared with control cells, conferring susceptibility to DNA damage. CONCLUSION: Our study identified novel TLK2 pathogenic variants, confirming and further expanding the MRD57-related phenotype. The molecular characterisation of missense variants increases our knowledge about TLK2 function and provides new insights into its role in neurodevelopmental disorders.

Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis.

Global analysis of protein phosphorylation by mass spectrometry proteomic techniques has emerged in the last decades as a powerful tool in biological and biomedical research. However, there are several factors that make the global study of the phosphoproteome more challenging than measuring non-modified proteins. The low stoichiometry of the phosphorylated species and the need to retrieve residue specific information require particular attention on sample preparation, data acquisition and processing to ensure reproducibility, qualitative and quantitative robustness and ample phosphoproteome coverage in phosphoproteomic workflows. Aiming to investigate the effect of different variables in the performance of proteome wide phosphoprotein analysis protocols, ProteoRed-ISCIII and EuPA launched the Proteomics Multicentric Experiment 11 (PME11). A reference sample consisting of a yeast protein extract spiked in with different amounts of a phosphomix standard (Sigma/Merck) was distributed to 31 laboratories around the globe. Thirty-six datasets from 23 laboratories were analyzed. Our results indicate the suitability of the PME11 reference sample to benchmark and optimize phosphoproteomics strategies, weighing the influence of different factors, as well as to rank intra and inter laboratory performance.

Identification and drug-induced reversion of molecular signatures of Alzheimer's disease onset and progression in AppNL-G-F, AppNL-F, and 3xTg-AD mouse models.

BACKGROUND: In spite of many years of research, our understanding of the molecular bases of Alzheimer's disease (AD) is still incomplete, and the medical treatments available mainly target the disease symptoms and are hardly effective. Indeed, the modulation of a single target (e.g., ß-secretase) has proven to be insufficient to significantly alter the physiopathology of the disease, and we should therefore move from gene-centric to systemic therapeutic strategies, where AD-related changes are modulated globally. METHODS: Here we present the complete characterization of three murine models of AD at different stages of the disease (i.e., onset, progression and advanced). We combined the cognitive assessment of these mice with histological analyses and full transcriptional and protein quantification profiling of the hippocampus. Additionally, we derived specific Aß-related molecular AD signatures and looked for drugs able to globally revert them. RESULTS: We found that AD models show accelerated aging and that factors specifically associated with Aß pathology are involved. We discovered a few proteins whose abundance increases with AD progression, while the corresponding transcript levels remain stable, and showed that at least two of them (i.e., lfit3 and Syt11) co-localize with Aß plaques in the brain. Finally, we found two NSAIDs (dexketoprofen and etodolac) and two anti-hypertensives (penbutolol and bendroflumethiazide) that overturn the cognitive impairment in AD mice while reducing Aß plaques in the hippocampus and partially restoring the physiological levels of AD signature genes to wild-type levels. CONCLUSIONS: The characterization of three AD mouse models at different disease stages provides an unprecedented view of AD pathology and how this differs from physiological aging. Moreover, our computational strategy to chemically revert AD signatures has shown that NSAID and anti-hypertensive drugs may still have an opportunity as anti-AD agents, challenging previous reports.

Nature-inspired dimerization as a strategy to modulate neuropeptide pharmacology exemplified with vasopressin and oxytocin.

Vasopressin (VP) and oxytocin (OT) are cyclic neuropeptides that regulate fundamental physiological functions via four G protein-coupled receptors, V1aR, V1bR, V2R, and OTR. Ligand development remains challenging for these receptors due to complex structure-activity relationships. Here, we investigated dimerization as a strategy for developing ligands with novel pharmacology. We regioselectively synthesised and systematically studied parallel, antiparallel and N- to C-terminal cyclized homo- and heterodimer constructs of VP, OT and dVDAVP (1-deamino-4-valine-8-d-arginine-VP). All disulfide-linked dimers, except for the head-to-tail cyclized constructs, retained nanomolar potency despite the structural implications of dimerization. Our results support a single chain interaction for receptor activation. Dimer orientation had little impact on activity, except for the dVDAVP homodimers, where an antagonist to agonist switch was observed at the V1aR. This study provides novel insights into the structural requirements of VP/OT receptor activation and spotlights dimerization as a strategy to modulate pharmacology, a concept also frequently observed in nature.

Furan warheads for covalent trapping of weak protein-protein interactions: cross-linking of thymosin ß4 to actin.

We describe furan as a triggerable 'warhead' for site-specific cross-linking using the actin and thymosin ß4 (Tß4)-complex as model of a weak and dynamic protein-protein interaction (PPI) with known 3D structure and with application potential in disease contexts. The identified cross-linked residues demonstrate that lysine is a target for the furan warhead. The presented in vitro validation of covalently acting 'furan-armed' Tß4-variants provides initial proof to further exploit furan-technology for covalent drug design targeting lysines.

Protamine Characterization by Top-Down Proteomics: Boosting Proteoform Identification with DBSCAN.

Protamines replace histones as the main nuclear protein in the sperm cells of many species and play a crucial role in compacting the paternal genome. Human spermatozoa contain protamine 1 (P1) and the family of protamine 2 (P2) proteins. Alterations in protamine PTMs or the P1/P2 ratio may be associated with male infertility. Top-down proteomics enables large-scale analysis of intact proteoforms derived from alternative splicing, missense or nonsense genetic variants or PTMs. In contrast to current gold standard techniques, top-down proteomics permits a more in-depth analysis of protamine PTMs and proteoforms, thereby opening up new perspectives to unravel their impact on male fertility. We report on the analysis of two normozoospermic semen samples by top-down proteomics. We discuss the difficulties encountered with the data analysis and propose solutions as this step is one of the current bottlenecks in top-down proteomics with the bioinformatics tools currently available. Our strategy for the data analysis combines two software packages, ProSight PD (PS) and TopPIC suite (TP), with a clustering algorithm to decipher protamine proteoforms. We identified up to 32 protamine proteoforms at different levels of characterization. This in-depth analysis of the protamine proteoform landscape of normozoospermic individuals represents the first step towards the future study of sperm pathological conditions opening up the potential personalized diagnosis of male infertility.

Characterization of Human Sperm Protamine Proteoforms through a Combination of Top-Down and Bottom-Up Mass Spectrometry Approaches.

Protamine 1 (P1) and protamine 2 (P2) family are extremely basic, sperm-specific proteins, packing 85-95% of the paternal DNA. P1 is synthesized as a mature form, whereas P2 components (HP2, HP3, and HP4) arise from the proteolysis of the precursor (pre-P2). Due to the particular protamine physical-chemical properties, their identification by standardized bottom-up mass spectrometry (MS) strategies is not straightforward. Therefore, the aim of this study was to identify the sperm protamine proteoforms profile, including their post-translational modifications, in normozoospermic individuals using two complementary strategies, a top-down MS approach and a proteinase-K-digestion-based bottom-up MS approach. By top-down MS, described and novel truncated P1 and pre-P2 proteoforms were identified. Intact P1, pre-P2, and P2 mature proteoforms and their phosphorylation pattern were also detected. Additionally, a +61 Da modification in different proteoforms was observed. By the bottom-up MS approach, phosphorylated residues for pre-P2, as well as the new P2 isoform 2, which is not annotated in the UniProtKB database, were revealed. Implementing these strategies in comparative studies of different infertile phenotypes, together with the evaluation of P1/P2 and pre-P2/P2 MS-derived ratios, would permit determining specific alterations in the protamine proteoforms and elucidate the role of phosphorylation/dephosphorylation dynamics in male fertility.

The histone code reader PHD finger protein 7 controls sex-linked disparities in gene expression and malignancy in Drosophila.

The notable male predominance across many human cancer types remains unexplained. Here, we show that Drosophila l(3)mbt brain tumors are more invasive and develop as malignant neoplasms more often in males than in females. By quantitative proteomics, we have identified a signature of proteins that are differentially expressed between male and female tumor samples. Prominent among them is the conserved chromatin reader PHD finger protein 7 (Phf7). We show that Phf7 depletion reduces sex-dependent differences in gene expression and suppresses the enhanced malignant traits of male tumors. Our results identify potential regulators of sex-linked tumor dimorphism and show that these genes may serve as targets to suppress sex-linked malignant traits.

Top-Down Proteomics Applied to Human Cerebrospinal Fluid.

Cerebrospinal fluid (CSF) is the fluid of choice to study pathologies and disorders of the central nervous system (CNS). Its composition, especially its proteins and peptides, holds the promise that it may reflect the pathological state of an individual. Traditionally, proteins and peptides in CSF have been analyzed using bottom-up proteomics technologies in the search of high proteome coverage. However, the limited protein sequence coverage of this technology means that information regarding post-translational modifications (PTMs) and alternative splice variants is lost. As an alternative technology, top-down proteomics offers low to medium proteome coverage, but high protein coverage enabling almost a full characterization of the proteins' primary structure. This allows us to precisely identify distinct molecular forms of proteins (proteoforms) as well as naturally occurring bioactive peptide fragments, which could be of critical biological relevance and would otherwise remain undetected with a classical proteomics approach.Here, we describe various strategies including sample preparation protocols, off-line intact protein prefractionation, and LC-MS/MS methods together with data analysis pipelines to analyze cerebrospinal fluid (CSF) by top-down proteomics. However, there is not a unique or standardized method and the selection of the top-down strategy will depend on the exact goal of the study. Here, we describe various top-down proteomics methods that enable rapid protein characterization and may be an excellent companion analytical workflow in the search for new protein biomarkers in neurodegenerative diseases.

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

Direct Evidence of the Presence of Cross-Linked Aß Dimers in the Brains of Alzheimer's Disease Patients.

Brain-derived amyloid-ß (Aß) dimers are associated with Alzheimer's disease (AD). However, their covalent nature remains controversial. This feature is relevant, as a covalent cross-link has been proposed to make brain-derived dimers (brain dimers) more synaptotoxic than Aß monomers and would also make them suitable candidates for biomarker development. To resolve this controversy, we here present a three-step approach. First, we validated a type of synthetic cross-linked Aß (CL Aß) dimers, obtained by means of the photoinduced cross-linking of unmodified proteins (PICUP) reaction, as well-defined mimics of putative brain CL Aß dimers. Second, we used these PICUP CL Aß dimers as standards to improve the isolation of brain Aß dimers and to develop state-of-the-art mass spectrometry (MS) strategies to allow their characterization. Third, we applied these MS methods to the analysis of brain Aß dimer samples allowing the detection of the CL [Aß(6-16)]2 peptide comprising a dityrosine cross-link. This result demonstrates the presence of CL Aß dimers in the brains of patients with AD and opens up avenues for establishing new therapeutic targets and developing novel biomarkers for this disease.

Structure of the homodimeric androgen receptor ligand-binding domain.

The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å2 large dimerization surface, which harbours over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor.

A multicentric study to evaluate the use of relative retention times in targeted proteomics.

Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. BIOLOGICAL SIGNIFICANCE: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups.