Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics.

Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometry-based proteomics approach to investigate the global dynamics of (phospho) protein expression during gastruloid differentiation. Our findings revealed many proteins with temporal expression and unique expression profiles for each germ layer, which we also validated using single-cell proteomics technology. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with single-cell RNA sequencing (scRNA-seq) identified a critical role for ZEB2 in mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.

Advances in experimental and computational methodologies for the study of microbial-surface interactions at different omics levels.

The study of the biological response of microbial cells interacting with natural and synthetic interfaces has acquired a new dimension with the development and constant progress of advanced omics technologies. New methods allow the isolation and analysis of nucleic acids, proteins and metabolites from complex samples, of interest in diverse research areas, such as materials sciences, biomedical sciences, forensic sciences, biotechnology and archeology, among others. The study of the bacterial recognition and response to surface contact or the diagnosis and evolution of ancient pathogens contained in archeological tissues require, in many cases, the availability of specialized methods and tools. The current review describes advances in in vitro and in silico approaches to tackle existing challenges (e.g., low-quality sample, low amount, presence of inhibitors, chelators, etc.) in the isolation of high-quality samples and in the analysis of microbial cells at genomic, transcriptomic, proteomic and metabolomic levels, when present in complex interfaces. From the experimental point of view, tailored manual and automatized methodologies, commercial and in-house developed protocols, are described. The computational level focuses on the discussion of novel tools and approaches designed to solve associated issues, such as sample contamination, low quality reads, low coverage, etc. Finally, approaches to obtain a systems level understanding of these complex interactions by integrating multi omics datasets are presented.

Miniaturised interaction proteomics on a microfluidic platform with ultra-low input requirements.

Essentially all cellular processes are orchestrated by protein-protein interactions (PPIs). In recent years, affinity purification coupled to mass spectrometry (AP-MS) has been the preferred method to identify cellular PPIs. Here we present a microfluidic-based AP-MS workflow, called on-chip AP-MS, to identify PPIs using minute amounts of input material. By using this automated platform we purify the human Cohesin, CCC and Mediator complexes from as little as 4 micrograms of input lysate, representing a 50─100-fold downscaling compared to regular microcentrifuge tube-based protocols. We show that our platform can be used to affinity purify tagged baits as well as native cellular proteins and their interaction partners. As such, our method holds great promise for future biological and clinical AP-MS applications in which sample amounts are limited.

Integrative multi-omics analysis of intestinal organoid differentiation.

Intestinal organoids accurately recapitulate epithelial homeostasis in vivo, thereby representing a powerful in vitro system to investigate lineage specification and cellular differentiation. Here, we applied a multi-omics framework on stem cell-enriched and stem cell-depleted mouse intestinal organoids to obtain a holistic view of the molecular mechanisms that drive differential gene expression during adult intestinal stem cell differentiation. Our data revealed a global rewiring of the transcriptome and proteome between intestinal stem cells and enterocytes, with the majority of dynamic protein expression being transcription-driven. Integrating absolute mRNA and protein copy numbers revealed post-transcriptional regulation of gene expression. Probing the epigenetic landscape identified a large number of cell-type-specific regulatory elements, which revealed Hnf4g as a major driver of enterocyte differentiation. In summary, by applying an integrative systems biology approach, we uncovered multiple layers of gene expression regulation, which contribute to lineage specification and plasticity of the mouse small intestinal epithelium.

Chromatin enrichment for proteomics.

During interphase, chromatin hosts fundamental cellular processes, such as gene expression, DNA replication and DNA damage repair. To analyze chromatin on a proteomic scale, we have developed chromatin enrichment for proteomics (ChEP), which is a simple biochemical procedure that enriches interphase chromatin in all its complexity. It enables researchers to take a 'snapshot' of chromatin and to isolate and identify even transiently bound factors. In ChEP, cells are fixed with formaldehyde; subsequently, DNA together with all cross-linked proteins is isolated by centrifugation under denaturing conditions. This approach enables the analysis of global chromatin composition and its changes, which is in contrast with existing chromatin enrichment procedures, which either focus on specific chromatin loci (e.g., affinity purification) or are limited in specificity, such as the analysis of the chromatin pellet (i.e., analysis of all insoluble nuclear material). ChEP takes half a day to complete and requires no specialized laboratory skills or equipment. ChEP enables the characterization of chromatin response to drug treatment or physiological processes. Beyond proteomics, ChEP may preclear chromatin for chromatin immunoprecipitation (ChIP) analyses.

Proteomics of a fuzzy organelle: interphase chromatin.

Chromatin proteins mediate replication, regulate expression, and ensure integrity of the genome. So far, a comprehensive inventory of interphase chromatin has not been determined. This is largely due to its heterogeneous and dynamic composition, which makes conclusive biochemical purification difficult, if not impossible. As a fuzzy organelle, it defies classical organellar proteomics and cannot be described by a single and ultimate list of protein components. Instead, we propose a new approach that provides a quantitative assessment of a protein's probability to function in chromatin. We integrate chromatin composition over a range of different biochemical and biological conditions. This resulted in interphase chromatin probabilities for 7635 human proteins, including 1840 previously uncharacterized proteins. We demonstrate the power of our large-scale data-driven annotation during the analysis of cyclin-dependent kinase (CDK) regulation in chromatin. Quantitative protein ontologies may provide a general alternative to list-based investigations of organelles and complement Gene Ontology.

A protein profile study to discriminate CIN lesions from normal cervical epithelium.

BACKGROUND: Cervical intraepithelial neoplasia (CIN), a frequently encountered disease caused by Human Papilloma Virus (HPV) is often diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. Since it is known that this procedure strongly affects the water-soluble proteins contained in the cervical tissue we decided to investigate whether a water-soluble protein-saving biopsy processing method can be used to support the diagnosis of normal and CIN. METHODS: Cervical punch biopsies from 55 women were incubated for 24 h at 4°C in RPMI1640 medium for protein analysis prior to usual FFPE processing and p16 and Ki67-supported histologic consensus diagnosis was assessed. The biopsy supernatants were subjected to surface-enhanced laser desorption-ionization time of flight mass spectrometry (SELDI-TOF MS) for identifying differentially expressed proteins. Binary logistic regression and classification and regression trees (CART) were used to develop a classification model. RESULTS: The age of the patients ranged from 26 to 40 years (median 29.7). The consensus diagnoses were normal cervical tissue (n = 10) and CIN2-3 (n = 45). The mean protein concentration was 1.00 and 1.09 mg/ml in the normal and CIN2-3 group, respectively. The peak detection and clustering process resulted in 40 protein peaks. Many of these peaks differed between the two groups, but only three had independent discriminating power. The overall classification results were 88%. CONCLUSIONS: Water-soluble proteins sampled from punch biopsies are promising to assist the diagnosis of normal and CIN2-3.

Interaction proteomics of the HMGA chromatin architectural factors.

The high mobility group A (HMGA) chromatin architectural transcription factors are a group of proteins involved in development and neoplastic transformation. They take part in an articulated interaction network, both with DNA and other nuclear proteins, organizing multimolecular complexes at chromatin level. Here, we report the development of a novel in vitro strategy for the identification of HMGA molecular partners based on the combination of an RP-HPLC prefractionation procedure, 2-DE gels, blot-overlay and MS. To demonstrate that our approach could be a reliable screening method we confirmed a representative number of interactions in vitro by GST pull-down and far-Western and in vivo by co-affinity purification. This approach allowed us to enlarge the HMGA molecular network confirming their involvement also in non-transcriptional-related processes such as RNA processing and DNA repair.