A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties

Improvement of the performance and reliability of a cell-surface untargeted proteomics workflow in the context of multiple myeloma antigen discovery

peer reviewedDisease management of multiple myeloma (MM) is challenging owing to patients’ multiple relapses and resistance to standard treatments. Therefore, the introduction of innovative cell therapies such as chimeric antigen receptor T-cells (CAR-T cells) opened new horizons in the outcome of severe refractory patients affected by aggressive forms of the disease. However, one of the limitations of the expansion of those treatments is the lack of specific MM tumor-associated antigens that could be targeted by current immunotherapies. Indeed, effectiveness of current CAR-T cells treatments could be hampered due to possible antigen-evasion strategies. Therefore, the discovery of new cell surface antigens could be an interesting approach to avoid occurrence of resistance that could lead to treatment failures. For this purpose, a sensitive and robust cell surface mass spectrometry (MS)-based proteomics workflow was implemented and improved to increase identification rate of proteins. In the analytical point of view, a sensitive microfluidic liquid chromatography-based chip coupled to high resolution MS was used in this study to analyze protein digests from a human MM cell line. Moreover, data-independent acquisition mediated by ion mobility was also applied to maximize proteome coverage. The performance of sample preparation is a critical part of the workflow that influences the output in terms of protein identification. Indeed, different steps of this procedure including biotinylation, elution, digestion and peptide clean-up were optimized using one million cells from a human MM cell line to yield the highest number of identification. Furthermore, the workflow was also applied to the analysis of cell line samples containing decreasing number of cells from one million to fifty thousands. The aim was to evaluate the performance of the workflow to analyze low number of cells due to the complexity to obtain primary cells from patients containing high number of cells. Performing the whole workflow, including sample preparation, MS analysis and data treatment, is time-consuming. The initial sample preparation protocol contains more than 50 steps of manual handling of the samples which emphasizes the complexity of the process. In order to ensure the reliability of the sample preparation procedure, the use of quality control (QC) metrics is relevant. Therefore, an approach was implemented in our workflow to remove samples that generated low quality data. This post-analysis method is based on the identification of “all process” QC protein of different intensities to determine the reliability of the sample preparation. This approach was developed using samples containing one million cells but was also evaluated for lower number of cells until fifty thousands. Moreover, the analysis of patient samples was performed using the developed workflow and the identification of QC proteins allowed to discard low quality samples from further analysis. In conclusion, a untargeted MS-based proteomics workflow to analyze MM cell lines and primary cells was successfully developed through the optimization of the sample preparation workflow. Additionally, the implementation of QC proteins allowed to increase the reliability of sample preparation

Potential of Single Pulse and Multiplexed Drift-Tube Ion Mobility Spectrometry Coupled to Micropillar Array Column for Proteomics Studies

Proteomics is one of the most significant methodologies to better understand the molecular pathways involved in diseases and to improve their diagnosis, treatment and follow-up. The investigation of the proteome of complex organisms is challenging from an analytical point of view, because of the large number of proteins present in a wide range of concentrations. In this study, nanofluidic chromatography, using a micropillar array column, was coupled to drift-tube ion mobility and time-of-flight mass spectrometry to identify as many proteins as possible in a protein digest standard of HeLa cells. Several chromatographic parameters were optimized. The high interest of drift-tube ion mobility to increase the number of identifications and to separate isobaric coeluting peptides was demonstrated. Multiplexed drift-tube ion mobility spectrometry was also investigated, to increase the sensitivity in proteomics studies. This innovative proteomics platform will be useful for analyzing patient samples to better understand unresolved disorders

Interest of CE-IM-MS as a complementary tool to chromatographic-based methods for cell surface antigen discovery: proof-of-concept using human myeloma LP-1 cell line.

peer reviewedDisease management of multiple myeloma is challenging owing to patients’ multiple relapses and resistance to standard treatments. Therefore, the introduction of innovative cell therapies such as chimeric antigen receptor T-cells (CAR-T cells) opened new horizons in the outcome of severe refractory patients affected by aggressive forms of the disease. However, one of the limitations of the expansion of those treatments is the lack of specific MM tumor-associated antigens that could be targeted by current immunotherapies. Indeed, effectiveness of current CAR-T cells treatments could be hampered due to possible antigen-evasion strategies. Therefore, the discovery of new cell surface antigens could be an interesting approach to avoid occurrence of resistance that could lead to treatment failures. For this purpose, the use of mass spectrometry (MS) proteomics-based methodologies was considered. Due to the high sample complexity, liquid chromatography (LC) is commonly used prior MS detection to maximize protein identifications. Similarly, capillary electrophoresis (CE) could be an alternative due to its ability to provide high efficiency and high throughput separation. However, CE-MS performance could be impaired by lower sensitivity due to poor design of CE-MS interface as well as low sample loading capacity. In this study, those issues were tackled by using an online preconcentration technique namely dynamic pH junction to enhance sensitivity as well as loading capacity. Indeed, different conditions were compared in order to increase loaded volume without compromising separation efficiency. Besides, neutral-coated capillary was used in order to increase peak capacity leading to a higher number of identified entities compared to uncoated capillaries. Since CE separation principle is orthogonal to the mechanism that drives separation in LC, the use of both techniques to analyze the same sample allowed the increase of overall information in terms of number of identified proteins. Indeed, the interest of combining electrophoretic to chromatographic approaches was confirmed for the identification of antigens at the surface of human myeloma LP-1 cell line. As a matter of fact, despite the high number of proteins identified using LC-MS, more than a half of the proteins identified in CE could not be detected by LC. The capability of ion-mobility (IM) was also exploited in this study. Indeed, the addition of IMS module between LC or CE and MS lead to better proteome coverage due to the additional dimension of separation. To the best of our knowledge, little attention has been paid to the potential orthogonality between CE and IMS in proteomic studies to date. In this study the combination of CE with IMS allowed the separation of isobaric and co-migrating peptides leading to the identification of a larger number of unique proteins, thus increasing the possibility of detecting new antigens. In conclusion, the proof of concept concerning the interest of CE-IM-MS for the discovery of cell surface antigens was achieved during this study

Contribution of proteomics to the development of new therapeutics

peer reviewe

Interest of capillary zone electrophoresis coupled to mass spectrometry for clinical proteomics analysis : how to improve sensitivity?

Multiple myeloma is an incurable hematological malignancy located in the bone marrow. Nowadays, disease management is still complicated due to drug resistance leading to relapses for patients. Therefore, it is of high interest to identify new cell surface antigens that could act as targets of several immunotherapeutic approaches including monoclonal antibodies as well as promising active therapies such as chimeric antigen receptor T cells (CART cells). Since clinical proteomics usually involves complex samples (cell proteome), the use of highly resolutive and sensitive instruments to perform comprehensive analysis of those complex proteomes is relevant. Capillary zone electrophoresis tandem mass spectrometry (CZE-MS/MS) is an interesting tool for proteomic analysis as the separation principle is different to liquid chromatography tandem mass spectrometry (LC-MS/MS). Therefore, the combination of both techniques can bring complementary information to enlarge proteome coverage. In this study, sample preconcentration techniques were investigated in order to improve sample loading and therefore sensitivity. The use of dynamic pH junction (DPJ) allowed the identification of more peptides and proteins compared to conventional injections. Besides, an approximate 100-fold gain in sensitivity was observed using DPJ. Then, a nanoflow sheath liquid interface (EMASS-II) was compared to the traditional coaxial sheath liquid interface (Triple tube). The use of EMASS-II interface allowed the identification of approximately two times more peptides and proteins thanks to an improvement in sensitivity. Indeed, compared to the Triple tube, peak height and peak area were improved by a factor of 12 and 23-fold, respectively. Finally, data obtained using CZE-MS/MS was compared to those acquired using LC-Chip-MS/MS. Interestingly, 50% of the identified proteins obtained with CZE were unique to this electrophoretic technique. Therefore, we consider that the interest of using CZE-MS/MS for clinical proteomic studies is high. In conclusion, we were able to overcome the lack of sensitivity resulting from CZE-MS coupling interface by using online preconcentration and a nanoflow sheath-liquid interface, namely EMASS-II. Besides, the results also proved that proteome coverage is enhanced when CZE and LC-Chip are used in combination owing to their orthogonality