Abstract
Hematopoiesis is a highly dynamic cellular process involving the multi-step differentiation of hematopoietic stem cells (HSCs) through various intermediate progenitor stages, ultimately giving rise to mature blood cells. Single-cell studies have revealed a remarkable molecular and functional heterogeneity within hematopoietic stem and progenitor cells (HSPCs). Single-cell transcriptome
profiling underlies these studies, while it is well known that transcript levels do not always accurately predict protein abundance due to variations in translation and degradation rates. As proteins are the effector molecules of the cells and directly influence the functional and phenotypic characteristics, a fundamental understanding of cell state heterogeneity requires direct measurement of the proteome. The progress in global proteomics studies has been limited by the
lack of methods suitable for analyzing low-input samples, including single cells, however, with rapid recent advances, mass spectrometry-based proteomics has emerged as a powerful tool to tackle this problem. In this study, we utilized novel single-cell and low-input (500 cells) proteomics to explore the continuous landscape of HSPCs in both healthy human and murine bone marrow (BM) hematopoiesis. We demonstrate the power of low-input proteomics in three different yet interconnected projects.
Initially, we established a multiplexed single-cell proteomics (scp-MS) workflow that involves FACS isolation into 384-well plates and applied it to a disrupted hematopoietic hierarchy, including leukemic stem cells (LSC), progenitors, and blasts. This workflow identified approximately 1,000 proteins per cell, revealing two differentiation routes; the classic progression from LSCs to progenitors and blasts, and a previously unidentified route going directly from LSCs
to blasts in a primary leukemia cell culture system.
Subsequently, the multiplexed scp-MS workflow was applied to a highly complex HSPC hierarchy derived from the BM of healthy individuals. This study marked the first application of scp-MS in a complex cellular hierarchy derived from primary BM samples. The study highlighted limitations in commonly used FACS isolation schemes and identified proteome-level factors not evident at the transcriptome level based on publicly available RNA-seq data. Among others, we identified metabolic proteins that are associated with HSC properties, highlighting the importance of metabolic regulation in the most immature stem cells.
In the final section, a label-free low-input proteomics workflow was developed, enabling a comprehensive proteomic map of various HSPC populations from the murine BM. This approach identified approximately 7,000 proteins from as few as 500 cells, resembling the number of proteins similar to studies with significantly more cells as input. The increased proteome coverage, compared to existing scp-MS capabilities, facilitated the identification of both well-known and novel transcriptional regulators along diverse differentiation trajectories in early hematopoiesis.
This study stands out as the first effort to generate a comprehensive proteomic map of HSPCs derived from the BM of a single mouse. This holds significance from two perspectives; firstly, it provides a high-quality resource dataset with relatively low technical variation, and secondly, it requires a minimal number of experimental mice for in-depth proteomic characterization of rare cell types. Our exploration of the most immature stem cells points towards a cell surface protein, ADP-ribosyltransferase ART4, as a potential marker that can distinguish long-term HSCs from multipotent progenitors. Both human scp-MS and mouse low-input proteomics datasets underscore the importance of metabolic regulation in earliest stem cells, highlighting the power of global proteomics in elucidating key metabolic processes underlying stem cell quiescence.
Collectively, this project advanced low-input proteomics methodologies as well as showcased their power in dissecting the complexities of human and murine hematopoietic hierarchies. The datasets generated here serve as a valuable resource for validating candidate proteins in functional studies.
While the primary focus was on the healthy hematopoietic system, the potential of low-input proteomics extends to perturbed hematopoiesis as well as diverse tissue types.
profiling underlies these studies, while it is well known that transcript levels do not always accurately predict protein abundance due to variations in translation and degradation rates. As proteins are the effector molecules of the cells and directly influence the functional and phenotypic characteristics, a fundamental understanding of cell state heterogeneity requires direct measurement of the proteome. The progress in global proteomics studies has been limited by the
lack of methods suitable for analyzing low-input samples, including single cells, however, with rapid recent advances, mass spectrometry-based proteomics has emerged as a powerful tool to tackle this problem. In this study, we utilized novel single-cell and low-input (500 cells) proteomics to explore the continuous landscape of HSPCs in both healthy human and murine bone marrow (BM) hematopoiesis. We demonstrate the power of low-input proteomics in three different yet interconnected projects.
Initially, we established a multiplexed single-cell proteomics (scp-MS) workflow that involves FACS isolation into 384-well plates and applied it to a disrupted hematopoietic hierarchy, including leukemic stem cells (LSC), progenitors, and blasts. This workflow identified approximately 1,000 proteins per cell, revealing two differentiation routes; the classic progression from LSCs to progenitors and blasts, and a previously unidentified route going directly from LSCs
to blasts in a primary leukemia cell culture system.
Subsequently, the multiplexed scp-MS workflow was applied to a highly complex HSPC hierarchy derived from the BM of healthy individuals. This study marked the first application of scp-MS in a complex cellular hierarchy derived from primary BM samples. The study highlighted limitations in commonly used FACS isolation schemes and identified proteome-level factors not evident at the transcriptome level based on publicly available RNA-seq data. Among others, we identified metabolic proteins that are associated with HSC properties, highlighting the importance of metabolic regulation in the most immature stem cells.
In the final section, a label-free low-input proteomics workflow was developed, enabling a comprehensive proteomic map of various HSPC populations from the murine BM. This approach identified approximately 7,000 proteins from as few as 500 cells, resembling the number of proteins similar to studies with significantly more cells as input. The increased proteome coverage, compared to existing scp-MS capabilities, facilitated the identification of both well-known and novel transcriptional regulators along diverse differentiation trajectories in early hematopoiesis.
This study stands out as the first effort to generate a comprehensive proteomic map of HSPCs derived from the BM of a single mouse. This holds significance from two perspectives; firstly, it provides a high-quality resource dataset with relatively low technical variation, and secondly, it requires a minimal number of experimental mice for in-depth proteomic characterization of rare cell types. Our exploration of the most immature stem cells points towards a cell surface protein, ADP-ribosyltransferase ART4, as a potential marker that can distinguish long-term HSCs from multipotent progenitors. Both human scp-MS and mouse low-input proteomics datasets underscore the importance of metabolic regulation in earliest stem cells, highlighting the power of global proteomics in elucidating key metabolic processes underlying stem cell quiescence.
Collectively, this project advanced low-input proteomics methodologies as well as showcased their power in dissecting the complexities of human and murine hematopoietic hierarchies. The datasets generated here serve as a valuable resource for validating candidate proteins in functional studies.
While the primary focus was on the healthy hematopoietic system, the potential of low-input proteomics extends to perturbed hematopoiesis as well as diverse tissue types.
| Original language | English |
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| Number of pages | 131 |
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| Publication status | Published - 5 Jan 2024 |