Understanding the biochemistry of the cell requires measurement of all the molecules it produces. Single-cell proteomics recently became possible through advances in microanalytical sample preparation, separation by nano-flow liquid chromatography (nanoLC) and capillary electrophoresis (CE), and detection using electrospray ionization (ESI) high-resolution mass spectrometry (HRMS). Here, we demonstrate capillary microsampling CE-ESI-HRMS to be scalable to proteomics across broad cellular dimensions. This study established proof-of-principle using giant, ∼250-µm-diameter cells from embryos of the frog Xenopuslaevis and small, ∼35-µm-diameter neurons in culture from the mouse hippocampus. From ∼18 ng, or ∼0.2% of the total cellular proteome, subcellular analysis of the ventral-animal midline (V11) and equatorial (V12) cells identified 1,133 different proteins in a 16-cell embryo. CE-HRMS achieved ∼20-times higher sensitivity and doubled the speed of instrumental measurements compared to nanoLC, the closest neighboring single-cell technology of choice. Microanalysis was scalable to 722 proteins groups from ∼5 ng of cellular protein digest from identified left dorsal-animal midline cell (D11), supporting sensitivity for smaller cells. Capillary microsampling enabled the isolation and transfer of individual neurons from the culture, identifying 37 proteins between three different cells. A total of 224 proteins were detected from 500 pg of neuronal protein digest, which estimates to a single neuron. Serial dilution returned 157 proteins from sample amounts estimating to about half a cell (250 pg protein) and 70 proteins from ca. a quarter of a neuron (125 pg protein), suggesting sufficient sensitivity for subcellular proteomics. CE-ESI-HRMS complements nanoLC proteomics with scalability, sensitivity, and speed across broad cellular dimensions.

Biological processes unfold across broad spatial and temporal dimensions, and measurement of the underlying molecular world is essential to their understanding. Interdisciplinary efforts advanced mass spectrometry (MS) into a tour de force for assessing virtually all levels of the molecular architecture, some in exquisite detection sensitivity and scalability in space‐time. In this review, we offer vignettes of milestones in technology innovations that ushered sample collection and processing, chemical separation, ionization, and 'omics analyses to progressively finer resolutions in the realms of tissue biopsies and limited cell populations, single cells, and subcellular organelles. Also highlighted are methodologies that empowered the acquisition and analysis of multidimensional MS data sets to reveal proteomes, peptidomes, and metabolomes in ever‐deepening coverage in these limited and dynamic specimens. In pursuit of richer knowledge of biological processes, we discuss efforts pioneering the integration of orthogonal approaches from molecular and functional studies, both within and beyond MS. With established and emerging community‐wide efforts ensuring scientific rigor and reproducibility, spatiotemporal MS emerged as an exciting and powerful resource to study biological systems in space‐time.

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