Dilute to Enrich for Deeper Proteomics: A Yolk-Depleted Carrier for Limited Populations of Embryonic (Frog) Cells.

Abundant proteins challenge deep mass spectrometry (MS) analysis of the proteome. Yolk, the source of food in many developing vertebrate embryos, complicates chemical separation and interferes with detection. We report here a strategy that enhances bottom-up proteomics in yolk-laden specimens by diluting the interferences using a yolk-depleted carrier (YODEC) proteome via isobaric multiplexing quantification. This method was tested on embryos of the South African Clawed Frog (Xenopus laevis), where a >90% yolk proteome content challenges deep proteomics. As a proof of concept, we isolated neural and epidermal fated cell clones from the embryo by dissection or fluorescence-activated cell sorting. Compared with the standard multiplexing carrier approach, YODEC more than doubled the detectable X. laevis proteome, identifying 5,218 proteins from D11 cell clones dissected from the embryo. Ca. ∼80% of the proteins were quantified without dropouts in any of the analytical channels. YODEC with high-pH fractionation quantified 3,133 proteins from ∼8,000 V11 cells that were sorted from ca. 2 embryos (1.5 µg total, or 150 ng yolk-free proteome), marking a 15-fold improvement in proteome coverage vs the standard proteomics approach. About 60% of these proteins were only quantifiable by YODEC, including molecular adaptors, transporters, translation, and transcription factors. While this study was tailored to limited populations of Xenopus cells, we anticipate the approach of "dilute to enrich" using a depleted carrier proteome to be adaptable to other biological models in which abundant proteins challenge deep MS proteomics.

A Sensitive and Controlled Data-Independent Acquisition Method for Proteomic Analysis of Cell Therapies.

Mass spectrometry (MS)-based proteomic measurements are uniquely poised to impact the development of cell and gene therapies. With the adoption of rigorous instrumental performance qualifications (PQs), large-scale proteomics can move from a research to a manufacturing control tool. Especially suited, data-independent acquisition (DIA) approaches have distinctive qualities to extend multiattribute method (MAM) principles to characterize the proteome of cell therapies. Here, we describe the development of a DIA method for the sensitive identification and quantification of proteins on a Q-TOF instrument. Using the improved acquisition parameters, we defined a control strategy and highlighted some metrics to improve the reproducibility of SWATH acquisition-based proteomic measurements. Finally, we applied the method to analyze the proteome of Jurkat cells that here serves as a model for human T-cells. Raw and processed data were deposited in PRIDE (PXD029780).

In Vivo Subcellular Mass Spectrometry Enables Proteo-Metabolomic Single-Cell Systems Biology in a Chordate Embryo Developing to a Normally Behaving Tadpole (X. laevis)*.

We report the development of in vivo subcellular high-resolution mass spectrometry (HRMS) for proteo-metabolomic molecular systems biology in complex tissues. With light microscopy, we identified the left-dorsal and left-ventral animal cells in cleavage-stage non-sentient Xenopus laevis embryos. Using precision-translated fabricated microcapillaries, the subcellular content of each cell was double-probed, each time swiftly (<5 s/event) aspirating <5 % of cell volume (≈10 nL). The proteins and metabolites were analyzed by home-built ultrasensitive capillary electrophoresis electrospray ionization employing orbitrap or time-of-flight HRMS. Label-free detection of ≈150 metabolites (57 identified) and 738 proteins found proteo-metabolomic networks with differential quantitative activities between the cell types. With spatially and temporally scalable sampling, the technology preserved the integrity of the analyzed cells, the neighboring cells, and the embryo. 95 % of the analyzed embryos developed into sentient tadpoles that were indistinguishable from their wild-type siblings based on anatomy and visual function in a background color preference assay.

Mass Spectrometry Advances and Perspectives for the Characterization of Emerging Adoptive Cell Therapies.

Adoptive cell therapy is an emerging anti-cancer modality, whereby the patient's own immune cells are engineered to express T-cell receptor (TCR) or chimeric antigen receptor (CAR). CAR-T cell therapies have advanced the furthest, with recent approvals of two treatments by the Food and Drug Administration of Kymriah (trisagenlecleucel) and Yescarta (axicabtagene ciloleucel). Recent developments in proteomic analysis by mass spectrometry (MS) make this technology uniquely suited to enable the comprehensive identification and quantification of the relevant biochemical architecture of CAR-T cell therapies and fulfill current unmet needs for CAR-T product knowledge. These advances include improved sample preparation methods, enhanced separation technologies, and extension of MS-based proteomic to single cells. Innovative technologies such as proteomic analysis of raw material quality attributes (MQA) and final product quality attributes (PQA) may provide insights that could ultimately fuel development strategies and lead to broad implementation.

Single-cell proteomics in complex tissues using microprobe capillary electrophoresis mass spectrometry.

Direct measurement of proteins produced by single cells promises to expand our understanding of molecular cell-to-cell differences (heterogeneity) and their contribution to normal and impaired development. High-resolution mass spectrometry (HRMS) is the modern technology of choice for the label-free identification and quantification of proteins, albeit usually in large populations of cells. Recent advances in microscale sample collection and processing, separation, and ionization have extended this powerful technology to single cells. This chapter describes a protocol based on microprobe capillary electrophoresis (CE) HRMS to enable the direct proteomic profiling of single cells embedded in complex tissues without the requirement for dissociation or whole-cell dissection. We here demonstrate the technology for identified individual cells in early developing embryos of Xenopus laevis and zebrafish as well as electrophysiologically identified single neurons in physiologically active brain slices from the mouse substantia nigra. Instructions are provided step-by-step to identify single cells using physiological or morphological cues, collect the content of the cells using microfabricated capillaries, and perform bottom-up proteomics using a custom-built CE electrospray ionization (ESI) mass spectrometer equipped with a quadrupole time-of-flight or orbitrap mass analyzer. Results obtained by this approach have revealed previously unknown differences between the proteomic state of embryonic cells and neurons. The data from single-cell proteomics by microprobe CE-ESI-HRMS complements those from single-cell transcriptomics, thereby opening exciting potentials to deepen our knowledge of molecular mechanisms governing cell and developmental processes.

A microanalytical capillary electrophoresis mass spectrometry assay for quantifying angiotensin peptides in the brain.

The renin-angiotensin system (RAS) of the brain produces a series of biologically active angiotensinogen-derived peptides involved in physiological homeostasis and pathophysiology of disease. Despite significant research efforts to date, a comprehensive understanding of brain RAS physiology is lacking. A significant challenge has been the limited set of bioanalytical assays capable of detecting angiotensin (Ang) peptides at physiologically low concentrations (2-15 fmol/g of wet tissue) and sufficient chemical specificity for unambiguous molecular identifications. Additionally, a complex brain anatomy calls for microanalysis of specific tissue regions, thus further taxing sensitivity requirements for identification and quantification in studies of the RAS. To fill this technology gap, we here developed a microanalytical assay by coupling a laboratory-built capillary electrophoresis (CE) nano-electrospray ionization (nano-ESI) platform to a high-resolution mass spectrometer (HRMS). Using parallel reaction monitoring, we demonstrated that this technology achieved confident identification and quantification of the Ang peptides at approx. 5 amol to 300 zmol sensitivity. This microanalytical assay revealed differential Ang peptide profiles between tissues that were micro-sampled from the subfornical organ and the paraventricular nucleus of the hypothalamus, important brain regions involved in thirst and water homeostasis and neuroendocrine regulation to stress. Microanalytical CE-nano-ESI-HRMS extends the analytical toolbox of neuroscience to help better understand the RAS.

Microsampling Capillary Electrophoresis Mass Spectrometry Enables Single-Cell Proteomics in Complex Tissues: Developing Cell Clones in Live Xenopus laevis and Zebrafish Embryos.

Label-free single-cell proteomics by mass spectrometry (MS) is currently incompatible with complex tissues without requiring cell culturing, single-cell dissection, or tissue dissociation. We here report the first example of label-free single-cell MS-based proteomics directly in single cells in live vertebrate embryos. Our approach integrates optically guided in situ subcellular capillary microsampling, one-pot extraction-digestion of the collected proteins, peptide separation by capillary electrophoresis, ionization by an ultrasensitive electrokinetically pumped nanoelectrospray, and detection by high-resolution MS (Orbitrap). With a 700 zmol (420 000 copies) lower limit of detection, this trace-sensitive technology confidently identified and quantified ∼750-800 protein groups (<1% false-discovery rate) by analyzing just ∼5 ng of protein digest, viz. <0.05% of the total protein content from individual cells in a 16-cell Xenopus laevis (frog) embryo. After validating the approach by recovering animal-vegetal-pole proteomic asymmetry in the frog zygote, the technology was applied to uncover proteomic reorganization as the animal-dorsal (D11) cell of the 16-cell embryo gave rise to its neural-tissue-fated clone in the embryo developing to the 32-, 64-, and 128-cell stages. In addition to enabling proteomics on smaller cells in X. laevis, we also demonstrated this technology to be scalable to single cells in live zebrafish embryos. Microsampling single-cell MS-based proteomics raises exciting opportunities to study cell and developmental processes directly in complex tissues and whole organisms at the level of the building block of life: the cell.

Proteomic Characterization of the Neural Ectoderm Fated Cell Clones in the Xenopus laevis Embryo by High-Resolution Mass Spectrometry.

The molecular program by which embryonic ectoderm is induced to form neural tissue is essential to understanding normal and impaired development of the central nervous system. Xenopus has been a powerful vertebrate model in which to elucidate this process. However, abundant vitellogenin (yolk) proteins in cells of the early Xenopus embryo interfere with protein detection by high-resolution mass spectrometry (HRMS), the technology of choice for identifying these gene products. Here, we systematically evaluated strategies of bottom-up proteomics to enhance proteomic detection from the neural ectoderm (NE) of X. laevis using nanoflow high-performance liquid chromatography (nanoLC) HRMS. From whole embryos, high-pH fractionation prior to nanoLC-HRMS yielded 1319 protein groups vs 762 proteins without fractionation (control). Compared to 702 proteins from dorsal halves of embryos (control), 1881 proteins were identified after yolk platelets were depleted via sucrose-gradient centrifugation. We combined these approaches to characterize protein expression in the NE of the early embryo. To guide microdissection of the NE tissues from the gastrula (stage 10), their precursor (midline dorsal-animal, or D111) cells were fate-mapped from the 32-cell embryo using a fluorescent lineage tracer. HRMS of the cell clones identified 2363 proteins, including 147 phosphoproteins (without phosphoprotein enrichment), transcription factors, and members from pathways of cellular signaling. In reference to transcriptomic maps of the developing X. laevis, 76 proteins involved in signaling pathways were gene matched to transcripts with known enrichment in the neural plate. Besides a protocol, this work provides qualitative proteomic data on the early developing NE.

Enhanced Peptide Detection Toward Single-Neuron Proteomics by Reversed-Phase Fractionation Capillary Electrophoresis Mass Spectrometry.

The ability to detect peptides and proteins in single cells is vital for understanding cell heterogeneity in the nervous system. Capillary electrophoresis (CE) nanoelectrospray ionization (nanoESI) provides high-resolution mass spectrometry (HRMS) with trace-level sensitivity, but compressed separation during CE challenges protein identification by tandem HRMS with limited MS/MS duty cycle. Here, we supplemented ultrasensitive CE-nanoESI-HRMS with reversed-phase (RP) fractionation to enhance identifications from protein digest amounts that approximate to a few mammalian neurons. An ~1 to 20 µg neuronal protein digest was fractionated on a RP column (ZipTip), and 1 ng to 500 pg of peptides were analyzed by a custom-built CE-HRMS system. Compared with the control (no fractionation), RP fractionation improved CE separation (theoretical plates ~274,000 versus 412,000 maximum, resp.), which enhanced detection sensitivity (2.5-fold higher signal-to-noise ratio), minimized co-isolation spectral interferences during MS/MS, and increased the temporal rate of peptide identification by up to ~57%. From 1 ng of protein digest (<5 neurons), CE with RP fractionation identified 737 protein groups (1,753 peptides), or ~480 protein groups (~1,650 peptides) on average per analysis. The approach was scalable to 500 pg of protein digest (~a single neuron), identifying 225 protein groups (623 peptides) in technical triplicates, or 141 protein groups on average per analysis. Among identified proteins, 101 proteins were products of genes that are known to be transcriptionally active in single neurons during early development of the brain, including those involved in synaptic transmission and plasticity and cytoskeletal organization. Graphical abstract ᅟ.

New-generation mass spectrometry expands the toolbox of cell and developmental biology.

Systems cell biology understanding of development requires characterization of all the molecules produced in the biological system. Decades of research and new-generation sequencing provided functional information on key genes and transcripts. However, there is less information available on how differential gene expression translates into the domains of functionally important proteins, peptides, and metabolites, and how changes in these molecules impact development. Mass spectrometry (MS) is the current technology of choice for the detection and quantification of large numbers of proteins and metabolites, because it requires no use of antibodies, functional probes, or a priori knowledge of molecules produced in the system. This review focuses on recent technologies that have improved MS sensitivity for proteins and metabolites and enabled new functionalities to assess their temporal and spatial changes during vertebrate embryonic development. This review highlights case studies, in which new-generation MS tools have enabled the study of hundreds-to-thousands of proteins and metabolites in tissues, cell populations, and single cells in model systems of vertebrate development, particularly the frog (Xenopus), zebrafish, and mouse. New-generation MS expands the toolbox of cell and developmental studies, raising exciting potentials to advance basic and translational research in the life sciences.

High-Sensitivity Mass Spectrometry for Probing Gene Translation in Single Embryonic Cells in the Early Frog (Xenopus) Embryo.

Direct measurement of protein expression with single-cell resolution promises to deepen the understanding of the basic molecular processes during normal and impaired development. High-resolution mass spectrometry provides detailed coverage of the proteomic composition of large numbers of cells. Here we discuss recent mass spectrometry developments based on single-cell capillary electrophoresis that extend discovery proteomics to sufficient sensitivity to enable the measurement of proteins in single cells. The single-cell mass spectrometry system is used to detect a large number of proteins in single embryonic cells in the 16-cell embryo of the South African clawed frog (Xenopus laevis) that give rise to distinct tissue types. Single-cell measurements of protein expression provide complementary information on gene transcription during early development of the vertebrate embryo, raising a potential to understand how differential gene expression coordinates normal cell heterogeneity during development.

Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS).

Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or <0.2% of the total protein contained in a blastomere in the 16-cell embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level.

Single-Cell Mass Spectrometry for Discovery Proteomics: Quantifying Translational Cell Heterogeneity in the 16-Cell Frog (Xenopus) Embryo.

We advance mass spectrometry from a cell population-averaging tool to one capable of quantifying the expression of diverse proteins in single embryonic cells. Our instrument combines capillary electrophoresis (CE), electrospray ionization, and a tribrid ultrahigh-resolution mass spectrometer (HRMS) to enable untargeted (discovery) proteomics with ca. 25 amol lower limit of detection. CE-µESI-HRMS enabled the identification of 500-800 nonredundant protein groups by measuring 20 ng, or <0.2% of the total protein content in single blastomeres that were isolated from the 16-cell frog (Xenopus laevis) embryo, amounting to a total of 1709 protein groups identified between n=3 biological replicates. By quantifying ≈150 nonredundant protein groups between all blastomeres and replicate measurements, we found significant translational cell heterogeneity along multiple axes of the embryo at this very early stage of development when the transcriptional program of the embryo has yet to begin.