Chimeric Antigen Receptor Designed to Prevent Ubiquitination and Downregulation Showed Durable Antitumor Efficacy.

Clinical evidence suggests that poor persistence of chimeric antigen receptor-T cells (CAR-T) in patients limits therapeutic efficacy. Here, we designed a CAR with recyclable capability to promote in vivo persistence and to sustain antitumor activity. We showed that the engagement of tumor antigens induced rapid ubiquitination of CARs, causing CAR downmodulation followed by lysosomal degradation. Blocking CAR ubiquitination by mutating all lysines in the CAR cytoplasmic domain (CARKR) markedly repressed CAR downmodulation by inhibiting lysosomal degradation while enhancing recycling of internalized CARs back to the cell surface. Upon encountering tumor antigens, CARKR-T cells ameliorated the loss of surface CARs, which promoted their long-term killing capacity. Moreover, CARKR-T cells containing 4-1BB signaling domains displayed elevated endosomal 4-1BB signaling that enhanced oxidative phosphorylation and promoted memory T cell differentiation, leading to superior persistence in vivo. Collectively, our study provides a straightforward strategy to optimize CAR-T antitumor efficacy by redirecting CAR trafficking.

Soluble FLT1 binds lipid microdomains in podocytes to control cell morphology and glomerular barrier function.

Vascular endothelial growth factor and its receptors, FLK1/KDR and FLT1, are key regulators of angiogenesis. Unlike FLK1/KDR, the role of FLT1 has remained elusive. FLT1 is produced as soluble (sFLT1) and full-length isoforms. Here, we show that pericytes from multiple tissues produce sFLT1. To define the biologic role of sFLT1, we chose the glomerular microvasculature as a model system. Deletion of Flt1 from specialized glomerular pericytes, known as podocytes, causes reorganization of their cytoskeleton with massive proteinuria and kidney failure, characteristic features of nephrotic syndrome in humans. The kinase-deficient allele of Flt1 rescues this phenotype, demonstrating dispensability of the full-length isoform. Using cell imaging, proteomics, and lipidomics, we show that sFLT1 binds to the glycosphingolipid GM3 in lipid rafts on the surface of podocytes, promoting adhesion and rapid actin reorganization. sFLT1 also regulates pericyte function in vessels outside of the kidney. Our findings demonstrate an autocrine function for sFLT1 to control pericyte behavior.

DeKinomics pulse-chases kinase functions in living cells.

Cellular context is crucial for understanding the complex and dynamic kinase functions in health and disease. Systematic dissection of kinase-mediated cellular processes requires rapid and precise stimulation ('pulse') of a kinase of interest, as well as global and in-depth characterization ('chase') of the perturbed proteome under living conditions. Here we developed an optogenetic 'pulse-chase' strategy, termed decaging kinase coupled proteomics (DeKinomics), for proteome-wide profiling of kinase-driven phosphorylation at second-timescale in living cells. We took advantage of the 'gain-of-function' feature of DeKinomics to identify direct kinase substrates and further portrayed the global phosphorylation of understudied receptor tyrosine kinases under native cellular settings. DeKinomics offered a general activation-based strategy to study kinase functions with high specificity and temporal resolution under living conditions.

Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology1,2. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance3-7. Furthermore, PSC activation occurs very early during PDAC tumorigenesis8-10, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy.

Functional and Clinical Proteomic Exploration of Pancreatic Cancer.

Pancreatic cancer, in most cases being pancreatic ductal adenocarcinoma (PDAC), is one of the most lethal cancers with a median survival time of less than 6 months. Therapeutic options are very limited for patients with PDAC, and surgery is still the most effective treatment, making improvements in early diagnosis critical. One typical characteristic of PDAC is the desmoplastic reaction of its stroma microenvironment, which actively interacts with cancer cells to orchestrate key components in tumorigenesis, metastasis, and chemoresistance. A global exploration of cancer-stroma crosstalk is essential to decipher PDAC biology and design intervention strategies. Over the past decade, the dramatic improvement in proteomics technologies has enabled the profiling of proteins, post-translational modifications (PTMs), and their protein complexes at unprecedented sensitivity and dimensionality. Here, starting with our current understanding of PDAC characteristics, including precursor lesions, progression models, tumor microenvironment, and therapeutic advancements, we describe how proteomics contributes to the functional and clinical exploration of PDAC, providing insights into PDAC carcinogenesis, progression, and chemoresistance. We summarize recent achievements enabled by proteomics to systematically investigate PTMs-mediated intracellular signaling in PDAC, cancer-stroma interactions, and potential therapeutic targets revealed by these functional studies. We also highlight proteomic profiling of clinical tissue and plasma samples to discover and verify useful biomarkers that can aid early detection and molecular classification of patients. In addition, we introduce spatial proteomic technology and its applications in PDAC for deconvolving tumor heterogeneity. Finally, we discuss future prospects of applying new proteomic technologies in comprehensively understanding PDAC heterogeneity and intercellular signaling networks. Importantly, we expect advances in clinical functional proteomics for exploring mechanisms of cancer biology directly by high-sensitivity functional proteomic approaches starting from clinical samples.

Chemical Proteomic Approach for In-Depth Glycosylation Profiling of Plasma Carcinoembryonic Antigen in Cancer Patients.

Carcinoembryonic antigen (CEA) of human plasma is a biomarker of many cancer diseases, and its N-glycosylation accounts for 60% of molecular mass. It is highly desirable to characterize its glycoforms for providing additional dimension of features to increase its performance in prognosis and diagnosis of cancers. However, to systematically characterize its site-specific glycosylation is challenging because of its low abundance. Here, we developed a highly sensitive strategy for in-depth glycosylation profiling of plasma CEA through chemical proteomics combined with multienzymatic digestion. A trifunctional probe was utilized to generate covalent bond of plasma CEA and its antibody upon UV irradiation. As low as 1 ng/ml CEA in plasma could be captured and digested with trypsin and chymotrypsin for intact glycopeptide characterization. Twenty six of 28 potential N-glycosylation sites were well identified, which were the most comprehensive N-glycosylation site characterization of CEA on intact glycopeptide level as far as we known. Importantly, this strategy was applied to the glycosylation analysis of plasma CEA in cancer patients. Differential site-specific glycoforms of plasma CEA were observed in patients with colorectal cancers (CRCs) and lung cancer. The distributions of site-specific glycoforms were different as the progression of CRC, and most site-specific glycoforms were overexpressed in stage II of CRC. Overall, we established a highly sensitive chemical proteomic method to profile site-specific glycosylation of plasma CEA, which should generally applicable to other well-established cancer glycoprotein biomarkers for improving their cancer diagnosis and monitoring performance.

N-linked glycoproteins and host proteases are involved in swine acute diarrhea syndrome coronavirus entry.

IMPORTANCE: Gaining insight into the cell-entry mechanisms of swine acute diarrhea syndrome coronavirus (SADS-CoV) is critical for investigating potential cross-species infections. Here, we demonstrated that pretreatment of host cells with tunicamycin decreased SADS-CoV attachment efficiency, indicating that N-linked glycosylation of host cells was involved in SADS-CoV entry. Common N-linked sugars Neu5Gc and Neu5Ac did not interact with the SADS-CoV S1 protein, suggesting that these molecules were not involved in SADS-CoV entry. Additionally, various host proteases participated in SADS-CoV entry into diverse cells with different efficiencies. Our findings suggested that SADS-CoV may exploit multiple pathways to enter cells, providing insights into intervention strategies targeting the cell entry of this virus.

Plasma proteomic characterization of colorectal cancer patients with FOLFOX chemotherapy by integrated proteomics technology.

BACKGROUND: Colorectal Cancer (CRC) is a prevalent form of cancer, and the effectiveness of the main postoperative chemotherapy treatment, FOLFOX, varies among patients. In this study, we aimed to identify potential biomarkers for predicting the prognosis of CRC patients treated with FOLFOX through plasma proteomic characterization. METHODS: Using a fully integrated sample preparation technology SISPROT-based proteomics workflow, we achieved deep proteome coverage and trained a machine learning model from a discovery cohort of 90 CRC patients to differentiate FOLFOX-sensitive and FOLFOX-resistant patients. The model was then validated by targeted proteomics on an independent test cohort of 26 patients. RESULTS: We achieved deep proteome coverage of 831 protein groups in total and 536 protein groups in average for non-depleted plasma from CRC patients by using a Orbitrap Exploris 240 with moderate sensitivity. Our results revealed distinct molecular changes in FOLFOX-sensitive and FOLFOX-resistant patients. We confidently identified known prognostic biomarkers for colorectal cancer, such as S100A4, LGALS1, and FABP5. The classifier based on the biomarker panel demonstrated a promised AUC value of 0.908 with 93% accuracy. Additionally, we established a protein panel to predict FOLFOX effectiveness, and several proteins within the panel were validated using targeted proteomic methods. CONCLUSIONS: Our study sheds light on the pathways affected in CRC patients treated with FOLFOX chemotherapy and identifies potential biomarkers that could be valuable for prognosis prediction. Our findings showed the potential of mass spectrometry-based proteomics and machine learning as an unbiased and systematic approach for discovering biomarkers in CRC.

Pixel-Level Classification of Five Histologic Patterns of Lung Adenocarcinoma.

Lung adenocarcinoma is the most common histologic type of lung cancer. The pixel-level labeling of histologic patterns of lung adenocarcinoma can assist pathologists in determining tumor grading with more details than normal classification. We manually annotated a dataset containing a total of 1000 patches (200 patches for each pattern) of 512 × 512 pixels and 420 patches (contains test sets) of 1024 × 1024 pixels according to the morphological features of the five histologic patterns of lung adenocarcinoma (lepidic, acinar, papillary, micropapillary, and solid). To generate an even large amount of data patches, we developed a data stitching strategy as a data augmentation for classification in model training. Stitched patches improve the Dice similarity coefficient (DSC) scores by 24.06% on the whole-slide image (WSI) with the solid pattern. We propose a WSI analysis framework for lung adenocarcinoma pathology, intelligently labeling lung adenocarcinoma histologic patterns at the pixel level. Our framework contains five branches of deep neural networks for segmenting each histologic pattern. We test our framework with 200 unclassified patches. The DSC scores of our results outpace comparing networks (U-Net, LinkNet, and FPN) by up to 10.78%. We also perform results on four WSIs with an overall accuracy of 99.6%, demonstrating that our network framework exhibits better accuracy and robustness in most cases.

Scaled-Down Thermal Profiling and Coaggregation Analysis of the Proteome for Drug Target and Protein Interaction Analysis.

Thermal proteome profiling (TPP), an experimental technique combining the cellular thermal shift assay (CETSA) with quantitative protein mass spectrometry (MS), identifies interactions of drugs and chemicals with endogenous proteins. Thermal proximity coaggregation (TPCA) profiling extended TPP to study the intracellular dynamics of protein complexes. In TPP and TPCA, samples are subjected to multiple denaturing temperatures, each requiring over 100 µg of proteins, which restricts their applications for rare cells and precious clinical samples. We developed a workflow termed STASIS (scaled-down thermal profiling and coaggregation analysis with SISPROT) that scales down the required protein to as low as 1 µg per temperature. This is achieved by heating and centrifugation using the same PCR tube, processing samples with the SISPROT technology (simple and integrated spintip-based proteomics technology), and tip-based manual fractionation of TMT-labeled peptides. We evaluate the STASIS workflow with starting protein quantities of 10, 5, and 1 µg per temperature prior to heating, identifying between 4000 and 5000 proteins with 6 h of acquisition time. Importantly, we observed a high correlation in the Tm of proteins with minimal difference in TPCA performance for predicting protein complexes. Moreover, STASIS could identify the targets of methotrexate and panobinostat with high precision with 1 µg of proteins per temperature. In conclusion, STASIS is a robust cost-effective technique for target deconvolution and extended TPCA to rare primary cells and precious clinical samples for the analysis of protein complexes.

Comprehensive Evaluation and Optimization of the Data-Dependent LC-MS/MS Workflow for Deep Proteome Profiling.

Data-dependent liquid chromatography-tandem mass spectrometry (LC-MS/MS) is widely used in proteomic analyses. A well-performed LC-MS/MS workflow, which involves multiple procedures and interdependent metrics, is a prerequisite for deep proteome profiling. Researchers have previously evaluated LC-MS/MS performance mainly based on the number of identified peptides and proteins. However, this is not a comprehensive approach. This motivates us to develop MSRefine, which aims to evaluate and optimize the performance of the LC-MS/MS workflow for data-dependent acquisition (DDA) proteomics. It extracts 47 kinds of metrics, scores the metrics, and reports visual results, assisting users in evaluating the workflow, locating problems, and providing optimizing strategies. In this study, we compared and analyzed multiple pairs of datasets spanning different samples, methods, and instruments and demonstrated that the comprehensive visual metrics and scores in MSRefine enable us to evaluate the performance of the various experiments and provide optimal strategies for the identification of more peptides and proteins.

Combination of automated sample preparation and micro-flow LC-MS for high-throughput plasma proteomics.

BACKGROUND: Non-invasive detection of blood-based markers is a critical clinical need. Plasma has become the main sample type for clinical proteomics research because it is easy to obtain and contains measurable protein biomarkers that can reveal disease-related physiological and pathological changes. Many efforts have been made to improve the depth of its identification, while there is an increasing need to improve the throughput and reproducibility of plasma proteomics analysis in order to adapt to the clinical large-scale sample analysis. METHODS: We have developed and optimized a robust plasma analysis workflow that combines an automated sample preparation platform with a micro-flow LC-MS-based detection method. The stability and reproducibility of the workflow were systematically evaluated and the workflow was applied to a proof-of-concept plasma proteome study of 30 colon cancer patients from three age groups. RESULTS: This workflow can analyze dozens of samples simultaneously with high reproducibility. Without protein depletion and prefractionation, more than 300 protein groups can be identified in a single analysis with micro-flow LC-MS system on a Orbitrap Exploris 240 mass spectrometer, including quantification of 35 FDA approved disease markers. The quantitative precision of the entire workflow was acceptable with median CV of 9%. The preliminary proteomic analysis of colon cancer plasma from different age groups could be well separated with identification of potential colon cancer-related biomarkers. CONCLUSIONS: This workflow is suitable for the analysis of large-scale clinical plasma samples with its simple and time-saving operation, and the results demonstrate the feasibility of discovering significantly changed plasma proteins and distinguishing different patient groups.

Dynamic crotonylation of EB1 by TIP60 ensures accurate spindle positioning in mitosis.

Spindle position control is essential for cell fate determination and organogenesis. Early studies indicate the essential role of the evolutionarily conserved Gαi/LGN/NuMA network in spindle positioning. However, the regulatory mechanisms that couple astral microtubules dynamics to the spindle orientation remain elusive. Here we delineated a new mitosis-specific crotonylation-regulated astral microtubule-EB1-NuMA interaction in mitosis. EB1 is a substrate of TIP60, and TIP60-dependent crotonylation of EB1 tunes accurate spindle positioning in mitosis. Mechanistically, TIP60 crotonylation of EB1 at Lys66 forms a dynamic link between accurate attachment of astral microtubules to the lateral cell cortex defined by NuMA-LGN and fine tune of spindle positioning. Real-time imaging of chromosome movements in HeLa cells expressing genetically encoded crotonylated EB1 revealed the importance of crotonylation dynamics for accurate control of spindle orientation during metaphase-anaphase transition. These findings delineate a general signaling cascade that integrates protein crotonylation with accurate spindle positioning for chromosome stability in mitosis.

Dynamic Phosphotyrosine-Dependent Signaling Profiling in Living Cells by Two-Dimensional Proximity Proteomics.

Tyrosine phosphorylation (pTyr)-dependent signaling pathways play a vital role in various biological processes, which are spatiotemporally assembled and dynamically regulated on a minute scale by pTyr in living cells. Studying these pTyr-mediated signaling complexes is therefore challenging due to the highly dynamic nature of the protein complexes and the low abundance of pTyr. In this study, we adopted minute-resolution APEX2-based proximity labeling (PL) in living cells and Src SH2 superbinder-based pTyr peptide enrichment for simultaneously profiling these protein complexes and associated pTyr sites from the same affinity-purified sample. Upon different time courses of EGF stimulation of the living cells stably expressing APEX2-FLAG-GRB2, we constructed two-dimensional time-course curves for both interactome and tyrosine phosphoproteome. Well-annotated pTyr signaling complexes in EGFR signaling and located at the endosome were quantified with tightly correlated time-course curves for both interacting proteins and pTyr sites. Importantly, the correlated time-course curves for EGFR and endosomal HGS were well validated by targeted-parallel reaction monitoring (PRM)-MS analysis. Taking advantage of the high sensitivity of the PRM assay, the low-abundant pTyr peptide EGFR pY1110, which cannot be identified in the data-dependent acquisition (DDA) analysis, could be well quantified. Collectively, this two-dimensional proximity proteomic strategy is promising for comprehensively characterizing pTyr-mediated protein complexes with high sensitivity in living cells.

Robust Capillary- and Micro-Flow Liquid Chromatography-Tandem Mass Spectrometry Methods for High-Throughput Proteome Profiling.

Capillary- and micro-flow liquid chromatography-tandem mass spectrometry (capLC-MS/MS and µLC-MS/MS) is becoming a valuable alternative to nano-flow LC-MS/MS due to its high robustness and throughput. The systematic comparison of capLC-MS/MS and µLC-MS/MS systems for global proteome profiling has not been reported yet. Here, the capLC-MS/MS (150 µm i.d. column, 1 µL/min) and µLC-MS/MS (1 mm i.d. column, 50 µL/min) systems were both established based on UltiMate 3000 RSLCnano coupled to an Orbitrap Exploris 240 by integrating with different flowmeters. We evaluated both systems in terms of sensitivity, analysis throughput, separation efficiency, and robustness. capLC-MS/MS was about 10 times more sensitive than µLC-MS/MS at different gradient lengths. Compared with capLC-MS/MS, µLC-MS/MS was able to achieve higher analysis throughput and separation efficiency. During the 7 days' long-term performance test, both systems showed good reproducibility of chromatographic full width (RSD < 3%), retention time (RSD < 0.4%), and protein identification (RSD < 3%). These results demonstrate that capLC-MS/MS is more suitable for high-throughput analysis of clinical samples with a limited starting material. When enough samples are available, µLC-MS/MS is preferred. Together, capLC and µLC coupled to Orbitrap Exploris 240 with moderate sensitivity should well meet the needs of large-cohort clinical proteomic analysis.

Integrated and High-Throughput Approach for Sensitive Analysis of Tyrosine Phosphoproteome.

Tyrosine phosphorylation (pTyr) regulates various signaling pathways under normal and cancerous states. Due to their low abundance and transient and dynamic natures, systematic profiling of pTyr sites is challenging. Antibody and engineered binding domain-based approaches have been well applied to pTyr peptide enrichment. However, traditional methods have the disadvantage of a long sample preparation process, which makes them unsuitable for processing limited amount of samples, especially in a high-throughput manner. In this study we developed a 96-well microplate-based approach to integrate all the sample preparation steps starting from cell culture to MS-compatible pTyr peptide enrichment in three consecutive 96-well microplates. By assembling an engineered SH2 domain onto a microplate, nonspecific adsorption of phosphopeptides is greatly reduced, which allows us to remove the Ti-IMAC purification and three C18 desalting steps (after digestion, pTyr enrichment, and Ti-IMAC purification) and, therefore, greatly simplifies the entire pTyr peptide enrichment workflow, especially when processing a large number of samples. Starting with 96-well microplate-cultured, pervanadate-stimulated cells, our approach could enrich 21% more pTyr sites than the traditional serial pTyr enrichment approach and showed good sensitivity and reproducibility in the range of 200 ng to 200 µg peptides. Importantly, we applied this approach to profile tyrosine kinase inhibitor-mediated EGFR signaling pathway and could well differentiate the distinct response of different pTyr sites. Collectively, the integrated 96-well microplate-based approach is valuable for profiling pTyr sites from limited biological samples and in a high-throughput manner.

Generic Plug-and-Play Strategy for High-Throughput Analysis of PTM-Mediated Protein Complexes.

Protein complexes mediated by various post-translational modifications (PTMs) play important roles in almost every aspect of biological processes. PTM-mediated protein complexes often have weak and transient binding properties, which limit their unbiased profiling especially in complex biological samples. Here, we developed a plug-and-play chemical proteomic approach for high-throughput analyis of PTM-mediated protein complexes. Taking advantage of the glutathione-S-transferase (GST) tag, which is the gold standard for protein purification and has wide access to a variety of proteins of interest (POIs), a glutathione (GSH) group- and photo-cross-linking group-containing trifunctional chemical probe was developed to tag POIs and assembled onto a streptavidin-coated 96-well plate for affinity purification, photo-cross-linking, and proteomics sample preparation in a fully integrated manner. Compared with the previously developed photo-pTyr-scaffold strategy, by assembling the tyrosine phosphorylation (pTyr) binding domain through covalent NHS chemistry, the new plug-and-play strategy using a noncovalent GST-GSH interaction has comparable enrichment efficiency for EGF stimulation-dependent pTyr protein complexes. To further prove its feasibility, we additionally assembled four pTyr-binding domains in the 96-well plate and selectively identified their pTyr-dependent interacting proteins. Importantly, we systematically optimized and applied the plug-and-play approach for exploring protein methylation-mediated protein complexes, which are difficult to be characterized due to their weak binding affinity and the lack of efficient enrichment strategies. We explored a comprehensive protein methylation-mediated interaction network assembled by five protein methylation binding domains including the chromo domain of MPP8, tandem tudor domain of KDM4A, full-length CBX1, PHD domain of RAG2, and tandem tudor domain of TP53BP1 and validated the chromo domain- and tudor domain-mediated interaction with histone H3. Collectively, this plug-and-play approach provides a convenient and generic strategy for exploring PTM-dependent protein complexes for any POIs with the GST tag.

A fully integrated sample preparation strategy for highly sensitive intact glycoproteomics.

A fully integrated sample preparation technology, termed Intact GlycoSISPROT, was developed for the highly sensitive analysis of site-specific glycopeptides. Through integrating all glycoproteomic sample preparation steps into a single spintip, Intact GlycoSISPROT provided a tool for site-specific glycosylation analysis with low micrograms to even nanograms of protein sample.

Fully integrated on-line strategy for highly sensitive proteome profiling of 10-500 mammalian cells.

Recent development in proteomic sample preparation using nanofluidic devices has made single-cell proteome profiling possible. However, these nanofluidic devices require special expertise and costly nanopipetting instruments. They are also specially designed for single cells, are not well-suited for profiling rare samples consisting of a few hundred mammalian cells, arguably a more common need that remains a great challenge. Herein, we developed an easy-to-use and scalable device for processing low-input samples, which combined the merits of previously reported rare cell proteomic reactor (RCPR) and mixed-mode simple and integrated spintip-based proteomics technology, as an alternative to nanofluidic devices. All steps of proteomics sample preparation, including protein preconcentration, impurity removal, reduction, alkylation, digestion, and desalting, were fully integrated in our workflow, and the device can be directly connected to online nanoLC-MS system after processing the rare samples. Using the developed 3-frit mixed-mode RCPR, we identified on average 946 ± 158, 2 998 ± 106, and 3 934 ± 85 protein groups in data-dependent acquisition (DDA) mode from 10, 100, and 500 fluorescence-activated cell sorting (FACS)-sorted 293T cells, respectively. As an illustrative application of this technology, we performed a label-free proteome comparison of 500 FACS-sorted mouse cochlear hair cells of two different ages. On average, 2 595 ± 230 and 2 042 ± 120 protein groups were quantified in the juvenile and the adult samples in DDA mode, respectively, achieving dynamic ranges of over 6 orders of magnitude for both.