Biology subjects

Nakajima, D.

Publications and source records attributed to Nakajima, D..

8 recordsLinked to original sources

bioRxiv2026

A High-Sensitivity and High-Throughput LC-MS Workflow for Low-Input and Single-Cell Proteomics

Single-cell proteomics (SCP) requires sample preparation that minimizes sample loss and highly sensitive LC-MS analysis because the amount of protein available from a single cell is extremely limited. Here, we developed an integrated workflow for low-input and single-cell proteomics based on single-cell proteomics by LMNG-assisted sample preparation (scpLASP), which enables microliter-scale sample preparation using standard laboratory consumables. Systematic optimization of peptide reconstitution conditions identified 0.01% n-dodecyl-{beta}-D-maltoside (DDM) combined with 0.01% lauryl maltose neopentyl glycol (LMNG) as the condition yielding the highest number of identified peptides among those tested for low-input samples. Optimization of the LC mobile-phase composition identified 0.5% acetic acid with 1% dimethyl sulfoxide (DMSO) as the optimal condition, increasing the mean number of identified protein groups from 6,767 with 0.1% formic acid to 7,409 for 250 pg HeLa digests analyzed using an Orbitrap Astral Zoom. Shortening the LC gradient from 11 to 5.5 min enabled higher analytical throughput while maintaining mean identification counts of 6,558 and 7,088 protein groups from 100 and 250 pg HeLa digests, respectively, with median protein-level coefficients of variation of 9.20% and 6.99%. When the optimized workflow was applied to HEK293-derived Expi293F single cells using the same 5.5-min LC gradient, an effective analytical throughput of 135 samples per day was achieved with an injection volume of 4.5 {micro}L, accounting for sample-loading time. This workflow identified a median of 5,655 protein groups per cell using an empirical spectral library generated from 20-cell samples. Overall, this study establishes a sensitive and high-throughput workflow for low-input and single-cell proteomics that combines low-loss microliter-scale sample preparation with optimized LC-MS analysis, achieving high proteome depth and analytical throughput without requiring specialized nanoliter-scale sample-preparation devices.

biochemistry↗

bioRxiv2026

Development of an Ethylenediaminetetraacetic Acid-Enhanced Deep Proteomic Profiling Method for Dried Blood Spots and Its Application in Mouse Disease Models

Dried blood spots (DBS) are well-established microsamples used in clinical testing and newborn screening. However, their use in deep proteomics is hindered by highly abundant blood proteins and inefficient protein recovery from filter paper matrices. The non-targeted analysis of non-specifically DBS-absorbed proteins (NANDA) workflow partially overcomes the impact of abundant blood proteins and has enabled the identification of over 5,000 proteins from DBS samples. Nonetheless, residual abundant proteins, including hemoglobin and fibrinogen, constrain deep proteomic analysis. Therefore, this study aimed to evaluate the effects of the metal chelator ethylenediaminetetraacetic acid (EDTA) on the depth of DBS proteomic analysis. An optimized EDTA-enhanced NANDA protocol that incorporated a 100 mM EDTA wash step was compatible with standard DBS collection procedures and required no modification of current clinical workflows, markedly enhancing the depletion of abundant proteins and facilitating its potential use in clinical and translational settings. When combined with Orbitrap Astral data-independent acquisition mass spectrometry, this approach enabled the single-shot identification of more than 7,000 proteins from DBS samples; to the best of our knowledge, this represents the deepest proteome coverage reported to date, and the workflow further supported high-throughput and highly reproducible analyses. Additionally, its application to mouse disease models revealed disease-specific systemic immune signatures from minimal blood volumes. Collectively, these results establish EDTA-enhanced NANDA as a practical and scalable workflow that overcomes longstanding limitations of DBS proteomics, thereby enabling deep, high-throughput, minimally invasive proteomic profiling across diverse biological and experimental contexts.

molecular biology↗

bioRxiv2026

TomAP-MS: an improved tomato lectin affinity purification-based mass spectrometry workflow enabling ultra-deep plasma proteomics

Plasma proteomics is increasingly important for biomarker discovery and disease stratification; however, comprehensive and high-throughput analysis remains challenging because of the extreme dynamic range of plasma proteins. We previously established tomato lectin affinity purification-based mass spectrometry (TomAP-MS), a workflow that enhances plasma proteome coverage via tomato lectin-mediated enrichment. The initial workflow depended on a 4% sodium dodecyl sulfate (SDS) elution, followed by SP3-based purification and digestion, which raised complexity and restricted throughput. In this study, we developed an improved TomAP-MS workflow incorporating lauryl maltose neopentyl glycol (LMNG)-assisted acid elution (LAcE), in which proteins are eluted under acidic conditions in the presence of LMNG. This process is followed by pH adjustment and direct tryptic digestion without SP3 cleanup. Compared with conventional acid elution and the original SDS/SP3 workflow, LAcE increased protein identifications while simplifying sample preparation and improving throughput. Using the optimized workflow, we identified more than 7,500 proteins from human plasma and demonstrated broader applicability in extracellular vesicle enrichment and protein interaction analysis workflows. We demonstrated that ethylenediaminetetraacetic acid plasma was the preferred specimen type, enabling the identification of over 5,000 proteins from just 1 {micro}L of plasma, with minimal impact on proteomic profiles after up to three freeze-thaw cycles. Additionally, the analysis of plasma from 200 healthy individuals reproducibly detected 4,117 proteins across all samples, including many proteins associated with inherited disorders. These findings establish TomAP-MS with LAcE as a practical platform for deep plasma proteomics, supporting its future application in proteomics-based screening and diagnostics.

biochemistry↗

bioRxiv2024

A novel newborn screening modality: Non-targeted proteome analysis using low-cost iron powders

In this study, we developed a simple protein extraction method for dried blood spots (DBS) that potentially meets the throughput required for newborn screening (NBS) and optimizes non-targeted proteomic analysis in combination with liquid chromatography coupled mass spectrometry in the data-independent-acquisition mode (DIA-LC-MS/MS). The developed pipeline, termed Non-targeted Analysis of Non-specifically DBS-Absorbed proteins (NANDA), successfully addressed the following three challenges: (1) processing of 96 3.2-mm DBS punches in parallel using low-cost iron powders with a robotic system, (2) identifying more than 5,000 proteins using DIA-LC-MS/MS, and (3) improving DIA-LC-MS/MS throughput to 40 samples/day with minimal compromise in protein coverage depth. The results imply that this pipeline can open new venues for conducting NBS using non-targeted quantitative proteome profiling, which has been a currently missing modality in NBS.

molecular biology↗

bioRxiv2024

Light-guided actin polymerization drives directed motility in protocells

Motility is a hallmark of lifes dynamic processes, enabling cells to actively chase prey, repair wounds, and shape organs. Recreating these intricate behaviors using well-defined molecules remains a major challenge at the intersection of biology, physics, and molecular engineering. Although the polymerization force of the actin cytoskeleton is characterized as a primary driver of cell motility, building a minimal platform to test this process in protocellular systems has proven elusive. The difficulty lies in the daunting task of distilling key components from motile cells and integrating them into model membranes in a physiologically relevant manner. To address this, we developed a method to optically control actin polymerization with high spatiotemporal precision within cell-mimetic lipid vesicles known as giant unilamellar vesicles (GUVs). Within these active protocells, the reorganization of actin networks triggered outward membrane extensions as well as the unidirectional movement of GUVs at speeds of up to 0.43 {micro}m/min, within the range of adherent mammalian cells. Notably, our findings reveal the requirements of both branched and linear actin networks for efficient membrane protrusions. This approach offers a powerful platform for unraveling the intricacies of cell migration, designing synthetic cells with active morphodynamics, and advancing bioengineering applications, such as self-propelled delivery systems and autonomous tissue-like materials.

synthetic biology↗

bioRxiv2024

Ultra-deep proteomics by Thin-diaPASEF with a 60-cm long column system

Proteomics using mass spectrometry (MS) has significantly advanced, offering deep insights into complex proteomes. The timsTOF MS platform with its parallel accumulation-serial fragmentation (PASEF) technology has achieved high scan speeds and high-quality spectra. Bukers timsTOF HT, which features TIMS-XR technology, offers an improved dynamic range and analysis depth, supporting high sample loadings. Moreover, various improvements to the data-independent acquisition method based on the PASEF technology (diaPASEF) have been reported. Despite these advancements, most high-level deep proteomic reports are based on the Orbitrap Astral and Orbitrap Exploris 480, and analytical systems using timsTOF MS still require improvement. Here, Brukers timsTOF HT was used to validate and optimize key diaPASEF parameters, leading to the development of a Thin-diaPASEF method. This method provides a high quantitative accuracy and consistency. In our validation, 9,400 proteins were identified in a single shot from HEK cells (strictly controlled Protein false discovery rate < 1%), the highest number analyzed by the timsTOF MS series using standard human cultured cells. Furthermore, by combining Thin-diaPASEF with an improved Lycopersicon esculentum lectin method, over 5,000 proteins were identified in a 24-sample/d analysis, and we succeeded in constructing a system with high proteome coverage that can be used for biomarker discovery.

biochemistry↗

bioRxiv2023

Universal pretreatment development for low-input proteomics using Lauryl Maltose Neopentyl Glycol

In recent years, the demand for low-input proteomics, most notably single-cell proteomics (SCP), has increased. In this study, we developed a Lauryl Maltose Neopentyl Glycol (LMNG)-assisted sample preparation (LASP) method that suppresses the loss of proteins and peptides in samples by adding LMNG, a surfactant, to the digested solution and removing the LMNG simply via reversed phase solid-phase extraction. The advantage of removing LMNG during sample preparation for general proteomic analysis is that it prevents mass spectrometry (MS) contamination. When the LASP method was applied to the low-input SP3 method and on-bead digestion in immunoprecipitation-MS, the recovery of the digested peptides was greatly improved. Furthermore, we established a simple and operationally easy sample preparation method for SCP based on the LASP method (scpLASP) and identified a median of 1,175 proteins from a single HEK239F cell using liquid chromatography (LC)-MS/MS with a throughput of 80 samples per day.

biochemistry↗

bioRxiv2022

TRIAC disrupts cerebral thyroid hormone action via a negative feedback loop and heterogenous distribution among organs

3,3,5-triiodothyroacetic acid (TRIAC) is a metabolite of endogenous thyroid hormones (THs) that can bind to and activate TH receptors. As TRIAC was previously detected in sewage effluent, we aimed to investigate exogenous TRIACs potential for endocrine disruption. We administered either TRIAC or 3,3,5-triiodo-L-thyronine (LT3) to both euthyroid mice and 6-propyl-2-thiouracil-induced hypothyroid mice. In hypothyroid mice, TRIAC alleviated growth retardation, suppressed the hypothalamus-pituitary-thyroid (HPT) axis, and upregulated TH-responsive genes in the pituitary gland, liver, and heart. We observed that, unlike LT3, TRIAC does not upregulate the expression of TH-responsive genes in the cerebrum. Measurement of organ-specific TRIAC levels suggested that TRIAC was not efficiently trafficked into the cerebrum. Furthermore, by analyzing euthyroid mice, we found that cerebral TRIAC levels did not increase despite TRIAC administration at higher concentrations, whereas serum and cerebral TH levels were substantially decreased. Hence, TH-responsive genes in the cerebrum appear to be downregulated by TRIAC. In summary, TRIAC administration decreases circulating TH levels by suppressing the HPT axis, while the consequent attenuation of TH actions was compensated by TRIAC in peripheral tissues but not in the cerebrum due to the relative impermeability of the blood-brain barrier towards TRIAC. We verified that exogenous TRIAC disrupts TH actions in the cerebrum. This disruption is apparently due to the additive effects of circulating endogenous THs being depleted via a negative feedback loop involving the HPT axis and heterogenous distribution of TRIAC among different organs. Our findings indicate that environmental TRIAC poses a potential neurodevelopmental risk.

ecology↗