Biology subjects

Kodera, Y.

Publications and source records attributed to Kodera, Y..

6 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

DIA-MS based plasma peptidomic workflow for profiling organ-derived peptides

Plasma contains diverse bioactive peptides that play crucial roles in maintaining homeostasis and regulating disease responses. However, the presence of peptides derived from high-abundance proteins such as albumin makes comprehensive analysis of native peptides secreted by organs challenging. This study aimed to establish a highly sensitive plasma peptidomic approach by combining data-independent acquisition (DIA) with spectral libraries of plasma and organs. First, peptides were extracted from plasma and eleven organ types using a high-yield peptide extraction method, the differential solubilization method. These peptides were then measured via data-dependent acquisition (DDA) analysis using a timsTOF HT for constructing empirical spectral library. Subsequently, DIA-MS data from plasma samples were measured and analyzed using this spectral library. This strategy achieved identification of, on average, over 5,500 peptides per run, with over 2,000 organ-derived peptides including 19 known bioactive peptides. The novel strategy proposed here enables highly sensitive quantitative analysis of organ-derived peptides in plasma, linking them to their secreting organs. It is expected to substantially contribute not only to the discovery of biomarkers and novel bioactive peptides but also to elucidating the pathophysiology of systemic diseases.

biochemistry↗

bioRxiv2025

2-Mercaptoethanol/DMSO workflow enables highly reproducible quantitative proteomics

Proteomics provides a systematic and high-throughput approach to comprehensively characterize protein networks, enabling insights into cellular functions and disease mechanisms. Carbamidomethylation using iodoacetamide (IAA), a common method for cysteine alkylation, is known to cause nonspecific modifications that increase MS spectral complexity and reduce quantitative accuracy. Here, we extended our 2-mercaptoethanol (2-ME) adduction method, enhanced by dimethyl sulfoxide (DMSO), from single-protein applications to a proteome-wide workflow. Mouse liver proteomes were processed using either 2-ME/DMSO or conventional IAA treatment, followed by LC-MS/MS analysis. The optimized 2-ME treatment increased the number of cysteine-modified peptides by 1.6-1.9-fold. Although total protein identifications were comparable, 77% of proteins exhibited improved sequence coverage. Quantitative reproducibility was also enhanced, with peptides quantified coefficient of variation (CV) [≤] 20% increasing from 61.5% (IAA) to 86.1% (2-ME), and proteins from 80.6% to 93.5%. Application of the workflow to an ovarian clear cell carcinoma reliably detected cisplatin-induced alterations. The 2-ME/DMSO workflow offers a simple and highly reproducible proteomics strategy for accurate quantitative proteomics.

biochemistry↗

bioRxiv2025

Structure-activity relationship of an all-α-helical prenyltransferase reveals a mechanism for indole prenylation.

Enzymes are involved in the biosynthesis of various secondary metabolites found in nature. The catalytic mechanism is regulated by the three-dimensional structure of the enzyme especially at the catalytic site, resulting in the natural products with complicated conformation derived from a regioselective, chemoselective, and stereoselective fashion of the enzyme reaction. Prenyltransferase (PT), which belongs to the prenylsynthase (PS) family, catalyzes the condensation of isoprene to an aromatic compound, consequently producing a terpenoid scaffold structure. Therefore, it plays an important role in which expands the chemical diversity of terpenoids. Although the three-dimensional structures of PS which categorized in the same family were resolved, the catalytic mechanism of the PT has been vailed. In this study, we determined the X-ray crystal structure of a novel prenyltransferase SxPT1 derived from marine Streptomyces. Here we described that SxPT1 analyzed the enzyme reactions and discussed its catalytic mechanism.

biochemistry↗

bioRxiv2024

Reducing Offsite Modification using 2-mercaptoethanol for Proteome Analysis.

Alkylation of the thiol group in cysteine (Cys) residues using halide reagents is a significant step in proteomics. However, non-specific modifications to the N-terminus and other amino acids are known. Thus promiscuous offsite alkylation in the peptide further complicated the MS spectra and thus made the difficulty of identification and quantification of all peptides. 2-mercaptoethanol (2-ME) is not only a regent for the reduction of the disulfide bond but also is bound to the Cys residue. Furthermore, it is known that dimethyl sulfoxide (DMSO) enhances the disulfide bond formation. Thus, based on these facts, we developed a method for specifical modification of Cys residues using 2-ME and DMSO. The specific modification of Cys residue by 2-ME were promoted by the concentration-dependent manner of DMSO with quite less offsite modification reaction compared with recent procedures. This Cys-specific modification technique may not only improve the quantification of peptides containing cysteine but also enhance the quantification accuracy of all peptides.

biochemistry↗

bioRxiv2021

Hidden pathway for cytokine receptor activation: Structural insights into a marine sponge-derived lectin that activates the thrombopoietin receptor via recognition of the fucose moiety

N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that constitutively activates the receptor. However, the molecular basis for this mechanism has not been studied because no external agonists existed. We describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC), a homodimerized lectin with calcium-dependent fucose-binding properties. ThC-induced activation persists due to limited receptor internalization. The strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. MPL is subject to sugar-mediated activation, where the kinetics differ from those of cytokines. This result suggests the presence of diverse receptor activation pathways in human thrombopoiesis. One-sentence summaryA marine sponge lectin catalyzes thrombopoietin receptor dimerization and activation, exhibiting strong synergy with thrombopoietin, and modulates internalization of the receptor.

cell biology↗