Biology subjects

Kanao, E.

Publications and source records attributed to Kanao, E..

10 recordsLinked to original sources

bioRxiv2026

Expanding N-terminomics Coverage by Extending N-terminal Peptide Length

In bottom-up proteomics, protease specificity determines peptide length and LC/MS/MS detectability; in N-terminomics, this is critical because each protein N-terminal peptide is unique and cannot be substituted by other peptides. CHromatographic AMplification of Protein N-terminal peptides (CHAMP-N) combines LysargiNase digestion with StageTip-based strong cation exchange chromatography (SCX), removing internal peptides by exploiting their newly generated N-terminal Lys/Arg residues and thereby selectively isolating protein N-terminal peptides. However, LysargiNase cleavage near the protein N-terminus can generate excessively short peptides. Here, we developed two complementary CHAMP-N approaches that modurate N-terminal peptide length while retaining the charge-based SCX separation principle. LysN digestion avoided cleavage at N-terminal-proximal Arg residues, whereas D3-acetylation blocked Lys cleavage by LysargiNase to produce ArgN-like specificity. Both approaches extended otherwise short N-terminal peptides while preserving N-terminal basic residues on internal peptides. Conventional LysargiNase, LysN, and ArgN-like CHAMP-N generated complementary sets of N-terminal peptides. Integration of the three workflows identified 2,552 protein N-termini, approximately twice the number identified by conventional CHAMP-N alone. The median relative standard deviations of N-terminal peptide intensities were approximately 15% for all three workflows, indicating comparable technical reproducibility. These results demonstrate that coordinating protease cleavage specificity with SCX selectivity reduces sequence-dependent losses and expands N-terminome coverage while preserving the simplicity and reproducibility of CHAMP-N.

biochemistry↗

bioRxiv2026

Direct Injection NanoHILIC/MS/MS Proteomics from Reversed-Phase StageTip Eluate

NanoHILIC/MS/MS provides high sensitivity for low-input peptide analysis, yet its use in bottom-up proteomics has been constrained by a persistent solvent mismatch: tryptic peptides exhibit poor solubility in the [≥]95% acetonitrile (ACN) required for nanoHILIC injection. Our previously reported two-step solubilization method [Anal Chem 2025, 97 (19), 10227-10235] alleviated this issue but required large dilution volumes, limiting the amount of sample that could be injected. Here, we introduce DiReCT (Dissolution from Reverse-Phase Chromatography Tips), a StageTip-based workflow that integrates peptide solubilization, desalting, and nanoHILIC-compatible elution into a single operation. During elution from RP-StageTips, residual water on the stationary phase is rapidly displaced by a small volume of high-ACN solvent, generating a transient mid-ACN environment that maximizes peptide solubility without drying. This mechanism enables high-recovery peptide concentration and allows direct injection of the entire eluate onto nanoHILIC/MS/MS. Using [~]0.25 ng of HeLa digest, DiReCT/nanoHILIC/MS/MS identified 1177 peptides and 410 proteins, representing 8.9- and 6.7-fold increases over nanoRPLC/MS/MS, respectively.

biochemistry↗

bioRxiv2026

High Recovery Stop-and-Go Extraction Tips Using Polytetrafluoroethylene Disks Embedded with Poly(styrene-divinylbenzene) Particles for Proteomics

The stop-and-go extraction tip (StageTip) is widely used for peptide purification in bottom-up proteomics, yet the original 3M EmporeTM disk is no longer available, prompting the need to evaluate current alternatives. Two types of commercially available poly(styrene-divinylbenzene) (SDB)-containing polytetrafluoroethylene (PTFE) disks were used to fabricate StageTips. Desalting was performed on 500 ng and 20 ng of HeLa tryptic peptides, and the nanoLC/MS/MS results were compared. Both StageTips demonstrated equivalent performance for the 500 ng sample, but for the 20 ng sample, one StageTip identified 1.8 times more peptides, particularly longer and more hydrophobic peptides. Physical characterization and scanning electron microscope imaging of these disks revealed that the high-performing disk contains more PTFE fibers, partially covering the SDB particle surface. This likely prevents hydrophobic peptides from being trapped in small mesopores, improving recovery from low-input samples. These findings demonstrate that disk material properties critically influence performance in trace proteomics.

biochemistry↗

bioRxiv2025

Rewiring of RNA-protein coupling in osteocytes in response to hyperglycemic levels of glucose

Osteocytes are central regulators of skeletal homeostasis, yet how their transcriptome and proteome jointly adapt to metabolic stress is unclear. Here, we combined RNA-sequencing with label-free quantitative proteomics in MLO-Y4 osteocytic cells exposed to hyperglycemic levels of glucose, to interrogate RNA-protein coupling at baseline and under hyperglycemic stress. Transcriptionally, high glucose and mannitol elicited overlapping osmoadaptive transcriptional programs indicative of metabolic remodeling, whereas high glucose alone induced mitochondrial and oxidative phosphorylation downregulation alongside selective activation of bone anabolic and inflammatory pathways. RNA-protein integration revealed moderate coupling at baseline, indicating that mRNA levels capture only part of the proteomic output. High glucose reorganized RNA-protein relationship, sorting genes into four patterns of matched or opposing RNA-protein responses. The four groups were enriched in distinct biological pathways that shaped cellular response to stress exposing significant post-transcriptional and translational control under high glucose levels. These data indicate that osteocytes adapt to stress through program-specific RNA-protein interactions in which post-transcriptional regulation of protein translation reshapes the proteome beyond what is predicted by mRNA levels.

bioinformatics↗

bioRxiv2024

Prediction of Protein Half-lives from Amino Acid Sequences by Protein Language Models

We developed a protein half-life prediction model, PLTNUM, based on a protein language model using an extensive dataset of protein sequences and protein half-lives from the NIH3T3 mouse embryo fibroblast cell line as a training set. PLTNUM achieved an accuracy of 71% on validation data and showed robust performance with an ROC of 0.73 when applied to a human cell line dataset. By incorporating Shapley Additive Explanations (SHAP) into PLTNUM, we identified key factors contributing to shorter protein half-lives, such as cysteine-containing domains and intrinsically disordered regions. Using SHAP values, PLTNUM can also predict potential degron sequences that shorten protein half-lives. This model provides a platform for elucidating the sequence dependency of protein half-lives, while the uncertainty in predictions underscores the importance of biological context in influencing protein half-lives.

bioinformatics↗

bioRxiv2024

High-Recovery Desalting Tip Columns for a Wide Variety of Peptides in Mass Spectrometry-based Proteomics

In mass spectrometry-based proteomics, loss-minimized peptide purification techniques play a key role in improving sensitivity and coverage. We have developed a desalting tip column packed with thermoplastic polymer-coated chromatographic particles, named ChocoTip, to achieve high recoveries in peptide purification by pipette-tip-based LC with centrifugation (tipLC). ChocoTip identified more than twice as many peptides from 20 ng of tryptic peptides from Hela cell lysate compared to a typical StageTip packed with chromatographic particles entangled in a Teflon mesh in tipLC. The high recovery of ChocoTip in tipLC was maintained for peptides with a wide variety of physical properties over the entire retention time range of the LC/MS/MS analysis, and was especially noteworthy for peptides with long retention times. These excellent properties are attributable to the unique morphology of ChocoTip, in which the thermoplastic polymer covers the pores, thereby inhibiting irreversible adsorption of peptides into mesopores of the chromatographic particles. ChocoTip is expected to find applications, especially in clinical proteomics and single-cell proteomics, where sample amounts are limited.

biochemistry↗

bioRxiv2023

A Straightforward Approach for Living Biomembrane Printing onto Nanoparticle

Biomembrane coating technologies have increasingly been pursued to grant natural dynamic bio-interfaces onto synthetic nanomaterials. Herein, we report a one-step method to coat "living" biomembrane on nanoparticle surfaces in a non-destructive manner. In our method, nanoparticles were efficiently coated with cell membranes without losing the structural integrity by mechanically facilitating the passage of nanoparticles to a concentration layer of living cells with simple centrifugation. This was similar to the exosome-releasing process via endocytosis and exocytosis. The biomembrane originating from living Raw264.7 cells was coated onto the silica nanoparticle prepared by our method, and proteome profiling with nanoflow liquid chromatography-tandem mass spectrometry demonstrated that it was constructed with proteins derived from the membranous component. This proteome profile was not observed in silica nanoparticles prepared with dead cells. Finally, the hybridized cell membrane effectively suppressed the phagocytic activity of Raw264.7 cells to silica nanoparticles and improved the uptake efficiency into cancer cells. We believe our simple and efficient method to coat living biomembranes should be useful in developing medical and pharmaceutical applications involving nanoparticles.

synthetic biology↗

bioRxiv2023

Centrifugal Gel Crushing for Gel-based Proteome Analysis

We have developed a centrifugal gel crushing method using a pipette tip. Polyacrylamide gel slices are extruded from the narrowing cavity of a pipette tip by centrifugation in a few minutes to crush them into pieces of appropriate size. The size of the crushed gel could be controlled by several parameters, including centrifugal force and pipette tip cavity. In shotgun proteomics, gel-based LC/MS/MS, so-called GeLC/MS/MS, involves the essential but tedious processes of pre-fractionation by SDS-PAGE, followed by dicing the entire gel lane into several parts, fine dicing, and in-gel digestion after the diced gel is manually transferred to a microtube. In this study, we developed an alternative way to crush the pre-fractionated gel slice into small and irregular-shaped gels by centrifugal extrusion of the sliced gel from the narrow cavity of a pipette tip. As a result, we observed an improved recovery and reproducibility of digested proteins compared to the conventional method of manual dicing. We believe that this simple and rapid method of crushing polyacrylamide gels, which allows for parallel operations and automation, is useful for GeLC/MS/MS analysis and applicable to other approaches including top-down proteomics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/545122v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@da02dcorg.highwire.dtl.DTLVardef@f1dee1org.highwire.dtl.DTLVardef@15fb780org.highwire.dtl.DTLVardef@10848b1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

One thousand samples per day capillary-flow LC/MS/MS for high-speed, high-sensitivity and in-depth proteomics

We developed a capillary-flow LC/MS/MS system with ultrahigh speed, enabling a throughput of 1,000 samples per day while maintaining high sensitivity and depth of analysis. In targeted LC/MS mode, 36 endogenous phosphopeptides in HeLa cells, including EphA2- derived phosphopeptide isomers, were successfully quantified with high selectivity and linearity by combining ion mobility separation. When 500 ng of HeLa cell digest was measured 100 times repeatedly in data-dependent acquisition mode, the coefficient of variation of retention time, peak intensity and number of identified peptides were on average 3.4%, 19.8%, and 6.0%, respectively. In data-independent acquisition mode, this system achieved the identification and quantification of 3,139 protein groups from a 100 ng HeLa cell digest and 2,145 protein groups from a sample of only 10 ng. The coefficient of variation of protein commonly quantified in the triplicate analysis ranged from 12 to 24% for HeLa digest samples ranging from 10 to 1000 ng. Finally, we applied this high-speed system to the spatial proteomics of the mouse brain, and succeeded in capturing the proteome distribution along a 96-sectioned brain structure in 135 minutes. This is the first LC/MS/MS system to achieve both more than 500 samples per day and more than 3000 identified protein groups ID with less than 100 ng human cultured cells simultaneously.

systems biology↗

bioRxiv2022

Classification of Extracellular Vesicles based on Surface Glycan Structures by Spongy-like Separation Media

Extracellular vesicles (EVs) are lipid bilayer vesicles that enclose various biomolecules. EVs hold promise as sensitive biomarkers to detect and monitor various diseases. However, they have heterogenous molecular compositions. The compositions of EVs from identical donor cells obtained using the same purification methods may differ, which is a significant obstacle for elucidating objective biological functions. Herein the potential of a novel lectin-based affinity chromatography (LAC) method to classify EVs based on their glycan structures is demonstrated. The proposed method utilizes a spongy-like monolithic polymer (spongy monolith, SPM), which consists of poly(ethylene-co-glycidyl methacrylate) with continuous micropores and allows an efficient in-situ protein reaction with epoxy groups. Two distinct lectins with different specificities, Sambucus sieboldiana agglutinin and concanavalin A, are effectively immobilized on SPM without impacting the binding activity. Moreover, high recovery rates of liposomal nanoparticles as a model of EVs are achieved due to the large flow-through pores (>10 m) of SPM. Finally, lectin-immobilized SPMs are employed to classify EVs based on the surface glycan structures and demonstrate different subpopulations by proteome profiling.

biochemistry↗