Biology subjects

Ishihama, Y.

Publications and source records attributed to Ishihama, Y..

At least 19 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

A Multi-Omics Framework Reveals Phosphorylation-Dependent Control of Protein Interactions

Protein phosphorylation regulates nearly every cellular process, yet most of the hundreds of thousands of human phosphosites remain functionally uncharacterized. Rather than prioritising phosphosites by conservation or structural features, here we use the abundance of an interaction partner as a readout of whether a phosphosite affects that interaction. This idea exploits the fact that subunits of stable complexes are often degraded when unbound. Here, we apply a nested linear regression model to pan-cancer data from 1,006 tumours, while controlling for transcriptional and other covariates. We identified 6,160 associations between 3,038 phosphosites and the abundance of interacting proteins, including several known interaction-regulating sites. Mapping these onto AlphaFold-predicted complexes placed 239 sites at interaction interfaces, while another 402 were linked to compartment-specific localisation, indicating that phosphorylation can also tune interactions by relocating proteins between compartments. Affinity-purification mass spectrometry of NKAP and NUF2 phosphosite mutants experimentally supported some of these predictions. Together, this framework reveals a widespread coupling between phosphorylation and interaction-dependent protein abundance and provides a prioritized, structure-informed resource for characterizing the human phosphoproteome

bioinformatics↗

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

Seamless workflow of hydroxy acid-modified metal oxide chromatography for rapid and sensitive phosphoproteomics sample preparation

Phosphoproteomics by liquid chromatography/tandem mass spectrometry requires efficient phosphopeptide enrichment, but conventional workflows are often time-consuming and prone to sample loss, particularly at low input. Here, we present Rapid Hydroxy Acid-Modified Metal Oxide Chromatography (Rapid HAMMOC), a streamlined, TiO2-based enrichment workflow that features three key improvements. First, we optimized the TiO2 column loading conditions and found that alternative pH-buffering agents and organic solvents, such as sodium bicarbonate combined with ethyl acetate, outperformed the commonly used Tris-based buffer with isopropanol. Second, to minimize sample loss and manual handling in desalting, phosphopeptides eluted under basic conditions were directly loaded onto a dual-membrane StageTip composed of stacked strong anion exchange (SAX) and reversed-phase styrene-divinylbenzene (SDB) membranes (SAX-SDB StageTip). Third, the addition of lauryl maltose neopentyl glycol (LMNG), which is readily removed during desalting, suppressed nonspecific adsorption. Rapid HAMMOC provided markedly improved sensitivity, identifying approximately 5,000 class I phosphosites from 5 g of K562 cell digests, with a median 7.9-fold increase in intensity compared to the original workflow. Rapid HAMMOC also identified, on average, approximately 8,000 class I phosphosites from as little as 0.5 g input from HeLa, A549, and HCT116 cells. Furthermore, coupling Rapid HAMMOC with anti-puromycin immunoprecipitation enabled single-day profiling of nascent polypeptides from ultra-low input samples, yielding 2,310 high-confidence co-translational phosphosites. Beyond providing a practical enrichment workflow, this study offers broadly applicable insights that can be extended to other TiO2-based phosphoproteomic methods.

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

Infraslow histaminergic dynamics govern priming states to gate moment-to-moment memory accessibility

Memory expression fluctuates even in response to identical cues, suggesting that ongoing brain states bias memory accessibility. However, the cellular and circuit principles governing these state-dependent fluctuations remain unclear. Here, we show that spontaneous pre-cue activity of histaminergic neurons in the hypothalamic tuberomammillary nucleus (TMN) modulates the expression of reward-associative memory in mice. TMN histaminergic activity exhibited infraslow dynamics (0.05-0.1 Hz) that closely tracked an integrated brain-body state. Closed-loop cue delivery during high histaminergic states enhanced memory expression. Brief optogenetic activation or inhibition of these neurons before the cue bidirectionally modulated memory expression, and direct activation of histaminergic terminals in the basolateral amygdala (BLA) was sufficient to enhance memory expression. Furthermore, histaminergic inhibition before the cue impaired the cue-evoked BLA population response. Thus, ongoing histaminergic activity exerts an infraslow, state-setting influence that primes BLA circuits for robust cue responses, and in turn, modulates moment-to-moment memory accessibility.

neuroscience↗

bioRxiv2025

Bottom-up proteomics under acidic conditions using protease type XIII from Aspergillus saitoi

Bottom-up proteomics is a powerful technique for comprehensive analysis of proteins by proteolytic cleavage followed by liquid chromatography/tandem mass spectrometry to identify the resulting peptides. Trypsin is the gold-standard protease for bottom-up proteomics, though its cleavage specificity limits peptide identification, depending on the protein sequence. In addition, its optimal pH is weakly alkaline, which can cause modification artifacts such as deamidation. We hypothesized that these limitations might be overcome by using protease type XIII (P13ase) from Aspergillus saitoi, which is active at low pH. P13ase has been used for protein structural analysis by hydrogen-deuterium exchange mass spectrometry, but its cleavage preferences have not been clarified. Here, we show that P13ase primarily cleaves the C-terminal side of Lys, Arg, and Leu, and the optimal P13ase digestion conditions for bottom-up proteomics are pH 3.5, 37{degrees}C for 60 min. Under these conditions, sequence coverage of more than 90% was achieved for several proteins in HeLa cell extracts, which is unachievable with trypsin. In addition, P13ase digestion reduced artifacts such as deamidation products generated by cyclization reactions and subsequent hydrolysis. These results indicate that P13ase is a promising new tool for precision proteomics.

biochemistry↗

bioRxiv2025

Detecting Protein Higher-Order Structural Changes Using Kinase as a Phospho-Labeler

We developed an approach to detect protein conformational changes on a proteome-wide scale by labeling proteins with phosphate groups through in vitro kinase reactions. This structural proteomics approach combines residue-specific labeling based on substrate recognition by protein kinases with quantitative phosphoproteomics using phosphopeptide enrichment, enabling proteome-wide and site-specific profiling of protein conformational changes. By performing in vitro phosphorylation of myoglobin with and without heat denaturation and of HEK293T cell extracts with and without protease treatment, we demonstrated that differences in substrate protein structure are reflected in in vitro phosphorylation efficiency. Moreover, we successfully identified protein conformational changes upon RNA digestion in non-denatured cell extract proteome samples on a proteome-wide scale with high sensitivity by comparing phosphorylation efficiencies. This approach enables residue-specific readout of structural dynamics within the intracellular proteome. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/652599v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@b2c6c8org.highwire.dtl.DTLVardef@118b159org.highwire.dtl.DTLVardef@2374adorg.highwire.dtl.DTLVardef@1c14880_HPS_FORMAT_FIGEXP M_FIG C_FIG MotivationMass spectrometry (MS)-based structural analysis of proteins often involves labeling of exposed amino acid residues, followed by digestion and identification of labeled peptides by MS. However, the heterogeneity of labeled peptides and the complexity of biological samples hinder proteome-wide analysis of protein higher-order structures. To address this issue, we devised an approach that utilizes site-specific labeling with phosphate groups, taking advantage of the substrate recognition provided by protein kinases. The phosphate groups serve as enrichment handles, enabling selective extraction and highly sensitive analysis of labeled sites. HighlightsO_LIProtein labeling with phosphate groups using in vitro kinase reaction C_LIO_LIQuantitative MS-based readout of in vitro phosphorylation efficiency C_LIO_LIIn vitro phosphorylation efficiency reflects protein conformational changes C_LIO_LIProteome-wide and site-specific identification of protein conformational changes C_LI

biochemistry↗

bioRxiv2024

UniScore, a unified and universal measure for peptide identification by multiple search engines

We propose UniScore as a metric for integrating and standardizing the outputs of multiple search engines in the analysis of data-dependent acquisition (DDA) data from LC/MS/MS-based bottom-up proteomics. UniScore is calculated from the annotation information attached to the product ions alone by matching the amino acid sequences of candidate peptides suggested by the search engine with the product ion spectrum. The acceptance criteria are controlled independently of the score values by using the false discovery rate based on the target-decoy approach. Compared to other rescoring methods that use deep learning-based spectral prediction, larger amounts of data can be processed using minimal computing resources. When applied to large-scale global proteome data and phosphoproteome data, the UniScore approach outperformed each of the conventional single search engines examined (Comet, X! Tandem, Mascot and MaxQuant). Furthermore, UniScore could also be directly applied to peptide matching in chimeric spectra without any additional filters.

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

Leveraging Pretrained Deep Protein Language Model to Predict Peptide Collision Cross Section

Collision cross section (CCS) of peptide ions provides an important separation dimension in liquid chromatography/tandem mass spectrometry-based proteomics that incorporates ion mobility spectrometry (IMS), and its accurate prediction is the basis for advanced proteomics workflows. This paper describes novel experimental data and a novel prediction model for challenging CCS prediction tasks including longer peptides that tend to have higher charge states. The proposed model is based on a pretrained deep protein language model. While the conventional prediction model requires training from scratch, the proposed model enables training with less amount of time owing to the use of the pretrained model as a feature extractor. Results of experiments with the novel experimental data show that the proposed model succeeds in drastically reducing the training time while maintaining the same or even better prediction performance compared with the conventional method. Our approach presents the possibility of prediction in a "greener" manner of various peptide properties in proteomic liquid chromatography/tandem mass spectrometry experiments.

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↗

bioRxiv2024

Highly Sequence-specific, Timing-controllable m6A Demethylation by Modulating RNA-binding Affinity of m6A Erasers

In recent years, significant progress has been made in developing tools that consist of programmable RNA binding proteins and m6A-erasers to selectively demethylate m6A at specific sites. Especially, timing-controllable demethylation tools using stimulus-inducible dimerization show promise in understanding the impact of individual m6A modifications on dynamic physiological processes. However, a critical issue with these technologies is the possible off-target effects, wherein wild-type m6A-erasers constituting the tools may decrease methylation levels at sites other than the intended target m6A. In this study, we addressed this issue by reducing the intrinsic RNA-binding ability of m6A-erasers to prevent off-target effects and by fusing with PUF RNA-binding protein to provide recognition ability of arbitrary RNA sequences. Taking advantage of the reduced RNA-binding ability of the m6A-erasers themselves, we employed a rapamycin-based switching system in the linker connecting the PUF and m6A-eraser domains. The resulting m6A-erasers exhibited superior precision in demethylating targeted sites while minimizing off-target effects, presenting a novel approach for precise temporal control over m6A modification dynamics.

biochemistry↗

bioRxiv2024

One-step N-terminomics based on isolation of protein N-terminal peptides from LysargiNase digests by tip-based strong cation exchange chromatography

We have developed a one-step isolation method for protein N-terminal peptides from LysargiNase digests by pipette tip-based strong cation exchange (SCX) chromatography. This CHAMP-N (CHromatographic AMplification of Protein N-terminal peptides) method using disposable and parallel-processable SCX tips instead of conventional HPLC SCX columns facilitates simple, sensitive and high-throughput N-terminomic profiling without sacrificing the high identification numbers and selectivity achieved by the HPLC-based method. By applying the CHAMP-N method to HEK293T cells, we identified novel cleavage sites for signal and transit peptides, and non-canonical translation initiation sites. Finally, for proteome-wide terminomics, we present a simple and comprehensive N-and C-terminomics platform employing three different tip-based approaches, including CHAMP-N, in which protease digestion and one-step isolation by tip LC are commonly used to achieve complementary terminome coverages.

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

Pathogenic mutations of human phosphorylation sites affect protein-protein interactions

Despite their lack of a defined 3D structure, intrinsically disordered regions (IDRs) of proteins play important biological roles. Many IDRs contain short linear motifs (SLiMs) that mediate protein-protein interactions (PPIs), which can be regulated by post-translational modifications like phosphorylation. 20% of pathogenic missense mutations are found in IDRs, and understanding how such mutations affect PPIs is essential for unraveling disease mechanisms. Here, we employed peptide-based interaction proteomics to investigate 36 disease-causing mutations affecting phosphorylation sites. Our results unveiled significant differences in interactomes between phosphorylated and non-phosphorylated peptides, often due to disrupted phosphorylation-dependent SLiMs. We focused on a mutation of a serine phosphorylation site in the transcription factor GATAD1, which causes dilated cardiomyopathy. We found that this phosphorylation site mediates interaction with 14-3-3 family proteins. Follow-up experiments revealed the structural basis of this interaction and suggest that 14-3-3 binding affects GATAD1 nucleocytoplasmic transport by masking a nuclear localisation signal. Our results demonstrate that pathogenic mutations of human phosphorylation sites can significantly impact protein-protein interactions, offering fresh insights into potential molecular mechanisms underlying pathogenesis.

systems 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↗