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Alternative splicing events are a major causal mechanism for complex traits, but they have been understudied due to the limitation of short-read sequencing. Here, we generate a full-length isoform annotation of human immune cells from an individual by long-read sequencing for 29 cell subsets. This contains a number of unannotated transcripts and isoforms such as a read-through transcript of TOMM40-APOE in the Alzheimer’s disease locus. We profile characteristics of isoforms and show that repetitive elements significantly explain the diversity of unannotated isoforms, providing insight into the human genome evolution. In addition, some of the isoforms are expressed in a cell-type specific manner, whose alternative 3’-UTRs usage contributes to their specificity. Further, we identify disease-associated isoforms by isoform switch analysis and by integration of several quantitative trait loci analyses with genome-wide association study data. Our findings will promote the elucidation of the mechanism of complex diseases via alternative splicing.

To improve the coverage in bottom-up proteomics, S-aminoethylation of cysteine residues (AE-Cys) was carried out with 2-bromoethylamine, followed by cleavage with lysyl endopeptidase (Lys-C) or Lys-C/trypsin. A model study with bovine serum albumin showed that the C-terminal side of AE-Cys was successfully cleaved by Lys-C. The frequency of side reactions at amino acids other than Cys was less than that in the case of carbamidomethylation of Cys with iodoacetamide. Proteomic analysis of A549 cell extracts in the data-dependent acquisition mode after AE-Cys modification afforded a greater number of identified protein groups, especially membrane proteins. In addition, label-free quantification of proteins in mouse nonsmall cell lung cancer (NSCLC) tissue in the data-independent acquisition mode after AE-Cys modification showed improved NSCLC pathway coverage and greater reproducibility. Furthermore, the AE-Cys method could identify an epidermal growth factor receptor peptide containing the T790 M mutation site, a well-established lung-cancer-related mutation site that has evaded conventional bottom-up methods. Finally, AE-Cys was found to fully mimic Lys in terms of collision-induced dissociation fragmentation, ion mobility separation, and cleavage by Lys-C/trypsin, except for sulfoxide formation during sample preparation., To improve the coverage in bottom-up proteomics, S-aminoethylation of cysteine residues (AE-Cys) was carried out with 2-bromoethylamine, followed by cleavage with lysyl endopeptidase (Lys-C) or Lys-C/trypsin. A model study with bovine serum albumin showed that the C-terminal side of AE-Cys was successfully cleaved by Lys-C. The frequency of side reactions at amino acids other than Cys was less than that in the case of carbamidomethylation of Cys with iodoacetamide. Proteomic analysis of A549 cell extracts in the data-dependent acquisition mode after AE-Cys modification afforded a greater number of identified protein groups, especially membrane proteins. In addition, label-free quantification of proteins in mouse nonsmall cell lung cancer (NSCLC) tissue in the data-independent acquisition mode after AE-Cys modification showed improved NSCLC pathway coverage and greater reproducibility. Furthermore, the AE-Cys method could identify an epidermal growth factor receptor peptide containing the T790 M mutation site, a well-established lung-cancer-related mutation site that has evaded conventional bottom-up methods. Finally, AE-Cys was found to fully mimic Lys in terms of collision-induced dissociation fragmentation, ion mobility separation, and cleavage by Lys-C/trypsin, except for sulfoxide formation during sample preparation.

Three representative protein kinases with different substrate preferences, ERK1 (Pro-directed), CK2 (acidophilic), and PKA (basophilic), were used to investigate phosphorylation sequence motifs in substrate pools consisting of the proteomes from three different cell lines, MCF7 (human mammary carcinoma), HeLa (human cervical carcinoma), and Jurkat (human acute T-cell leukemia). Specifically, recombinant kinases were added to the cell-extracted proteomes to phosphorylate the substrates in vitro. After trypsin digestion, the phosphopeptides were enriched and subjected to nanoLC/MS/MS analysis to identify their phosphorylation sites on a large scale. By analyzing the obtained phosphorylation sites and their surrounding sequences, phosphorylation motifs were extracted for each kinase-substrate proteome pair. We found that each kinase exhibited the same set of phosphorylation motifs, independently of the substrate pool proteome. Furthermore, the identified motifs were also consistent with those found using a completely randomized peptide library. These results indicate that cell-extracted proteomes can provide kinase phosphorylation motifs with sufficient accuracy, even though their sequences are not completely random, supporting the robustness of phosphorylation motif identification based on phosphoproteome analysis of cell extracts as a substrate pool for a kinase of interest.

Mitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, which shapes the oxidative phosphorylation complexes essential for cellular energy metabolism. Despite the importance of mitochondrial translation (MT) control, it is challenging to identify and quantify the mitochondrial-encoded proteins because of their hydrophobic nature and low abundance. Here, we introduce a mass spectrometry–based proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture. Our method provides the highest protein identification rate with the shortest measurement time among currently available methods, enabling us to quantify 12 of the 13 mitochondrial-encoded proteins. We applied this method to uncover the global picture of (post-)translational regulation of both mitochondrial- and nuclear-encoded subunits of oxidative phosphorylation complexes. We found that inhibition of MT led to degradation of orphan nuclear-encoded subunits that are considered to form subcomplexes with the mitochondrial-encoded subunits. This method should be readily applicable to study MT programs in many contexts, including oxidative stress and mitochondrial disease.

We have successfully developed a bioinertized nanoflow liquid chromatography/tandem mass spectrometry (nanoLC/MS/MS) system for the highly sensitive analysis of phosphopeptides by depleting metal ions from the mobile phase. We found that not only direct contact of phosphopeptides with metal components, but also indirect contact with nanoLC pumps through the mobile phase causes significant losses during the recovery of phosphopeptides. Moreover, electrospray ionization was adversely affected by the mobile phase containing multiple metal ions as well as by the sample solvents contaminated with metal ions used in immobilized metal ion affinity chromatography for phosphopeptide enrichment. To solve these problems, metal ions were depleted by inserting an on-line metal ion removal device containing metal-chelating membranes between the gradient mixer and the autosampler. As a result, the peak areas of the identified phosphopeptides increased an average of 9.9-fold overall and 77-fold for multiply phosphorylated peptides with the insertion of the on-line metal ion removal system. This strategy would be applicable to highly sensitive analysis of other phosphorylated biomolecules by microscale-LC/MS/MS.

Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging, especially when analyzing tiny sample amounts. To address this, we present a hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem mass spectrometry (MSx) scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmark hybrid-DIA against state-of-the-art targeted MS methods (i.e., SureQuant) using EGF-stimulated HeLa cells and find the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiles the global phosphoproteome. To demonstrate the robustness, sensitivity, and biomedical potential of hybrid-DIA, we profile chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluate the phospho-signaling difference of cancer cells in 2D vs 3D culture.

Trapped ion mobility spectrometry (TIMS) has been considered as a promising tool in structural biology, proteomics and many other analytical applications. This dataset consists of cell lysates digested with each of seven proteases (trypsin, LysargiNase, Lys-C, Lys-N, Glu-C, Asp-N, and chymotrypsin), fractionated using SCX-StageTip, and analyzed by nanoLC/TIMS/Q/TOF. The data accompanying this paper have been deposited to jPOST with the identifiers JPST000959, JPST001017 and JPST001176.

Cellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the level of elongating nascent polypeptide chains (NPCs) and associated co-translational events. Here, we describe pSNAP, a method for proteome-wide profiling of NPCs by affinity enrichment of puromycin- and stable isotope-labeled polypeptides. pSNAP does not require ribosome purification and/or chemical labeling, and captures bona fide NPCs that characteristically exhibit protein N-terminus-biased positions. We applied pSNAP to evaluate the effect of silmitasertib, a potential molecular therapy for cancer, and revealed acute translational repression through casein kinase II and mTOR pathways. We also characterized modifications on NPCs and demonstrated that the combination of different types of modifications, such as acetylation and phosphorylation in the N-terminal region of histone H1.5, can modulate interactions with ribosome-associated factors. Thus, pSNAP provides a framework for dissecting co-translational regulations on a proteome-wide scale.

Herein, we explore the hidden molecular recognition abilities of ZnO nanowires uniformly grown on the inner surface of an open tubular fused silica capillary via liquid chromatography. Chromatographic evaluation revealed that ZnO nanowires showed a stronger intermolecular interaction with phenylphosphoric acid than any other monosubstituted benzene. Furthermore, ZnO nanowires specifically recognized the phosphate groups present in nucleotides even in the aqueous mobile phase, and the intermolecular interaction increased with the number of phosphate groups. This discrimination of phosphate groups in nucleotides was unique to the rich (10[1 with combining macron]0) m-plane of ZnO nanowires with a moderate hydrophilicity and negative charge. The discrimination could be evidenced by the changes in the infrared bands of the phosphate groups on nucleotides on ZnO nanowires. Finally, as an application of the molecular recognition, nucleotides were separated by the number of phosphate groups, utilizing optimized gradient elution on ZnO nanowire column. Thus, the present results elucidate the unique and versatile molecular selectivity of well-known ZnO nanostructures for the capture and separation of biomolecules.

Identifying cellular phosphorylation pathways based on kinase-substrate relationships is a critical step to understanding the regulation of physiological functions in cells. Mass spectrometry-based phosphoproteomics workflows have made it possible to comprehensively collect information on individual phosphorylation sites in a variety of samples. However, there is still no generic approach to uncover phosphorylation networks based on kinase-substrate relationships in rare cell populations. Here, we describe a motif-centric phosphoproteomics approach combined with multiplexed isobaric labeling, in which in vitro kinase reactions are used to generate targeted phosphopeptides, which are spiked into one of the isobaric channels to increase detectability. Proof-of-concept experiments demonstrate selective and comprehensive quantification of targeted phosphopeptides by using multiple kinases for motif-centric channels. More than 7, 000 tyrosine phosphorylation sites were quantified from several tens of micrograms of starting materials. This approach enables the quantification of multiple phosphorylation pathways under physiological or pathological regulation in a motif-centric manner.