OryzaPG-DB: Rice Proteome Database based on Shotgun Proteogenomics

<p>Abstract</p> <p>Background</p> <p>Proteogenomics aims to utilize experimental proteome information for refinement of genome annotation. Since mass spectrometry-based shotgun proteomics approaches provide large-scale peptide sequencing data with high throughput, a data repository for shotgun proteogenomics would represent a valuable source of gene expression evidence at the translational level for genome re-annotation.</p> <p>Description</p> <p>Here, we present OryzaPG-DB, a rice proteome database based on shotgun proteogenomics, which incorporates the genomic features of experimental shotgun proteomics data. This version of the database was created from the results of 27 nanoLC-MS/MS runs on a hybrid ion trap-orbitrap mass spectrometer, which offers high accuracy for analyzing tryptic digests from undifferentiated cultured rice cells. Peptides were identified by searching the product ion spectra against the protein, cDNA, transcript and genome databases from Michigan State University, and were mapped to the rice genome. Approximately 3200 genes were covered by these peptides and 40 of them contained novel genomic features. Users can search, download or navigate the database per chromosome, gene, protein, cDNA or transcript and download the updated annotations in standard GFF3 format, with visualization in PNG format. In addition, the database scheme of OryzaPG was designed to be generic and can be reused to host similar proteogenomic information for other species. OryzaPG is the first proteogenomics-based database of the rice proteome, providing peptide-based expression profiles, together with the corresponding genomic origin, including the annotation of novelty for each peptide.</p> <p>Conclusions</p> <p>The OryzaPG database was constructed and is freely available at <url>http://oryzapg.iab.keio.ac.jp/</url>.</p

Validity of the cell‐extracted proteome as a substrate pool for exploring phosphorylation motifs of kinases

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

Peptide Retention in Hydrophilic Strong Anion Exchange Chromatography Is Driven by Charged and Aromatic Residues

Hydrophilic strong anion exchange chromatography (hSAX) is becoming a popular method for the prefractionation of proteomic samples. However, the use and further development of this approach is affected by the limited understanding of its retention mechanism and the absence of elution time prediction. Using a set of 59 297 confidentially identified peptides, we performed an explorative analysis and built a predictive deep learning model. As expected, charged residues are the major contributors to the retention time through electrostatic interactions. Aspartic acid and glutamic acid have a strong retaining effect and lysine and arginine have a strong repulsion effect. In addition, we also find the involvement of aromatic amino acids. This suggests a substantial contribution of cation−π interactions to the retention mechanism. The deep learning approach was validated using 5-fold cross-validation (CV) yielding a mean prediction accuracy of 70% during CV and 68% on a hold-out validation set. The results of this study emphasize that not only electrostatic interactions but rather diverse types of interactions must be integrated to build a reliable hSAX retention time predictor

Exploring the landscape of ectodomain shedding by quantitative protein terminomics

膜タンパク質が「はさみ分子」によって切断される部位を大規模に解明 --細胞間コミュニケーションの制御機構解明に向けて--. 京都大学プレスリリース. 2021-03-30.Ectodomain shedding is a proteolytic process that regulates the levels and functions of membrane proteins. Dysregulated shedding is linked to severe diseases, including cancer and Alzheimer's disease. However, the exact cleavage sites of shedding substrates remain largely unknown. Here, we explore the landscape of ectodomain shedding by generating large-scale, cell-type-specific maps of shedding cleavage sites. By means of N- and C-terminal peptide enrichment and quantitative mass spectrometry, we quantified protein termini in the culture media of 10 human cell lines and identified 489 cleavage sites on 163 membrane proteins whose proteolytic terminal fragments are downregulated in the presence of a broad-spectrum metalloprotease inhibitor. A major fraction of the presented cleavage sites was identified in a cell-type-specific manner and mapped onto receptors, cell adhesion molecules, and protein kinases and phosphatases. We confidently identified 86 cleavage sites as metalloprotease substrates by means of knowledge-based scoring

Immunoreactivity profiling of Anti-Chinese hamster ovarian host cell protein antibodies by isobaric labeled affinity purification-mass spectrometry reveals low-recovery proteins

We evaluated the immunoreactivity profiles of eight commercial anti-host cell protein (anti-HCP) antibodies from different host animals and their antigens used for immunization by an isobaric labeled affinity purification-mass spectrometry (AP-MS) method. As a result, 34 proteins with high abundance but low recovery from harvest cell culture fluid were identified. Since they are likely to be underestimated in biopharmaceutical quality assessment, the features common to these proteins were investigated. Compared to other immunoprecipitated HCP proteins, proteins exhibiting lower molecular weight (ΔMW = -14600), lower isoelectric point (ΔpI = -0.86), and lower hydrophobicity (ΔGRAVY = -0.13) were enriched. This AP-MS method provides important information for HCP control strategies using immunological methods and is expected to contribute to the development of safe biopharmaceutics

Motif-centric phosphoproteomics to target kinase-mediated signaling pathways

細胞内リン酸化修飾の大規模計測に成功 --極微量試料からのリン酸化経路解析も可能に--. 京都大学プレスリリース. 2022-02-01.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