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

TurboID-EV: Proteomic Mapping of Recipient Cellular Proteins Proximal to Small Extracellular Vesicles

細胞外小胞の軌跡を照らす --細胞外小胞の標的細胞への取り込み機構の解明に貢献--. 京都大学プレスリリース. 2023-09-15.Extracellular vesicles (EVs), including exosomes, have been recognized as key mediators of intercellular communications through donor EV and recipient cell interaction. Until now, most studies have focused on the development of analytical tools to separate EVs and their applications for the molecular profiling of EV cargo. However, we lack a complete picture of the mechanism of EV uptake by the recipient cells. Here, we developed the TurboID-EV system with the engineered biotin ligase TurboID, tethered to the EV membrane, which allowed us to track the footprints of EVs during and after EV uptake by the proximity-dependent biotinylation of recipient cellular proteins. To analyze biotinylated recipient proteins from low amounts of input cells (corresponding to ∼10 μg of proteins), we developed an integrated proteomic workflow that combined stable isotope labeling with amino acids in cultured cells (SILAC), fluorescence-activated cell sorting, spintip-based streptavidin affinity purification, and mass spectrometry. Using this method, we successfully identified 456 biotinylated recipient proteins, including not only well-known proteins involved in endocytosis and macropinocytosis but also other membrane-associated proteins such as desmoplakin and junction plakoglobin. The TurboID-EV system should be readily applicable to various EV subtypes and recipient cell types, providing a promising tool to dissect the specificity of EV uptake mechanisms on a proteome-wide scale

Bioinertization of NanoLC/MS/MS Systems by Depleting Metal Ions From the Mobile Phases for Phosphoproteomics

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