Hookeun Lee

Ensuring the accuracy and reliability of liquid chromatography–mass spectrometry (LC–MS/MS)-based proteomic analysis requires robust quality control (QC) strategies. However, widely used cell line-based QC materials present challenges in reproducibility, scalability, and ethical compliance. In this study, we developed a microalgae-derived QC material based on Synechocystis sp. PCC6803 and evaluated its suitability for LC–MS/MS system performance monitoring. The Synechocystis-based QC samples demonstrated high identification reproducibility, with 76% of the protein groups being consistently detected across five replicates and maintaining a median coefficient of variation (CV) of 6.7% in quantitative precision. Furthermore, QC peptides exhibited excellent linearity (R² ≥ 0.98) and reproducibility (CV ≤ 12.06%) across varying injection amounts. These results indicate that Synechocystis-derived QC materials provide a reproducible and scalable approach that complements conventional QC workflows. They are particularly well suited for routine LC–MS/MS system performance monitoring in proteomics workflows involving non-human or mixed biological samples.

The pathogenesis of primary acquired nasolacrimal duct obstruction (PANDO) remains unclear, with several factors implicated, including anatomical structures, hormones, and tear components. This study explored tear proteins to better understand PANDO etiology by comparing protein expression in tears from patients with PANDO and healthy controls. Tear samples were collected from 22 patients with PANDO (mucous and membranous types) and 8 controls using Weck-Cel sponges. Protein analysis was conducted using LC-MS/MS to identify and quantify tear proteins. Female patients with PANDO had higher numbers of differentially expressed proteins (DEPs) compared with males. Certain DEPs associated with inflammatory pathways or the lacrimal duct epithelium, including SERPINB1, SERPINA3, CTSG, SLPI, and EZR, were identified in male patients. Although this is a preliminary study, our results offer insights into the pathogenesis of PANDO, with potential to distinguish between mucous and membranous subtypes. The potential biomarkers identified in this study could enhance early diagnosis and treatment, shedding light on inflammatory and immune processes in PANDO.

A novel approach was developed for the first time by combining ultrasmall Au₂₂ nanoclusters with boronic acid (BA) affinity chemistry, achieving highly efficient enrichment of early-stage glycated peptides.

Axonal degeneration resulting from optic nerve damage can lead to the progressive death of retinal ganglion cells (RGCs), culminating in irreversible vision loss. We contrasted two methods for inducing optic nerve damage: optic nerve compression (ONCo) and optic nerve crush (ONCr). These were assessed for their respective merits in simulating traumatic optic neuropathies and neurodegeneration. We also administered neural progenitor cells (NPCs) into the subtenon space to validate their potential in mitigating optic nerve damage. Our findings indicate that both ONCo and ONCr successfully induced optic nerve damage, as shown by increased ischemia and expression of genes linked to neuronal regeneration. Post NPCs injection, recovery in the expression of neuronal regeneration-related genes was more pronounced in the ONCo model than in the ONCr model, while inflammation-related gene expression saw a better recovery in ONCr. In addition, proteomic analysis of R28 cells in hypoxic conditions identified Vps35 and Syntaxin12 genes. Vps35 preserved mitochondrial function in ONCo, while Syntaxin12 appeared to restrain inflammation via the Wnt/β-catenin signaling pathway in ONCr. NPCs managed to restore damaged RGCs by elevating neuroprotection factors and controlling inflammation through mitochondrial homeostasis and Wnt/β-catenin signaling in hypoxia-injured R28 cells and in both animal models. Our results suggest that ischemic injury and crush injury cause optic nerve damage via different mechanisms, which can be effectively simulated using ONCo and ONCr, respectively. Moreover, cell-based therapies such as NPCs may offer promising avenues for treating various optic neuropathies, including ischemic and crush injuries.

Liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based proteomics is a powerful technique for profiling proteomes of cells, tissues, and body fluids. Typical bottom-up proteomic workflows consist of the following three major steps: sample preparation, LC–MS/MS analysis, and data analysis. LC–MS/MS and data analysis techniques have been intensively developed, whereas sample preparation, a laborious process, remains a difficult task and the main challenge in different applications. Sample preparation is a crucial stage that affects the overall efficiency of a proteomic study; however, it is prone to errors and has low reproducibility and throughput. In-solution digestion and filter-aided sample preparation are the typical and widely used methods. In the past decade, novel methods to improve and facilitate the entire sample preparation process or integrate sample preparation and fractionation have been reported to reduce time, increase throughput, and improve reproducibility. In this review, we have outlined the current methods used for sample preparation in proteomics, including on-membrane digestion, bead-based digestion, immobilized enzymatic digestion, and suspension trapping. Additionally, we have summarized and discussed current devices and methods for integrating different steps of sample preparation and peptide fractionation.

Pacific abalone (Haliotis discus hannai) is a commercially important mollusk; therefore, improvement of its growth performance and quality has been emphasized. During embryonic development, abalones undergo a series of distinct larval stages, including swimming veliger larvae, juveniles, and mature individuals, and their biomolecular composition varies depending on the developmental stage. Therefore, in the present study, we performed untargeted lipid profiling of abalone tissues at different developmental stages as well as the hemolymph of mature female and male abalones using ultrahigh‐performance liquid chromatography‐tandem mass spectrometry. These profiles can provide meaningful information to understand compositional changes in lipids through abalone metamorphosis and development. A total of 132 lipids belonging to 15 classes were identified from abalone tissues at different developmental stages. Moreover, 21 lipids belonging to 8 classes were identified from the hemolymph of mature abalones. All data were processed following strict criteria to provide accurate information. Triglycerides and phosphatidylcholines were the major lipid components identified in both tissues and hemolymph, accounting for, respectively, 27% and 15% of all lipids in tissues and, respectively, 24% and 38% of all lipids in the hemolymph. Of note, lysophosphatidylcholine was only detected in the tissues of mature abalones, paving the way for further analyses of abalone lipids based on developmental stages. The present findings offer novel insights into the lipidome of abalone tissues and hemolymph at different developmental stages, building a foundation for improving the efficiency and quality of abalone aquaculture.

The standard bottom‐up proteomic workflow is comprised of sample preparation, data acquisition, and data analysis. While the latter two parts have made considerable advances in the last decade, sample preparation has remained an important challenge within the workflow due to the multi‐step nature of complex biological samples, and still requires much development. Several sample preparation methods have been developed and used in the last two decades, including in‐gel, in‐solution, on‐bead, filter‐aided sample preparation, and suspension trapping, to improve reproducibility, efficiency, scalability, and reduce the handling time of this process. One of the most recent methods developed and applied in proteomics studies in recent years is suspension trapping, which combines rapid detergent removal, reactor‐type protein digestion, and peptide clean‐up in a tip or spin column. Suspension trapping is a simple, rapid, and reproducible digestion method that can effectively handle proteins in low microgram or sub‐microgram amounts. This review discusses the benefits of the suspension trapping digestion method in relation to its development and application in bottom‐up proteomics studies. We also discuss recent applications of suspension trapping digestion to different sample types and the features of the suspension trapping digestion method compared with other sample preparation methods.

Glycation is a spontaneous post-translational modification of lysine, arginine, and the N-terminus of proteins. Protein glycation is closely related to the pathogenesis of human diseases, including diabetes, Alzheimer’s disease, renal disease, and cancer. The levels of advanced glycation end products (AGEs) are positively correlated with the progression of many diseases. However, it remains challenging to analyze glycation-related products, such as reactive carbonyl species, Schiff bases, Amadori compounds, and AGEs, because of their high heterogeneity. Many analysis methods, such as fluorescence detection, immunoassays, and liquid chromatography-tandem mass spectrometry, have attempted to correlate glycation products with diseases. Some enrichment methods have been used to increase the probability of detection of glycated proteins due to their low abundance in blood plasma. This review summarizes the enrichment and analysis methods that are currently used to identify glycation as a disease biomarker in exploratory studies.