醣胺聚醣（glycosaminoglycans， GAGs）是一類帶負電荷的線性多醣，在細胞外基質結構維持、細胞訊息傳遞及疾病進程中扮演重要角色。在黏多醣貯積症（mucopolysaccharidoses， MPS）中，負責降解 GAGs 的溶酶體酵素缺乏或功能異常會導致特定 GAGs 累積於細胞與組織中，進而引發多系統性且逐漸惡化的臨床症狀，例如骨骼畸形、關節僵硬、器官腫大、心肺併發症，以及部分類型中的神經系統損傷。因此，GAGs 分析對於評估 GAGs 累積情形、輔助 MPS 診斷及監測疾病進展具有重要意義。目前基於質譜的 GAGs 分析方法通常結合酵素解聚或化學解聚，例如 methanolysis 或 butanolysis，並搭配液相層析串聯質譜（LC-MS/MS）進行分析。然而，這些方法通常需要較複雜的樣品前處理步驟，且可能造成原始 GAG 鏈結構相關資訊的流失。 為了解決上述限制，本研究旨在建立一種結合靜電作用奈米材料富集與質譜分析的 GAGs 分析策略，並評估降低對化學解聚步驟依賴及直接偵測完整 GAG 鏈的可行性。本研究首先以 heparin（HP）標準品作為模型 GAG，評估帶正電的聚精氨酸修飾奈米鑽石（polyarginine-coated nanodiamonds， PA-NDs）與聚乙烯亞胺磁性奈米粒子（poly(ethylenimine) magnetic nanoparticles， PEI-MNPs）的富集能力。經富集後，樣品先進行 acid-catalyzed butanolysis，再以 LC-MS/MS 分析，可觀察到 HP 衍生雙醣訊號，證實兩種奈米材料皆能捕捉 HP。進一步的吸附評估顯示，PEI-MNPs 具有良好的 HP 吸附表現；PA-NDs 亦可觀察到 HP 吸附，但由於重複實驗的變異較大，目前主要作為定性結果。整體結果支持兩種奈米材料應用於 GAG 富集的可行性。 為了進一步保留完整 GAG 鏈資訊，本研究測試不同洗脫條件，嘗試將完整 GAG 鏈從奈米粒子上釋放，並以 MALDI-TOF MS 進行直接分析。以 GAG 四糖標準品進行測試時，經 PEI-MNPs 富集後直接進行 MALDI-TOF MS 分析，可觀察到部分四糖相關訊號。進一步以高分子量 HP 測試時，當 PEI-MNPs 搭配 ammonium hydroxide/ammonium acetate 混合液作為洗脫條件時，可觀察到部分與 HP 標準品訊號重疊的訊號，顯示奈米粒子確實可提供分子富集之功能。然而，這些訊號與預期之高分子量HP分子組成有所差異，其來源尚待鑑定，猜測其結構可能受到鹽類加合物形成、反離子組成改變、樣品前處理條件或 MALDI 誘導片段化等因素影響。在多數其他洗脫條件下，未觀察到明顯的完整 GAG 相關訊號。儘管如此，將收集到的洗脫液進一步進行 acid-catalyzed butanolysis 並以 LC-MS/MS 分析後，仍可偵測到 HP 衍生雙醣訊號，表示部分 HP 相關成分可能已從奈米粒子上釋放，但未能以完整 GAG 鏈形式被 MALDI-TOF MS 有效偵測。 本研究進一步將奈米材料富集策略應用於細胞樣品。經 PEI-MNPs 或 PA-NDs 富集後，將樣品進行 acid-catalyzed butanolysis，再以 LC-MS/MS 進行分析，仍可觀察到細胞樣品中的 HP 衍生雙醣訊號。與未經富集的樣品相比，富集後樣品的化學背景干擾降低，其中 PA-NDs 在降低背景干擾方面表現出較 PEI-MNPs 更明顯的效果。由於此測試的定量分析正在進行優化中，基質效應對於內標的影響也將進一步評估，以提供未來臨床應用的重要基礎。此外，未來亦需進一步改善洗脫、回收、除鹽及高分子量 GAGs 的樣品前處理條件，以降低基質干擾、保存完整多醣鏈結構，並提升利用 MALDI-TOF MS 直接偵測高分子量 GAGs 的可行性。 整體而言，本研究顯示 PA-NDs 與 PEI-MNPs 皆具有捕捉 GAGs 的能力，並具應用於複雜生物樣品分析的潛力。其中，PEI-MNPs 具有良好的 HP 吸附表現，並在特定條件下展現直接分析完整 GAG 鏈的潛力。在細胞樣品中，兩種奈米材料富集後皆可降低化學背景干擾，其中 PA-NDs 的效果較為明顯。整體結果支持帶正電奈米材料應用於 GAG 富集與質譜分析的可行性，可作為未來開發快速 GAGs 分析方法及 MPS 相關生物標記研究之基礎。;Glycosaminoglycans (GAGs) are negatively charged linear polysaccharides that play important roles in extracellular matrix organization， cell signaling， and disease progression. In mucopolysaccharidoses (MPS)， deficiencies or dysfunctions of lysosomal enzymes responsible for GAG degradation lead to the accumulation of specific GAGs in cells and tissues， causing progressive multisystemic symptoms such as skeletal deformities， joint stiffness， organ enlargement， cardiac and respiratory complications， and， in some types， neurological impairment. Therefore， GAG analysis is important for evaluating GAG accumulation， supporting MPS diagnosis， and monitoring disease progression. Current mass spectrometry-based methods for GAG analysis often rely on LC-MS/MS combined with enzymatic or chemical depolymerization， such as methanolysis or butanolysis. However， these methods usually require relatively complex sample preparation and may result in the loss of information related to the original GAG chain structures. To address these limitations， this study aimed to establish a GAG analysis strategy combining electrostatic nanomaterial-based enrichment with mass spectrometric analysis， and to evaluate the feasibility of reducing reliance on chemical depolymerization and directly detecting intact GAG chains. Heparin (HP) standard was first used as a model GAG to evaluate the enrichment abilities of positively charged polyarginine-coated nanodiamonds (PA-NDs) and poly(ethylenimine) magnetic nanoparticles (PEI-MNPs). After enrichment and acid-catalyzed butanolysis， HP-derived disaccharide signals were detected by LC-MS/MS， confirming that both nanomaterials could capture HP. Further evaluation demonstrated effective HP adsorption by PEI-MNPs， while HP adsorption by PA-NDs was qualitatively confirmed， supporting their potential for GAG enrichment. To preserve intact GAG chain information， different elution conditions were further tested for direct MALDI-TOF MS analysis. Some tetrasaccharide-related signals were observed after PEI-MNP enrichment and direct analysis. For high-molecular-weight HP， several signals partially overlapping with the HP standard were observed when PEI-MNPs were used with an ammonium hydroxide/ammonium acetate mixture as the elution condition， suggesting that the nanoparticles may provided an enrichment effect. However， these signals differed from the expected molecular composition of high-molecular-weight HP， and their identities remain to be determined. They may be influenced by salt adduct formation， changes in counterion composition， sample preparation conditions， or MALDI-induced fragmentation. Direct MALDI-TOF MS detection of intact high-molecular-weight GAG chains therefore remained challenging under most tested conditions. The nanomaterial-based enrichment strategy was further applied to cell-derived samples. Following enrichment with PEI-MNPs or PA-NDs， the samples were subjected to acid-catalyzed butanolysis and LC-MS/MS analysis， and HP-derived disaccharide signals remained detectable. Both nanomaterials reduced chemical background interference compared with non-enriched samples， with PA-NDs showing a more pronounced reduction under the tested conditions. As the quantitative workflow is still being optimized， matrix effects on internal standard performance will be further evaluated to improve quantitative reliability and support future clinical applications. Further optimization of elution， recovery， desalting， and sample pretreatment for high-molecular-weight GAGs is also required to reduce matrix interference， preserve intact polysaccharide structures， and facilitate their direct detection by MALDI-TOF MS. Overall， both PA-NDs and PEI-MNPs demonstrated the ability to capture GAGs and showed potential for application to complex biological samples. PEI-MNPs exhibited effective HP adsorption and showed potential for direct mass spectrometric analysis of intact GAG chains under specific conditions. In cell-derived samples， enrichment with both nanomaterials reduced chemical background interference， with a more pronounced reduction observed for PA-NDs. Overall， these results support the feasibility of using positively charged nanomaterials for GAG enrichment and mass spectrometric analysis， providing a basis for the development of rapid GAG analysis methods and MPS-related biomarker research.
