我們通過正常（pH 8）或老化（pH 12）形式的BSA自行氧化還原合成出螢光牛血清白蛋白（BSA）包覆金（Au）奈米團簇（BSA-AuNCs），其顯示分別是藍色和紅色螢光，並提出新的結構見解。
首先使用MALDI-TOF可以得到低質量區域（<5000 Da）的Au簇信號（間距197 Da的訊號峰值）以及完整的BSA-AuNCs訊號（老化形式:MW~71k Da~25 Au和正常形式:MW~68k Da ~8 Au），再透過ESCA以及螢光光譜進一步確認形成了BSA包覆的Au奈米團簇。接著利用質譜和蛋白質體學技術發現甲硫胺酸、組胺酸和半胱胺酸為BSA-AuNCs中的主要氧化胺基酸，表明這三種胺基酸是BSA中主要負責還原Au離子的重要角色。
BSA-AuNCs的金奈米團簇核心進一步通過酵素水解搭配10k Da分子篩分離進行結構的深入探討。我們發現通過TCEP打斷雙硫鍵時BSA-AuNCs_red螢光猝滅，表明其核心主要受這些二硫鍵的保護。在Cys.86，Cys.125，Cys.301，Cys.302，Cys.484，Cys.499這些位點的特異性氧化比例相當高，可以確定雙硫鍵在合成時斷裂 ; 相反，未氧化的半胱胺酸殘基：Cys 99，Cys 339，Cys 537則在BSA-AuNCs_red的核心部分中，數據表明伴隨蛋白質構型變化，誘導Au離子的氧化半胱胺酸造成雙硫鍵斷裂，打開BSA基板的空間使Au連接到完整的雙硫鍵片段並成核。與紅色AuNCs相比，藍色AuNCs具有較低程度的半胱胺酸氧化程度，且藍色螢光也未被TCEP猝滅，以上顯示金團簇核心主要不受半胱胺酸殘基的保護。我們的數據揭示了BSA-AuNCs在胜肽層級上的結構見解，這是以前從未報導過的，證實BSA的多個雙硫鍵在BSA-AuNCs_red的形成及穩定中起到重要作用。

In this paper, we reported new structural insights on the fluorescent bovine serum albumin (BSA)−gold (Au) nanoclusters (BSA-AuNCs) synthesized by optimized method. Using MALDI-TOF, Au cluster signals (peaks with 197 Da-spacing) in the low mass region (<5000 Da) as well as intact ion signals (M.W.~71k Da ~25 Au for the aged form and M.W.~68k Da ~8 Au for the normal form) were detected, suggesting the formation of BSA-capsulated Au nanoclusters. Fluorescence and ESCA results also provide evidence of formation.
Using mass spectrometry and proteomics techniques, methionine, histidine, and cysteine were identified to be main oxidized amino acid in BSA-AuNCs, suggesting these three amino acids were primarily responsible for the reduction of Au ion by BSA. By observing the oxidation state of Cys. site, we can find some disulfide bond fragments in the process of synthesis will break.
The ligand protected nanocluster cores were further isolated by enzyme digestion and 10 kDa cut-off filtering. Site-specific oxidization was considered to break the disulfide linkage well as on the free Cys58 of the red BSA-AuNCs were identified mainly from the non-core fraction. In contrast, multiple non-oxidized cysteine residues: Cys99, Cys339, Cys537 were enriched in the core fraction of BSA-AuNCs_red which fluorescence was quenched upon disulfide cleavage by TCEP, suggesting BSA-AuNCs_red were mainly protected by these disulfide linkages. The data indicated protein conformational changes accompanied with Au ion-induced cysteine oxidation and disulfide cleavages make room for Au attachment to disulfide group. Compared to BSA-AuNCs_red, BSA-AuNCs_blue have lesser degree of cysteine oxidation and the Au core was not mainly protected by cysteine residues. The blue fluorescence was not quenched by TCEP either.
Our data revealed structural insights of BSA-AuNCs at the residue level which has never been gained before. Multiple disulfide linkages of BSA were confirmed to play important roles in the red BSA-AuNCs formation and stabilization.

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