兒茶酚雌激素 (Catechol estrogens, CEs) 是具有遺傳毒性的代謝產物，因其在血液中低濃度和高變化性而檢測上具有挑戰性。透過對添加CEs的溶血產物進行完整蛋白測定，可以發現內源性CEs主要 (> 99％) 以血紅蛋白 (Hemoglobin, Hb) 加成物形式存在於紅血球中。為了檢測內源性CE-Hb加成物，我們開發了一種兩步法，其中涉及以蛋白質沉澱和固相萃取從紅血球中純化Hb，然後將該方法與蛋白質體學結合，以液相層析串聯質譜法分析之。從自下而上蛋白質體學 (Bottom-up proteomics) 和標準添加法(Standard additions) 得到的結果中我們確定Hb-"β" 上的C93和C112是Hb被CEs修飾的主要位點。透過平行反應監測 (Parallel reaction monitoring, PRM) 靶向測定相同Hb-"β" 序列的非加成物胰蛋白酶肽、加成物胰蛋白酶肽和氧化胰蛋白酶肽，以確定紅血球中的CE修飾程度。我們達到了低於內源性CE-Hb修飾程度的定量極限 (S/N >8) ，相對標準誤範圍為5-22％，並將其應用於臨床樣本上。結果指出乳癌患者的內源性CE-Hb 修飾程度高於健康對照組，並且它們之間存在顯著差異（p=3.44 x10-6），表明預測乳腺癌風險的準確率接近 100%。本項研究報導了在蛋白質體學基礎上開發一種測定血紅蛋白加成物的有效樣品製備方法。
然而在測定 CE-Hb 修飾程度的整個過程，包括建立校正曲線和酵素消化，都非常耗時。在微滴中，許多反應的速率可以被極大地加速。我們研究並驗證利用微滴加速 CEs 加成反應和胰蛋白酶消化反應，其中Hb-? 和 Hb-β 的序列覆蓋率達到 100%，從而節省了大量建立校正曲線和酵素消化的時間。此外，我們也驗證了有機溶劑對微滴酵素消化的影響。其中ACN 的存在增強了肽的信號，而沒有顯著降低酵素消化的效率，表明微滴消化與液相層析結合的高可行性。更重要的是，當 Hb 加入高濃度的 CEs 時，在微滴質譜條件下觀察到具有 CEs 修飾的肽的訊號。然而如果添加低劑量的 CEs ，則由於離子抑制效應而無法檢測到CEs 修飾的肽的訊號。

Estrogen exposure is known as one of the main risk factors for development of breast cancer.Catechol estrogens (CEs), the genotoxic metabolites, are challenging targets to be measured due to the low abundance and high variability in blood. Since CEs were reported to conjugate with blood protein (Anal. Chem. 2019, 91, 15922−1593), it is of great importance to study the level of CEs in red blood cells (RBCs, also known as erythrocytes), the most abundant type of blood cell in blood. Hemoglobin (Hb), a major protein in RBCs, was chosen as the target protein to study the level of CE in circulating blood, as it can reflect estrogen exposure
level in a long time. A two-step method coupled with proteomics using liquid
chromatography-tandem mass spectrometry was developed to purify Hb from RBCs and to determine the CEs level in blood. The CE-Hb adduct was demonstrated to increase with the added CEs using standard additions and intact protein measurement. Moreover, two cysteine residues, namely C93-Hb-β and C112-Hb-β, were identified as the main adduction site of Hb using bottom-up proteomics and standard additions. To determine the adduction level in RBCs, the cysteine-containing peptides, including those with and without CE modification
and oxidation, were targeted by parallel reaction monitoring. The calibration curves with added CE in low and high concentration ranges were established. A quantitation limit (S/N > 8) was achieved below the adduction level of endogenous CE-Hb with a relative standard error ranging from 5% to 22% and applied to clinical samples. The CE-Hb adduction levels of patients were observed to be higher than that of healthy controls, and a significant difference between them (p=3.44 x10-6
) was presented, indicating a near 100% accuracy in predicting the risk of breast cancer. Our study reported an efficient sample preparation method for proteomics-based Hb adducts.
However, the whole process to determine CE-Hb adduction level, including establishment of calibration curves and enzymatic digestion, is time-consuming. In microdroplets, rates of many reactions can be extremely accelerated. We investigated accelerated CEs adduction reaction and trypsin digestion in microdroplets. The ultrafast digestion of Hb and hemolysates in microdroplets was demonstrated, with the sequence coverage of 100% for both Hb-? and Hb-β. Furthermore, the impact of ACN on microdroplet digestion was verified. The presence of ACN enhanced the peptides’ signals without significantly decreasing the efficiency of digestion, indicating the high feasibility of coupling the microdroplet digestion with HPLC. CEs adduction reaction was shown to be accelerated in microdroplets, saving a lot of time on establishing the calibration curves. More importantly, the peptides with CEs modification were observed under microdroplet-MS conditions when Hb was spiked with high concentration of CE. However, the peptides of interest were not detected due to ion suppression if the amount of added CEs was low.

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