Comparison of Different Mass Spectrometry Workflows for the Proteomic Analysis of Tear Fluid

The tear film is a multi-layer fluid that covers the corneal and conjunctival epithelia of the eye and provides lubrication, nutrients, and protection from the outside environment. Tear fluid contains a high concentration of proteins and has thus been recognized as a potential source of biomarkers for ocular disorders due to its proximity to disease sites on the ocular surface and the non-invasive nature of its collection. This is particularly true in the case of dry eye disease, which directly impacts the tear film and its components. Proteomic analysis of tear fluid is challenging mainly due to the wide dynamic range of proteins and the small sample volumes. However, recent advancements in mass spectrometry have revolutionized the field of proteomics enabling unprecedented depth, speed, and accuracy, even with small sample volumes. In this study using the Orbitrap Fusion Tribrid mass spectrometer, we compared four different mass spectrometry workflows for the proteomic analysis of tear fluid collected via Schirmer strips. We were able to establish a method of in-strip protein digestion that identified >3000 proteins in human tear samples from 11 healthy subjects. Our method offers a significant improvement in the number of proteins identified compared to previously reported methods without pooling samples.     

Prodynorphin and Proenkephalin in Cerebrospinal Fluid of Sporadic Creutzfeldt–Jakob Disease

Proenkephalin (PENK) and prodynorphin (PDYN) are endogenous opioid peptides mainly produced in the striatum and, to a lesser extent, in the cerebral cortex. Dysregulated metabolism and altered cerebrospinal fluid (CSF) levels of PENK and PDYN have been described in several neurodegenerative diseases. However, no study to date investigated these peptides in the CSF of sporadic Creutzfeldt–Jakob disease (sCJD). Using liquid chromatography-multiple reaction monitoring mass spectrometry, we evaluated the CSF PDYN- and PENK-derived peptide levels in 25 controls and 63 patients with sCJD belonging to the most prevalent molecular subtypes (MM(V)1, VV2 and MV2K). One of the PENK-derived peptides was significantly decreased in each sCJD subtype compared to the controls without a difference among subtypes. Conversely, PDYN-derived peptides were selectively decreased in the CSF of sCJD MV2K, a subtype with a more widespread overall pathology compared to the sCJD MM(V)1 and the VV2 subtypes, which we confirmed by semiquantitative analysis of cortical and striatal neuronal loss and astrocytosis. In sCJD CSF PENK and PDYN were associated with CSF biomarkers of neurodegeneration but not with clinical variables and showed a poor diagnostic performance. CSF PDYN and PENK-derived peptides had no significant diagnostic and prognostic values in sCJD; however, the distinct marker levels between molecular subtypes might help to better understand the basis of phenotypic heterogeneity determined by divergent neuronal targeting.     

光谱法研究甲啶铂与牛血清白蛋白的相互作用

甲啶铂(picoplatin)是一种新型的抗癌药物,但其在体内的作用机制尚不明确.利用荧光光谱技术研究模拟人生理条件(Tris-HCl缓冲溶液体系,pH7.4)下甲啶铂与牛血清白蛋白(BSA)的相互作用,甲啶铂对BSA的内源荧光(336 nm)有猝灭作用.在室温296 K和模拟人体温度310 K条件下,甲啶铂对BSA的...     

基于质谱的蛋白质绝对定量研究策略和建议

科学家已经研究获得了大量的蛋白质/多肽潜在生物标志物,这些生物标志物需要经过验证和确证才能进一步转化到临床应用.针对蛋白质/多肽的绝对定量研究在标志物验证和确证过程中起到关键作用.传统的蛋白质定量方法,如酶联免疫吸附试验(ELISA)技术存在蛋白质抗体难以获得、不同抗体批次之间存在差异、基于抗体的检测存在交叉反应等问题...     

前列腺特异性抗体电化学免疫传感器的研制

利用自组装的方法在金电极上制得巯基丁二胺铜(Ⅱ)/纳米金胶/前列腺特异性抗体(抗PSA)免疫修饰电极。用该修饰电极对PSA进行检测,发现其循环伏安图的氧化还原峰电流都随PSA浓度的增高而降低,峰电位没有变化。其最佳实验条件包括:pH5.2的0.1mol/L磷酸盐缓冲液作为底液,以及用示差脉冲方式进行定量测定。结果显示:该传感器的氧化峰电流减少值与PSA浓度在0.005-0.48μg/mL范围内成线性关系,检测下限为2ng/mL.在40 ng/mLPSA浓度下八次测量相对标准偏差为2.9%,该免疫传感器的稳定性和抗干扰性都较好。对血清中的PSA进行检测,获得满意的结果。     

Ficolin-2 Plasma Level Assesses Liver Fibrosis in Non-Alcoholic Fatty Liver Disease

Fibrosis is the strongest predictor for disease-specific mortality in non-alcoholic fatty liver diseases (NAFLD), but the need for liver biopsy limits its diagnosis. We assessed the performance of plasma ficolin-2 (FCN-2) as a biomarker of fibrosis identified by an in silico discovery strategy. Two hundred and thirty-five morbidly obese (MO) subjects with biopsy-proven NAFLD stratified by fibrosis stage (F0, n = 44; F1, n = 134; F2, n = 46; F3/F4, n = 11) and 40 cirrhotic patients were enrolled. The cohort was subdivided into discovery (n = 76) and validation groups (n = 159). The plasma level of FCN-2 and other candidate markers was determined. FCN-2 was inversely correlated with the stage of liver fibrosis (ρ = −0.49, p < 0.001) independently of steatosis (p = 0.90), inflammation (p = 0.57), and ballooning (p = 0.59). In the global cohort, FCN-2 level decreased significantly in a stepwise fashion from F0/F1 (median 4753 ng/mL) to F2–F3–F4 (2760 ng/mL) and in cirrhotic subjects (1418 ng/mL). The diagnostic performance of FCN-2 in detecting F ≥ 2 was higher than other indexes (APRI, FIB-4) (AUROC 0.82, 0.68, and 0.6, respectively). The accuracy improved when combined with APRI score and HDL values (FCNscore, AUROC 0.85). Overall, the FCN-2 plasma level can accurately discriminate liver fibrosis status (minimal vs. moderate/advanced) significantly improving the fibrosis diagnostic algorithms.     

超高效液相色谱-高分辨质谱测定大鼠血清中神经递质及其在电离辐射脑损伤动物模型中的应用

建立了超高效液相色谱四极杆静电场轨道阱高分辨质谱(UPLC-Q-Orbitrap HRMS)法快速分析大鼠血清中10种神经递质.以0.1％甲酸-乙腈为沉淀剂,采用蛋白沉淀法进行血清中蛋白质大分子的沉淀和目标分析物的高效萃取.建立目标分析物的精确质量数据库(质荷比、保留时间、同位素峰分布及特征碎片离子)和二级质谱库进行精...     

The Signaling Pathway of TNF Receptors: Linking Animal Models of Renal Disease to Human CKD

Chronic kidney disease (CKD) has been recognized as a global public health problem. Despite the current advances in medicine, CKD-associated morbidity and mortality remain unacceptably high. Several studies have highlighted the contribution of inflammation and inflammatory mediators to the development and/or progression of CKD, such as tumor necrosis factor (TNF)-related biomarkers. The inflammation pathway driven by TNF-α, through TNF receptors 1 (TNFR1) and 2 (TNFR2), involves important mediators in the pathogenesis of CKD. Circulating levels of TNFRs were associated with changes in other biomarkers of kidney function and injury, and were described as predictors of disease progression, cardiovascular morbidity, and mortality in several cohorts of patients. Experimental studies describe the possible downstream signaling pathways induced upon TNFR activation and the resulting biological responses. This review will focus on the available data on TNFR1 and TNFR2, and illustrates their contributions to the pathophysiology of kidney diseases, their cellular and molecular roles, as well as their potential as CKD biomarkers. The emerging evidence shows that TNF receptors could act as biomarkers of renal damage and as mediators of the disease. Furthermore, it has been suggested that these biomarkers could significantly improve the discrimination of clinical CKD prognostic models.     

Pt(Ⅱ)与甲胎蛋白的相互作用机理研究及其分析应用

采用电化学和荧光方法研究了Pt(Ⅱ) 水合物与甲胎蛋白(AFP)相互作用的机理,发现它们具有较强的相互结合能力.文中对Pt(Ⅱ)- AFP体系的适宜反应条件、影响因素及信号强度与AFP浓度的关系进行了研究.结果表明,在一定Pt(Ⅱ)浓度范围内,AFP浓度与其荧光峰电流强度成正比.本方法有较高的灵敏度,对AFP的检出限为10.2ng/mL.考察了共存物质的干扰影响,并对实际血清样品中的AFP进行分析,结果满意.     

The relationship between laboratory-based outcome measures and mortality in end-stage renal disease: A systematic review

Despite data that traditional laboratory-based outcome measures in dialysis are improving over time, population-based data indicate that mortality rates are not improving in parallel. With increased focus on performance measures based on laboratory-based outcomes (e.g., hematocrit, albumin, and parathyroid hormone), less emphasis has been placed on other markers, some of which may be stronger predictors of mortality. We performed a systematic review to interpret the predictive value of laboratory-based outcome measures in dialysis. We identified studies with data regarding the predictive value of laboratory-based outcomes for mortality in dialysis. We calculated the sample size-weighted pooled relative risk of death with dichotomized "high" vs. "low" levels of each measure. We rank-ordered predictors by scaling the pooled relative risk of each measure by its pooled standard deviation. There were 5171 titles, of which 128 (representing 44 laboratory-based outcomes) were selected. Nine were significantly associated with mortality, in order of decreasing scaled effect size: (1) tumor necrosis factor-α, (2) hematocrit, (3) interleukin-6, (4) troponin T, (5) Kt/V_urea, (6) prealbumin, (7) urea reduction ratio, (8) serum albumin, and (9) C-reactive protein. Other oft-cited measures such as calcium phosphate product and parathyroid hormone were not significantly associated with mortality in pooled analysis. Quality improvement efforts to improve traditional laboratory-based outcomes in end-stage renal disease are necessary, but likely insufficient, to improve overall mortality in dialysis. Renewed consideration of cardiovascular, inflammatory, and nutritional markers that are especially strong predictors of mortality may have important implications for risk stratification and targeted therapeutic interventions.