Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples

Serum proteins may often serve as indicators of disease and is a rich source for biomarker discovery. However, the large dynamic range of proteins in serum makes the analysis very challenging because high-abundant proteins tend to mask those of lower abundance. A prefractionation step, such as depletion of a few high-abundant proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may prove to be informative biomarkers. In the present study, five different depletion columns were investigated considering efficiency, specificity, and reproducibility. Our research included quantitative determination of total protein, albumin, and immunoglobulin G (IgG) concentrations, one- and two-dimensional gels and mass spectrometric analysis of the serum samples before and after the depletion step. Our results showed that all five depletion columns tested removed albumin and IgG with high efficiency. We found that based on reproducibility and binding specificity, the Multiple Affinity Removal Column that removed a total of six high-abundant proteins (albumin, IgG, antitrypsin, IgA, transferring, and haptoglobin) offered the most promising depletion approach. Among the disposable (single-use) products, the ProteoExtract Albumin/IgG Removal kit displayed the best results. Depleted serum from the Multiple Affinity Removal column was further evaluated by 2-D gel electrophoresis (2-DE) analysis, and the results indicated increased resolution and improved intensity of low-abundant proteins in a reproducible fashion. Our study provides a comprehensive investigation of commercially available depletion columns and will be of high importance for future proteomic studies on serum samples.     

血清长链非编码RNA MALAT-1 在卵巢癌诊断中的临床意义

目的:探讨血清长链非编码RNA MALAT-1检测在卵巢癌患者中的诊断作用。方法:收集187例在我院行卵巢肿瘤切除术患者术前血清。应用实时定量PCR检测血清中MALAT-1 m RNA的表达。MALAT-1 m RNA的拷贝数使用绝对定量(标准曲线)计算。MALAT-1的诊断价值采用ROC-AUC曲线和logistic回归分析并与血清CA125进行比较。结果:1本研究有效样本比例为88.77%(166/187)。卵巢良性肿瘤和恶性肿瘤比例分别为62.05%(103/166)和37.95%(63/166)。卵巢恶性肿瘤患者MALAT-1明显高于良性肿瘤患者(p0.001)。经ROC曲线分析,血清CA125和MALAT-1曲线下面积分别为0.726和0.844,且血清MALAT-1的诊断价值明显优于血清CA125(p=0.023)。经logistic回归分析,应用血清MALAT-1和CA125的预测准确性分别为78.92%和68.07%,应用血清MALAT-1可提高预测准确性10.85%。进一步的分析得出,取血清MALAT-1截断点为36.5时,其预测的敏感性和特异性分别高达84.1%和67.0%。结论:血清长链非编码RNA MALAT-1检测对卵巢肿瘤的性质具有良好的预测作用,可作为卵巢癌早期诊断的分子标志物。本研究结果需大样本前瞻性的研究进一步验证。     

The role of copper in drug-resistant murine and human tumors

Multidrug resistance (MDR) is still a major threat to successful clinical application of cancer chemotherapy. Copper plays an important role in biological systems, and copper is also involved in carcinogenesis. In the present investigation, we addressed the question whether metal copper might be involved in drug resistance of murine and human tumors. By means of atomic absorption spectroscopy, we determined serum copper concentrations. We found that the blood serum of tumor-bearing mice contained higher amounts of copper than healthy mice with tumors. Secondly, mice bearing doxorubicin-resistant Ehrlich ascites carcinoma- or cyclophosphamide-resistant Lewis lung carcinoma contained more copper in their serum than mice bearing the corresponding drug-sensitive parental tumors. Furthermore, the analysis of patients with breast cancer, colon carcinoma or lung cancer showed that the serum copper contents were higher in patients not responding to chemotherapy when compared to patients whose tumors responded to treatment. The copper levels in serum of healthy volunteers were lower than in cancer patients irrespective of their response to chemotherapy. Our results imply that the level of serum copper may be considered as a biomarker for treatment response.     

肺癌患者血清TGF-β1浓度检测及临床意义分析

目的 探讨检测肺癌患者血清中转化生长因子β1( transforming growth factorβtype1,TGF-β1)的临床应用价值.方法 收集98例肺癌患者血清标本及40例健康对照者血清标本,运用酶联免疫吸附试验(ELISA)检测两组标本血清TGF-β1浓度.分析二者之间的差异及其与肺癌患者临床特征之间的关系.结果 肺癌患者的血清TGF-β1浓度明显高于健康对照者,差异有统计学意义(66848 pg/mL±37178 pg/mL vs 48790 pg/mL±23111 pg/mL,P＜0.01);肺癌患者血清TGF-β1浓度与TNM分期,淋巴结转移,病理分型无相关性(P＞0.05);手术前后,化疗前后血清TGF-β1浓度差异无统计学意义(P＞0.05).结论 血清TGF-β1对肺癌的辅助诊断有一定的临床价值.     

Assessment of macrophage migration inhibitory factor in humans: protocol for accurate and reproducible levels

The analytical validation of a possible biomarker is the first step in the long translational process from basic science to clinical routine. Although the chemokine-like cytokine macrophage migration inhibitory factor (MIF) has been investigated intensively in experimental approaches to various disease conditions, its transition into clinical research is just at the very beginning. Because of its presence in preformed storage pools, MIF is the first cytokine to be released under various stimulation conditions. In the first proof-of-concept studies, MIF levels correlated with the severity and outcome of various disease states. In a recent small study with acute coronary syndrome patients, elevation of MIF was described as a new factor for risk assessment. When these studies are compared, not only MIF levels in diseased patients differ, but also MIF levels in healthy control groups are inconsistent. Blood MIF concentrations in control groups vary between 0.56 and 95.6 ng/ml, corresponding to a 170-fold difference. MIF concentrations in blood were analyzed by ELISA. Other than the influence of this approach due to method-based variations, the impact of preanalytical processing on MIF concentrations is unclear and has not been systematically studied yet. Before large randomized studies are performed to determine the impact of circulating MIF on prognosis and outcome and before MIF is characterized as a diagnostic marker, an accurate protocol for the determination of reproducible MIF levels needs to be validated. In this study, the measurement of MIF in the blood of healthy volunteers was investigated focusing on the potential influence of critical preanalytical factors such as anticoagulants, storage conditions, freeze/thaw stability, hemolysis, and dilution. We show how to avoid pitfalls in the measurement of MIF and that MIF concentrations are highly susceptible to preanalytical factors. MIF serum concentrations are higher than plasma concentrations and show broader ranges. MIF concentrations are higher in samples processed with latency than in those processed directly and strongly correlate with hemoglobin in plasma. Neither storage temperature nor storage length or dilution or repeated freezing and thawing influenced MIF concentrations in plasma. Preanalytical validation of MIF is essential. In summary, we suggest using plasma and not serum samples when determining circulating MIF and avoiding hemolysis by processing samples immediately after blood drawing.     

Opsonic Activity of Sera and Blister Fluid from Severely Burned Patients Evaluated by a Chemiluminescence Method

Burn wound sepsis is the most common and severe complication in the patients with severe burn. To know the systemic and local defect in immunity of burned patients, we measured the luminol-enhanced chemiluminescence (CL) response of normal polymorphonuclear leukocytes (PMNs) upon exposure to zymosan particles, bacteria or Candida albicans that were opsonized with any of patient's serum, blister fluid of burn wound or pooled normal serum (blood type AB). Sera from patients exhibited lower opsonic activities than those of pooled normal serum in the early postburn days. The levels of serum immunoglobulins, complement components and plasma fibronectin were found to correlate well with opsonin-index (OI), which was determined based on the CL response data obtained during the course of infusion therapy with fresh frozen plasma. Furthermore, patient's blister fluid showed much lower opsonic activity against bacteria such as Pseudomonas aeruginosa than patient's own serum. These results indicate that blister fluid is also not effective to opsonize bacteria because of the marked depression of the levels of immunoglobulins and complement components. Destruction of the skin barrier by thermal injury and impairment of systemic or local humoral immunity may predispose these patients to burn wound sepsis.     

Preliminary characterizations of a serum biomarker for sarcoidosis by comparative proteomic approach with tandem-mass spectrometry in ethnic Han Chinese patients

Background

The diagnosis of sarcoidosis is still a significant challenge in China because of the need to exclude other diseases including granulomatous infections and malignancies that may be clinically and radiographically similar. The specific aim of the study is to search for serum protein biomarkers of sarcoidosis and to validate their clinical usefulness in differential diagnosis.

Methods

Serum samples were collected from patients with sarcoidosis (n = 37), and compared to those from patients with tuberculosis (n = 20), other pulmonary diseases (n = 20), and healthy volunteers (n = 20) for determination of sarcoidosis-specific or -associated protein expression profiles. The first part of this study focused on proteomic analysis of serum from patients with sarcoidosis to identify a pattern of peptides capable of differentiating the studied populations using the ClinProt profiling technology based on mass spectrometry. Enzyme Linked Immunosorbent Assay (ELISA) was then used to verify corresponding elevation of the serum protein concentration of the potential biomarkers in the same patients sets. Receiver operating characteristic curve (ROC) analyses was performed to determine the optimal cutoff value for diagnosis. Immunohistochemistry was carried out to further confirm the protein expression patterns of the biomarkers in lung tissue.

Results

An unique protein peak of M/Z 3,210 Daltons (Da) was found to be differentially expressed between the sarcoidosis and control groups and was identified as the N-terminal peptide of 29 amino acids (94-122) of serum amyloid A (SAA). ELISA confirmed that the serum SAA level was significantly higher in the sarcoidosis group than that of the other 3 control groups (p < 0.05). The cutoff for serum SAA concentration determined by ROC analysis was 101.98 ng/ml, with the sensitivity and specificity of 96.3% and 52.5%, respectively. Immunohistochemical staining showed that the SAA depositions in lung tissue of the sarcoidosis patients were also significantly more intense than in non-sarcoid lung tissue (p < 0.05).

Conclusion

This is the first study to investigate serum protein markers in Chinese subjects with sarcoidosis. This study shows that the serum SAA expression profiles were different between the sarcoidosis and non-sarcoidosis groups. SAA may be a potential serum biomarker for ruling-out the diagnosis of sarcoidosis in Chinese subjects.     

Ovarian cancer marker of 11.7 kDa detected by proteomics is a serum amyloid A1

In this study, to reduce the number of major plasma components, we examined thermostable plasma fractions to search for a biomarker of ovarian cancer. An apparent cancer biomarker of 11.7 kDa was detected in these fractions using ProteinChip SELDI-TOF mass spectrometry system. This peak invariably appeared with another close peak of about 11.5 kDa, suggesting that it is a derivative of a larger mass molecule. Of 27 cancer plasma specimens, 15 (55.6%) demonstrated this peak pair, whereas only 2 of 34 controls specimens (5.8%) were shown to express it with low intensity. Using a method involving cysteine modification by 4-vinylpyridine (4-VP), 2-DE and HPLC, these peaks were identified by mass spectrometry as serum amyloid A1 (11.68 kDa) and its N-terminal arginine-truncated form (11.52 kDa).     

Mutant p21ras in vinyl chloride exposed workers

The production of mutations in cellular oncogenes such as ras is involved in the development of many human cancers. These mutations result in the expression of mutant forms of the encoded p21 protein which can potentially serve as a biomarker for this carcinogenic process. Workers exposed to vinyl chloride (VC) who are at risk for the development of the sentinel neoplasm angiosarcoma of the liver (ASL) represent a model population for the study of such a mutant p21ras biomarker, since VC is known to cause a specific ras mutation in ASL. In order to determine the relationship between VC exposure and this biomarker, serum samples from a cohort of 225 French VC workers and 111 age-sex-race-smoking-drinking matched unexposed controls were examined for the presence of mutant p21ras by immunoblotting with a mouse monoclonal antibody specific for the mutant protein. Stratifying the exposed workers by degree of VC exposure in estimated ppm-years by quartiles yielded a statistically significant trend for increasing odds ratio for sero-positivity of the p21ras biomarker with increasing exposure. These results suggest that this serum biomarker is related to VC exposure and may be an early indicator of carcinogenic risk in exposed individuals.     

血清miRNA-93、miRNA-223联合miRNA-324-3p可作为诊断PCOS的生物标记物

本研究拟基于最近的miRNA报道,结合前期关于肌肉生长抑素(myostatin, MSTN)的相关研究,探究多囊卵巢综合症(ploycystic ovary syndrome, PCOS)患者血清miRNA和生长抑素的表达及其相关临床病理特征。本研究选取160例在湘南学院附属医院妇科就诊的女性为研究对象,通过PCR、ELISA等方法比较PCOS患者血清miRNA和生长抑素的表达情况,并且分析其相关临床病理特征。结果显示,通过PCR的方法发现mi RNA-93、mi RNA-223在PCOS患者的血清中显著升高,而mi RNA-4522、mi RNA-6767-5p和mi RNA-324-3p则表达下降。本研究建立了3个miRNA的预测模型,并证实预测模型在筛选组和验证组中有很好的敏感性和特异性,可以有效区分PCOS患者和正常人群,但研究结果也发现,miRNA模型结合MSTN没有更好的诊断价值。     

油松与赤松疱锈病病原及转主的研究

油松与赤松疱锈病是两针松类枝干上的重要病害,从油松和赤松主干病皮上取锈孢子分别向山芍药、芍药、返顾马先蒿、轮花马先蒿、长白茶藨子、东北茶藨子、香茶藨子和刺李进行室内和室外人工接种试验,终于在山芍药、芍药上接种成功,产生与自然状态下相同的典型症状。同样方法用夏孢子接种也都成功,在两种芍药上所产生的夏孢子堆与冬孢子柱以及夏孢子与冬孢子的形态也基本相同。由此可以肯定,油松疱锈病与赤松疱锈病是同一病原,病原菌为松芍柱锈菌[cronartium flaccidum(Alb·et Schw·)Wint]。取锈孢子用同样方法经多次在返顾马先蒿、轮花马先蒿、长白茶藨子、东北茶藨子、香茶藨子和刺李上接种,均未成功,因此认为这些植物与此两种疱锈病的发生无关,两种疱锈病在辽宁的转主寄主是山芍药(Paeonia obovata Haxim.)和芍药(P.lactiflora Pall.)。     

Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma

A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease-related proteins and peptides. This study describes enrichment of serum by denaturing ultrafiltration, which enables an efficient profiling and identification of peptides up to 5 kDa. We consistently detect several hundred peptide-peaks in MALDI-TOF and SELDI-TOF spectra of enriched serum. The sample preparation is fast and reproducible with an average CV for all 276 peaks in the MALDI-TOF spectrum of 11%. Compared to unenriched serum, the number of peaks in enriched spectra is 4 times higher at an S/N ratio of 5 and 20 times higher at an S/N ratio of 10. To demonstrate utility of the methods, we compared 20 enriched sera of patients with hepatocellular carcinoma (HCC) and 20 age-matched controls using MALDI-TOF. The comparison of 332 peaks at p < 0.001 identified 45 differentially abundant peaks that classified HCC with 90% accuracy in this small pilot study. Direct TOF/TOF sequencing of the most abundant peptide matches with high probability des-Ala-fibrinopeptide A. This study shows that enrichment of the low molecular weight fraction of serum facilitates an efficient discovery of peptides that could serve as biomarkers for detection of HCC as well as other diseases.     

The biochemistry of blister fluid from pediatric burn injuries: proteomics and metabolomics aspects

Burn injury is a prevalent and traumatic event for pediatric patients. At present, the diagnosis of burn injury severity is subjective and lacks a clinically relevant quantitative measure. This is due in part to a lack of knowledge surrounding the biochemistry of burn injuries and that of blister fluid. A more complete understanding of the blister fluid biochemistry may open new avenues for diagnostic and prognostic development. Burn insult induces a highly complex network of signaling processes and numerous changes within various biochemical systems, which can ultimately be examined using proteome and metabolome measurements. This review reports on the current understanding of burn wound biochemistry and outlines a technical approach for ‘omics’ profiling of blister fluid from burn wounds of differing severity.     

Proteomics-based identification of autoantibody against CDC25B as a novel serum marker in esophageal squamous cell carcinoma

This study was aimed to identify tumor proteins that elicit a humoral response in patients with esophageal squamous cell carcinoma (ESCC). Autologous sera of 15 newly diagnosed patients with ESCC and age- and gender-matched 15 healthy controls were analyzed individually for antibody-based reactivity against proteins from 15 homogenized ESCC tissue mixture resolved by two-dimensional PAGE. One protein spot, which reacted with sera from ESCC patients but not with those from controls, was identified to be CDC25B by mass spectrometry and Western blotting. High expression of CDC25B was detected in ESCC cell lines and primary tumor tissues, but not in normal esophageal tissues. In addition, CDC25B expression was significantly higher in tumor tissue of patients with sera positive CDC25B-Abs than that of patients without CDC25B-Abs. Finally, anti-CDC25B antibodies were readily detectable in sera from 45 of 124 (36.29%) patients with ESCC, 13 of 150 (8.67%) patients with other types of cancer and 0 of 102 (0%) of healthy individuals. Thus, CDC25B autoantibodies may have clinical utility in ESCC screening and diagnosis.