Proteomics: the next revolution in laboratory medicine?

BACKGROUND: The identification of specific genetic alterations and protein profiles associated with disease offers a unique opportunity to develop proteomics-based assays for early diagnosis. By identifying proteins in serum/plasma, a minimally invasive tool is used to assess the presence of disease and to monitor response to treatment and/or disease progression. The potential clinical applications of this tool are broad-based, including the diagnosis not only of cancer but also cardiovascular and neuromuscular diseases, organ transplantation associated conditions, and infertility. METHODS: A number of competing chromatographic techniques have been proposed for overcoming the complexity and labor-intensive manipulations associated with the traditional technique for proteomic analysis, which is based on two-dimensional gel electrophoretic techniques. However, mass spectrometry has now assumed a central role in most proteomic workflows, and several combinations of ionization sources, analyzers and fragmentations devices have been described and developed. RESULTS: Thanks to proteomic applications in the diagnosis of cancer, several research groups have identified proteomic patterns associated with ovarian, prostatic, colorectal and other cancers. While the sensitivity and specificity of these patterns are highly satisfactory, there are still some open questions concerning the standardization, reproducibility, and inter-laboratory agreement of these data. CONCLUSIONS: Proteomics, and, in particular, serum mass spectroscopic proteomic pattern diagnostics, is a rapid expanding field of research. The plasma proteoma has an important position at the intersection between genes and diseases, and clinical laboratories must adapt to a new era of tests based on proteomics and genomics. In the future, mass spectrometry will become an essential tool in the clinical laboratory.     

可变钠和超滤曲线联合应用预防透析症状低血压的护理

目的 观察联合应用可变钠和超滤曲线透析对透析中低血压（intradialytic hypotension,IDH）及相关症状发生率的影响.方法 选择既往常规血液透析有IDH倾向的患者40例,采用可变钠和超滤曲线透析6周,监测血压、体重、超滤量,分别于2、4、6周完成透析相关症状问卷.结果 联合方法透析2、4、6周后IDH、头晕、肌肉抽搐和头痛发生率均显著低于常规方法症状发生率（P＜0.05）.结论 联合采用可变钠和超滤曲线有助于减少IDH发生,增加患者的耐受力,该方法不以增加患者钠负荷为代价,不造成透析间期体重增重过多.     

The clinical application of proteomics

BACKGROUND: Proteomics is defined as a scientific approach used to elucidate all protein species within a cell or tissue, and many researchers are taking advantage of proteomic technology to elucidate protein changes between healthy and diseased states. METHODS: The application of proteomic techniques and strategies to the field of medicine is slowly transforming the way biomarker discovery is conducted. However, the complexity of serum is the source of both its promise to clinical applications and its challenge to proteomic analysis. Like any new technology when it is first introduced, proteomics has been touted with much hope and promise. RESULTS AND CONCLUSIONS: We provide a review of the clinical application of proteomics with the emphasis on current practical issues and challenges facing proteomic research.     

Enzymes and related proteins as cancer biomarkers: a proteomic approach

BACKGROUND: The discovery of cancer biomarkers has become a major focus of cancer research, which holds promising future for early detection, diagnosis, monitoring disease recurrence and therapeutic treatment efficacy to improve long-term survival of cancer patients. Most of the functional information of the cancer-associated genes resides in the proteome. Since cancer is a complex disease, it might require a panel of multiple biomarkers in order to achieve sufficient clinical efficacy. METHODS: Serum/plasma is the most accessible biological specimen collected from patients. Therefore, serum proteomic diagnostics would be the most promising new test for cancer. With the advent of new and improved proteomic technologies, such as protein chips and mass spectrometry coupled with advanced bioinformatic tools, it is possible to develop potential cancer biomarkers. However, specimen collection, handling, study design and data analysis are essential components for successful biomarker discovery and validation. Multi-center case control study should be conducted with extensive clinical validation to minimize the impact of possible confounding variables (non-biological). CONCLUSIONS: Enzymes and related proteins, such as inhibitors, are promising candidates for cancer diagnostics.     

白细胞介素13及变异体刺激呼吸道平滑肌后差异蛋白分析

目的 应用双向电泳及质谱技术对野生型及变异型白细胞介素13(IL-13)刺激后的ASMC总蛋白进行差异比较研究,借此分析差异表达蛋白与支气管哮喘的关系,为哮喘的治疗提供新的靶标.方法 用固相pH双向电泳分离,分别给予野生型IL-13及变异型IL-13刺激的ASMC总蛋白组成分,找出异常IL-13处理后与野生型IL-13处理后有明显差别的蛋白斑点,用基质辅助激光解吸电离飞行时间串联质谱(MALDI-TOF)进行鉴定部分差异蛋白质.结果 获得了分辨率高,重复性好的电泳图谱,在野生型IL-13刺激组和变异型IL-13刺激组分别平均有(840±21)个和(892±17)个蛋白点(n=3)被检测,匹配的点数为(685±19)个,选取5个未匹配点做质谱分析,鉴定其中3个分别为stathmin 1,Ribosomal protein p0,NADH dehydrogenase.结论 本研究结果显示不同处理组的ASMC总蛋白表达存在差异,这些差异蛋白有可能为阐明野生型IL-13及其变异体引发哮喘机制提供新的实验证据.     

小鼠肺爆震伤蛋白质组学研究

目的:通过检测不同时间点肺爆震伤小鼠肺蛋白质组学变化,探究肺爆震伤损伤机制。方法:60只健康雄性C57BL/6小鼠,随机（随机数字法）分为对照组、爆震后12 h组、24 h组、48 h组、72 h组及1周组（每组10只）。在北部战区总医院动物实验室进行实验,利用自主研发的精准爆震装置建立小鼠肺爆震伤模型;通过大体观察和...     

Placental metabolic profiling in gestational diabetes mellitus: An important role of fatty acids

AimGestational diabetes mellitus (GDM) is the most common metabolic disorder during pregnancy. Accumulating studies have reported metabolites that are significantly associated with the development of GDM. However, studies on the metabolism of placenta, the most important organ of maternal‐fetal energy and material transport, are extremely rare. This study aimed to identify and discuss the relationship between differentially expressed metabolites (DEM) and clinical parameters of the mothers and newborns.MethodsIn this study, metabolites from 63 placenta tissues (32 GDM and 31 normal controls) were assayed by ultra‐performance liquid chromatography‐high resolution mass spectrometry (UPLC‐HRMS).ResultsA total of 1297 annotated metabolites were detected, of which 87 significantly different in GDM placenta. Lipids and lipid‐like molecules accounted for 62.1% of DEM as they were significantly enriched via the “biosynthesis of unsaturated fatty acids” and “fatty acid biosynthesis” pathways. Linoleic acid and α‐linolenic acid appeared to be good biomarkers for the prediction and diagnosis of GDM. In addition, the level of PC(14:0/18:0) was negatively correlated with neonatal weight. 14 metabolites significantly different in male and female offspring, with the most increase in female newborns.ConclusionEven if maternal blood glucose level is well controlled, there are still metabolic abnormalities in GDM. Lipids and lipid‐like molecules were the main differential metabolites, especially unsaturated fatty acids.     

Tree analysis of mass spectral urine profiles discriminates transitional cell carcinoma of the bladder from noncancer patient

BACKGROUND: Recent advances in proteomic profiling technologies, such as surface-enhanced laser desorption/ionization mass spectrometry (SELDI), have allowed preliminary profiling and identification of tumor markers in biological fluids in several cancer types and establishment of clinically useful diagnostic computational models. We developed a bioinformatics tool and used it to identify proteomic patterns in urine that distinguish transitional cell carcinoma (TCC) from noncancer. METHODS: Proteomic spectra were generated by mass spectroscopy (surface-enhanced laser desorption and ionization). A preliminary "training" set of spectra derived from analysis of urine from 46 TCC patients, 32 patients with benign urogenital diseases (BUD), and 40 age-matched unaffected healthy men were used to train and develop a decision tree classification algorithm that identified a fine-protein mass pattern that discriminated cancer from noncancer effectively. A blinded test set, including 38 new cases, was used to determine the sensitivity and specificity of the classification system. RESULTS: The algorithm identified a cluster pattern that, in the training set, segregated cancer from noncancer with sensitivity of 84.8% and specificity of 91.7%. The discriminatory pattern correctly identified. A sensitivity of 93.3% and a specificity of 87.0% for the blinded test were obtained when comparing the TCC vs. noncancer. CONCLUSIONS: These findings justify a prospective population-based assessment of proteomic pattern technology as a screening tool for bladder cancer in high-risk and general populations.     

High-throughput profiling for discovery of non-coding RNA biomarkers of lung disease

In respiratory medicine there is a need for clinical biomarkers for diagnosis, prognosis and assessment of response to therapy. Noncoding RNA (ncRNA) is expressed in all human cells; two major classes – long ncRNA and microRNA – are detectable extracellularly in the circulation and other biofluids. Altered ncRNA expression is associated with lung disease; collectively this indicates that ncRNA represents a potential biomarker class. This article presents and compares existing platforms for detection and quantification of ncRNA, specifically hybridization, qRT-PCR and RNA sequencing, and outlines methods for data interpretation and normalization. Each approach has merits and shortcomings, which can affect the choice of method when embarking on a biomarker study. Biomarker properties and pre-analytical considerations for ncRNA profiling are also presented. Since a variety of profiling approaches are available, careful study and experimental design are important. Finally, challenges and goals for reliable, standardized high-throughput ncRNA profiling in biofluids as lung disease biomarkers are reviewed.     

Comparison of three preparatory methods for detection of bacteremia by MALDI-TOF mass spectrometry

We evaluated 3 preparatory methods for processing positive blood culture bottle broths prior to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis-differential centrifugation, 10% sodium dodecyl sulfate (SDS), and the Sepsityper kit. Initial evaluation used genus and species level cutoff scores of 1.700-1.999 and ≥ 2.000, respectively. Processing of an initial 79 blood bottle cultures by differential centrifugation yielded correct identifications of 51 (65%) to the genus and 34 (43%) to the species levels. One hundred and one different blood bottles were simultaneously processed with 10% SDS and Sepsityper. Both yielded genus-level identifications for 77 (76%), and the 2 yielded species-level identifications for 49 (49%) and 54 (54%) of bottles, respectively. Adjustment of the score cutoff criteria for genus (1.500-1.699) and species (≥ 1.700) improved identification percentages, particularly for species-level identifications (P < 0.05).     

A rapid test for the diagnosis of thrombotic thrombocytopenic purpura using surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF)-mass spectrometry

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP), a life-threatening thrombotic microangiopathy, requires immediate diagnosis and plasma exchange therapy. Development of TTP is related to functional deficiency of ADAMTS-13 protease that leads to the accumulation of ultra large von Willebrand factor (VWF) and subsequent platelet thrombosis. Currently no clinical test is available for the rapid detection of ADAMTS-13 activity. OBJECTIVES: The goal is to devise a novel method to rapidly detect functional activity of ADAMTS-13 and improve clinical outcome. METHODS AND RESULTS: A recombinant VWF substrate containing the ADAMTS-13 cleavage site and a 6X Histidine tag was cleaved by ADAMTS-13 in a dose-dependent manner, generating approximately 7739 Da peptide containing a 6X Histidine tag. This cleaved peptide, bound to an IMAC/Nickel ProteinChip, was quantified using Surface Enhanced Laser Desorption/Ionization Time-of-flight Mass Spectrometry (SELDI-TOF-MS). The assay is capable of quantifying ADAMTS-13 activity as low as 2.5% in plasma within 4 h. When the cleaved peptide was quantified as a ratio of an internal control peptide, the test displayed good reproducibility, with an average inter-assay coefficient of variation (CV) of < 33%. Further validation revealed a mean ADAMTS-13 activity of 92.5% +/- 16.6% in 39 healthy donors. Sixteen patients with idiopathic TTP displayed mean ADAMTS-13 activity of 1.73% +/- 3.62%. Further utility of this novel method includes determining the inhibitory titer of ADAMTS-13 antibody in cases of acquired TTP. CONCLUSIONS: We have devised a novel SELDI-TOF-MS assay that offers a rapid, cost-effective, and functionally relevant test for timely diagnosis and management of TTP.     

基于电感耦合等离子体质谱法血清钙离子检测候选参考方法的建立

目的基于电感耦合等离子体质谱法(ICP-MS),建立一种检测血清钙离子的候选参考方法。方法方法学建立.将从医院收集到的血清标本以0.3%硝酸溶液直接稀释100倍,在血清标本基质溶液中添加不同浓度钙标准溶液,配制含血清基质的标准品溶液。以锗(Ge)为内标,采用标准加入法,计算血清钙离子浓度。对所建立的候选参考方法进行线性、精密度、正确度的性能评估和方法比对。结果血清钙离子浓度在0.000~20.400 mmol/L内(稀释后浓度为0.000~0.204 mmol/L)线性良好(R2>0.9999);批内不精密度为0.22%~0.47%,批间不精密度为0.64%~0.77%,总不精密度为0.98%~1.09%;检测3个浓度SRM 956d,结果均在证书要求的不确定度范围内,相对偏移分别为-0.16%,0.04%,0.23%;该方法参加2017年参考实验室外部质量评价计划(RELA),比对通过。与检验医学溯源联合委员会(JCTLM)所列参考方法进行比较,结果一致性良好。本研究所建立的候选参考方法与临床常规电极方法进行比较,具有良好的相关性。结论成功建立血清钙离子检测候选参考方法,线性范围宽、具有良好的精密度与准确度。     

气相色谱-质谱联用检测尿液8种有机酸实验室间质量的调查

目的评价2018年遗传代谢病气相色谱-质谱联用检测尿液有机酸实验室间质量调查结果,以改进和提高新生儿遗传代谢病筛查或相关实验室气相色谱-质谱联用检测质量。方法 2018年3月向全国19家开展气相色谱-质谱联用尿液有机酸检测的实验室发放2个批号质控尿液样品(批号201811和201812)。实验室自愿参加此次调查活动并按照规定格式上报结果、测定方法、仪器和试剂等相关信息。组织者用Clinet EQA程序和Microsoft Excel 2010等软件分析各实验室检测结果及检验前、检验中及检验后过程。结果实验室回报率为94.7%(18/19)。2个批号样品(批号201811和批号201812)的检测中,各参加实验室部分有机酸的检测结果相对稳定;相比高浓度而言,低浓度有机酸的检测水平相对较好。质控品稳定性检测结果显示,苯丙酮酸(稳健变异系数99.48%)和尿黑酸(稳健变异系数67.56%)的结果差异较大。结论相比高浓度,低浓度有机酸的检测水平相对较好;稳定性差的苯丙酮酸和尿黑酸不适合作为室间质量评价质控品,应选用更稳定的化合物作为质控品以评价实验室质量。     

A comparison of a Bayesian vs. a frequentist method for profiling hospital performance

The objective of this study was to compare the classification of hospitals as outcomes outliers using a commonly implemented frequentist statistical approach vs. an implementation of Bayesian hierarchical statistical models, using 30-day hospital-level mortality rates for a cohort of acute myocardial infarction patients as a test case. For the frequentist approach, a logistic regression model was constructed to predict mortality. For each hospital, a risk-adj usted mortality rate was computed. Those hospitals whose 95% confidence interval, around the risk-adjusted mortality rate, excludes the mean mortality rate were classified as outliers. With the Bayesian hierarchical models, three factors could vary: the profile of the typical patient (low, medium or high risk), the extent to which the mortality rate for the typical patient departed from average, and the probability that the mortality rate was indeed different by the specified amount. The agreement between the two methods was compared for different patient profiles, threshold differences from the average and probabilities. Only marginal agreement was shown between the Bayesian and frequentist approaches. In only five of the 27 comparisons was the kappa statistic at least 0.40. The remaining 22 comparisons demonstrated only marginal agreement between the two methods. Within the Bayesian framework, hospital classification clearly depended on patient profile, threshold and probability of exceeding the threshold. These inconsistencies raise questions about the validity of current methods for classifying hospital performance, and suggest a need for urgent research into which methods are most meaningful to clinicians, managers and the general public.     

Rapid micro-scale assay for homocysteine by liquid chromatography-tandem mass spectrometry

OBJECTIVES: Increased total homocysteine (tHcy) level is an independent risk factor for atherosclerosis and cardiovascular disease. Here, we describe a rapid tHcy micro-scale assay. METHODS: We developed an easy sample preparation and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of tHcy in human serum and plasma, respectively. RESULTS: The tHcy assay was linear up to 61.6 micromol/L, the detection limit was 1.0 micromol/L, the lower limit of quantification was 1.8 micromol/L, the imprecision in the range of 9.30-25.9 micromol/L was less than 5.9% and the analytical recovery was 94.7 +/- 6.3%. CONCLUSIONS: Our novel tHcy LC-MS/MS assay is a quick, precise and robust method for tHcy determination in routine diagnostics.     

Surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS): a new proteomic urinary test for patients with urolithiasis

SELDI-TOF-MS is a highly sensitive protein-analysis tool capable of detecting minute protein profile differences between biological samples. As proteins have been associated with urinary tract calculi, protein-based urinalysis may offer insights into their diagnosis. The purpose of this study was to evaluate SELDI-TOF-MS as a potential method for identifying urinary biomarkers of urolithiasis. Midstream sterile urine samples were obtained from 25 male patients with a confirmed diagnosis of urolithiasis (test group) and 25 male subjects with no known history of the disease (controls). Urinary levels of oxalate, total protein, albumin, and osteopontin were determined. Protein profiles were generated using SELDI-TOF-MS.SELDI-TOF-MS profiling revealed a relationship between protein peak intensities at 67 and 24 kDa that differed between the two groups. The ratio of p67:p24 was found to be less than 1.0 in all of the control samples (mean 0.26), while 18 out of 25 (72%) of the test group samples displayed a ratio greater than 1.0 (total group mean 4.75, P<0.001). Albumin, total protein, and oxalate levels were higher in the test group than the controls.Although SELDI-TOF-MS is not yet in widespread use in hospital and diagnostic laboratories, this system represents a promising new method for rapidly identifying patients with urolithiasis.     

基于MRM-MS技术的蛋白质生物标志物研究的前景

随着蛋白质组学技术的兴起,疾病生物标志物的研究发展迅速,但筛选出的海量候选标志物却极少进入临床应用.近几年,基于MRM-MS技术的方法和策略凭借其低成本、耗时短、高灵敏度、高准确性等优势引起了国内外检验医学界的广泛关注.基于MRM-MS的技术,特别是SISCAPA技术,可能在不远的将来将逐步发展成为蛋白质生物标志物研究的快车道.本文分析了当前生物标志物研究领域的瓶颈,系统阐述了MRM-MS技术的原理和特点,以及近年来该技术在蛋白质生物标志物研究领域的主要应用现状,并展望其发展前景.

Abstract：

With the development of proteomic technologies, large numbers of putative biomarkers have been screened out but few of them are put into the clinical application.Recently, MRM-MS based approaches and strategies have aroused extensive concern in domestic and overseas clinical laboratory community because of their cost-efficiency, time-saving, high analytical sensitivity and accuracy.MRM-MS based approaches, especially SISCAPA, may gradually become a key technology for the protein biomarker research in the near future.In this review, the "bottleneck" existing in the field of protein biomarkers research, and features as well as applications of MRM-MS are discussed and the future trend of research is prospected.