Dissecting the human plasma proteome and inflammatory response biomarkers

A central focus of clinical proteomics is to search for biomarkers in plasma for diagnostic and therapeutic use. We studied a set of plasma proteins accessed from the Healthy Human Individual's Integrated Plasma Proteome (HIP²) database, a larger set of curated human proteins, and a subset of inflammatory proteins, for overlap with sets of known protein biomarkers, drug targets, and secreted proteins. Most inflammatory proteins were found to occur in plasma, and over three times the level of biomarkers were found in inflammatory plasma proteins and their interacting protein neighbors compared to the sets of plasma and curated human proteins. Percentage overlaps with Gene Ontology terms were similar between the curated human set and plasma protein set, yet the set of inflammatory plasma proteins had a distinct ontology‐based profile. Most of the major hub proteins within protein‐protein interaction networks of tissue‐specific sets of inflammatory proteins were found to occur in disease pathways. The present study presents a systematic approach for profiling a plasma subproteome's relationship to both its potential range of clinical application and its overlap with complex disease.     

Proteomic approaches in the search for disease biomarkers

Significant technological advances in protein chemistry, physics and computer sciences in the last two decades have greatly improved protein separation methodologies, such as electrophoresis and chromatography, and have established mass spectrometry (MS) as an indispensable tool for protein study. The goal of this review is to provide a brief overview of the recent improvements in these methodologies and present examples from their application in proteome analysis and search for disease biomarkers.     

Increased level of phosphorylated desmin and its degradation products in heart failure

Although several risk factors such as infarct size have been identified, the progression/severity of heart failure (HF) remains difficult to predict in clinical practice. Using an experimental rat model of ischemic HF and phosphoproteomic technology, we found an increased level of phosphorylated desmin in the left ventricle (LV) of HF-rats. The purpose of the present work is to assess whether desmin is a circulating or only a tissue biomarker of HF. We used several antibodies in order to detect desmin, its proteolytic fragments and its phosphorylated form in LV and plasma by western blot, phosphate affinity electrophoresis, mass spectrometry and immunofluorescence. Plasma was treated with combinatorial peptide ligand library or depleted for albumin and immunoglobulins to increase the sensitivity of detection. We found a 2-fold increased serine-desmin phosphorylation in the LV of HF-rats, mainly in the insoluble fraction, suggesting the formation of desmin aggregates. Desmin cleavage products were also detected in the LV of HF rats, indicating that the increased phosphorylation of desmin results in more susceptibility to proteolytic activity, likely mediated by calpain activity. The native desmin and its degradation products were undetectable in the plasma of rat, mouse or human. These data suggest the potential of serine-phosphorylated form of desmin and its degradation products, but not of desmin itself, as tissue but not circulating biomarkers of HF.     

Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory

Whole-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640–12000 m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates.     

Biomarkers in heart failure with preserved ejection fraction

Biomarkers are widely used and studied in heart failure. Most studies have described the utility and performance of biomarkers in sub-studies of randomised clinical trials, where the vast majority of the patients suffered from heart failure with reduced ejection fraction (HFrEF), and not with preserved ejection fraction (HFpEF). As a result, there is a scarcity of data describing the levels, dynamics, clinical and biochemical correlates, and biology of biomarkers in patients suffering from HFpEF, whereas HFpEF is in fact a very frequent clinical entity. This article discusses the value of different biomarkers in HFpEF. We describe various aspects of natriuretic peptide measurements in HFpEF patients, with a focus on diagnosis, prognosis and the risk prediction of developing heart failure. Further, we will discuss several emerging biomarkers such as galectin-3 and suppression of tumorigenicity 2, and recently discovered ones such as growth differentiation factor-15 and syndecan-1.     

Cancer associated proteins in blood plasma: Determining normal variation

Protein biomarkers have the potential to improve diagnosis, stratification of patients into treatment cohorts, follow disease progression and treatment response. One distinct group of potential biomarkers comprises proteins which have been linked to cancer, known as cancer associated proteins (CAPs). We determined the normal variation of 86 CAPs in 72 individual plasma samples collected from ten individuals using SRM mass spectrometry. Samples were collected weekly during 5 weeks from ten volunteers and over one day at nine fixed time points from three volunteers. We determined the degree of the normal variation depending on interpersonal variation, variation due to time of day, and variation over weeks and observed that the variation dependent on the time of day appeared to be the most important. Subdivision of the proteins resulted in two predominant protein groups containing 21 proteins with relatively high variation in all three factors (day, week and individual), and 22 proteins with relatively low variation in all factors. We present a strategy for prioritizing biomarker candidates for future studies based on stratification over their normal variation and have made all data publicly available. Our findings can be used to improve selection of biomarker candidates in future studies and to determine which proteins are most suitable depending on study design.     

慢性充血性心力衰竭血浆血管活性水平及临床意义

目的：探讨慢性心力衰竭患者血浆B型利钠肽（BNP）、心钠素（ANF）、胱抑素C（CysC）的水平及临床意义。方法：选择70例CHF患者,按NYHA分级;20例健康者作为对照。采用放射免疫法检查BNP和ANF水平;采用免疫比浊法检查CysC。比较不同心衰等级以及不同BNP水平的上述指标的变化。结果：CHF患者血浆BNP、ANF、CysC水平与对照比较显著升高（P〈0．05）,并随NYHA等级增高而升高（P〈0．05）。与BNP〈400pg／ml组比较,400pg／ml≤BNP〈800pg／ml组和BNP≥800pg／ml组心力衰竭患者Cr、BUN水平显著增高（P〈0．05）;相反地,HDL水平显著降低。结论：血浆B型利钠肽（BNP）、心钠素（ANF）及胱抑素c参与．了CHF的发生发展过程．联合测定箕合号的变化．对CHF患者的诊断、评估及詹．险分层具有一定的临床意义．     

imzML--a common data format for the flexible exchange and processing of mass spectrometry imaging data

The application of mass spectrometry imaging (MS imaging) is rapidly growing with a constantly increasing number of different instrumental systems and software tools. The data format imzML was developed to allow the flexible and efficient exchange of MS imaging data between different instruments and data analysis software. imzML data is divided in two files which are linked by a universally unique identifier (UUID). Experimental details are stored in an XML file which is based on the HUPO-PSI format mzML. Information is provided in the form of a 'controlled vocabulary' (CV) in order to unequivocally describe the parameters and to avoid redundancy in nomenclature. Mass spectral data are stored in a binary file in order to allow efficient storage. imzML is supported by a growing number of software tools. Users will be no longer limited to proprietary software, but are able to use the processing software best suited for a specific question or application. MS imaging data from different instruments can be converted to imzML and displayed with identical parameters in one software package for easier comparison. All technical details necessary to implement imzML and additional background information is available at www.imzml.org.     

慢性充血性心力衰竭血浆血管活性水平及临床意义

目的:探讨慢性心力衰竭患者血浆B型利钠肽(BNP)、心钠素(ANF)、胱抑素C(CysC)的水平及临床意义。方法:选择70例CHF患者,按NA分级;20例健康者作为对照。采用放射免疫法检查BNP和ANF水平;采用免疫比浊法检查Cys C。比较不同心衰等级以及不同BNP水平的上述指标的变化。结果:CHF患者血浆BNP、ANF、CysC水平与对照比较显著升高(P<0.05),并随NYHA等级增高而升高(P<0.05)。与BNP<400pg/ml组比较,400pg/ml≤BNP<800pg/ml组和BNP≥800pg/ml组心力衰竭患者Cr、BUN水平显著增高(P<0.05);相反地,HDL水平显著降低。结论:血浆B型利钠肽(BNP)、心钠素(ANF)及胱抑素C参与了CHF的发生发展过程,联合测定其含量的变化,对CHF患者的诊断、评估及危险分层具有一定的临床意义。     

Potential of resveratrol in the treatment of heart failure

The concept of food has expanded beyond its traditional role of survival and hunger satisfaction, to include a role in the prevention and treatment of disease. Polyphenols are classes of compounds that are synthesized by plants to serve a wide variety of functions including growth pollination and defense. These compounds have recently received increased attention in medical research. In this group, one of the most studied has been resveratrol (3,5,4,-trihydroxystilbene), a polyphenol, which is found predominantly in grapes and berries. Over the past two decades, researchers have studied the ability of resveratrol to prevent or reverse the development of abnormalities in heart structure and function in animal models of heart disease and heart failure. The results from animal studies have been promising, and very recently, this knowledge has been translated into examining the efficacy of resveratrol in humans with heart disease/failure. In this review we will discuss the current status of resveratrol research on cardioprotection.     

The predictive value of plasma catestatin for all-cause and cardiac deaths in chronic heart failure patients

Catestatin (CST) is a proteolytic fragment of Chromogranin A with a broad spectrum of activities in the cardiovascular system. The level of plasma CST increases in chronic heart failure patients, but its potential relationship to patient prognosis is unknown. In this study, we measured plasma CST levels in 202 chronic heart failure patients and followed them for a median of 52.5 months. The plasma CST level was higher in patients with all-cause death and cardiac death than in survivors. According to univariate COX regression, higher plasma CST levels predicted increased risk of all-cause and cardiac death. After adjustment for other confounding factors, plasma CST was an independent risk factor for both outcomes, and the hazard ratios (HRs) were 1.84 (95% CI: 1.02–3.32, p = 0.042) and 2.41 (95% CI: 1.26–4.62, p = 0.008) for all-cause death and cardiac death, respectively. The new risk-predictive model considering CST was superior to the previous model for both outcomes by ANOVA and likelihood ratio tests (p = 0.040 and p = 0.008, respectively). Concurrent increases in plasma BNP (B-type natriuretic peptide) and CST levels predicted the highest risk for both all-cause and cardiac deaths [HR = 5.18 (95% CI: 1.94–13.87, p = 0.001) and HR = 9.19 (95% CI: 2.75–30.78, p < 0.001), respectively]. Large-scale studies are needed to further assess the value of plasma CST in predicting heart failure prognosis.     

Feature extraction in the analysis of proteomic mass spectra

Feature extraction or biomarker selection is a critical step in disease diagnosis and knowledge discovery based on protein MS. Many studies have discussed the classification methods applied in proteomics; however, few could be found to address feature extraction in detail. In this paper, we developed a systematic approach for the extraction of mass spectrum peak apex and peak area with special emphasis on noise filtration and peak calibration. Application to a head and neck cancer data generated at the Eastern Virginia Medical School [Wadsworth, J. T., Somers, K. D., Cazares, L. H., Malik, G. et al.., Clin. Cancer Res. 2004, 10, 1625-1632] revealed that the new feature extraction method would yield consistent and highly discriminatory biomarkers.     

Alignment of high resolution mass spectra: development of a heuristic approach for metabolomics

One of the challenges of using mass spectrometry for metabolomic analyses of samples consisting of thousands of compounds is that of peak identification and alignment. This paper addresses the issue of aligning mass spectral data from different samples in order to determine average component m/z peak values. The alignment scheme developed takes the instrument m/z measurement error into consideration in order to heuristically align two or more samples using a technique comparable to automated visual inspection and alignment. The results obtained using mass spectral profiles of replicate human urine samples suggest that this heuristic alignment approach is more efficient than other approaches using hierarchical clustering algorithms. The output consists of an average m/z and intensity value for the spectral components together with the number of matches from the different samples. One of the major advantages of using this alignment strategy is that it eliminates the boundary problem that occurs when using predetermined fixed bins to identify and combine peaks for averaging and the efficient runtime allows large datasets to be processed quickly.     

Patients with heart failure with preserved ejection fraction and low levels of natriuretic peptides

Aims

Heart failure with preserved ejection fraction (HFpEF) is common and its management remains difficult. B-type natriuretic peptide (BNP) levels are used to diagnose heart failure, and as an entry criterion for inclusion into trials. We investigated a population of HFpEF patients who had been randomised into a study based on clinical parameters, and compared those with low BNP levels to those with elevated BNP levels.

Methods

We examined patients who had been enrolled in the Coordinating study evaluating Outcomes of Advising and Counselling in Heart Failure (COACH), with preserved left ventricular ejection fraction (LVEF ≥ 40 %), and compared those with low BNP (< 100 pg/ml; n = 30) to those with elevated BNP (≥ 100 pg/ml; n = 127). Baseline characteristics, comorbidities, biomarkers, quality of life, and outcome parameters (hospitalisations and death) were compared between the groups. To validate our findings, we repeated all analyses for NT-proBNP (< 300 pg/ml and ≥ 300 pg/ml).

Results

Patients were similar with regard to most clinical characteristics (including age, sex, and LVEF), biomarkers, and comorbidities. In contrast, patients with a low BNP had higher body mass index levels (31 kg/m² vs. 27 kg/m²; p < 0.01) and lower cardiac troponin I (9 pg/ml vs. 15 pg/ml; p = 0.02). In addition, these patients were less frequently prescribed diuretics and beta-blockers. No differences in quality of life, heart failure related symptoms and the primary and secondary outcomes were observed between these groups. These observations were confirmed for NT-proBNP.

Conclusion

Among the patients with clinically diagnosed HFpEF, those with low BNP are strikingly similar to those with elevated BNP levels, except for BMI, which was significantly higher in these patients.

Electronic supplementary material

The online version of this article (doi:10.1007/s12471-016-0816-8) contains supplementary material, which is available to authorized users.     

Antithrombotic therapy in heart failure

Patients with congestive heart failure have a significant risk of stroke due to thromboembolism from the dilated left ventricle. Two relatively small trials suggest that oral anticoagulation with vitamin-K antagonists may reduce this risk when compared with placebo, aspirin or clopidogrel. However, more studies are eagerly awaited. So far, physicians seeing patients with heart failure should decide who needs antithrombotic prophylaxis on a case-by-case basis, especially since most heart failure patients have significant comorbidity precluding the use of oral anticoagulant.     

Development of a coupled-column liquid chromatographic–tandem mass spectrometric method for the direct determination of betamethasone in urine

Different hyphenated liquid chromatographic (LC) and mass spectrometric (MS) techniques were investigated in order to set-up a method for the fast, direct analysis of betamethasone in hydrolysed and non-hydrolysed urine using large-volume sample injection. After the optimisation of the LC parameters using a traditional UV detector and of the thermospray and mass spectrometric parameters by flow injection, urine samples (0.5 ml) were submitted to analysis by either LC combined with tandem mass spectrometry (MS–MS), coupled-column LC (LC–LC) combined with single quadrupole MS, and LC–LC–MS–MS. Both the three-step configurations (LC–MS–MS and LC–LC–MS) did not provide satisfactory results: loss of sensitivity was noted in the case of LC–MS–MS (likely due to reduced efficiency in the ionisation of betamethasone in the thermospray owing to the presence of large amounts of matrix interference), while in the case of LC–LC–MS a high chemical noise resulting in insufficient selectivity of detection was observed. On the contrary, LC–LC–MS–MS analysis proved to meet the demand of high speed of analysis (sample throughput, 4.5 h⁻¹), selectivity, and sensitivity (LOQ, 1 ng/ml; LOD, 0.2 ng/ml). Notwithstanding the complex analytical system adopted, the developed procedure was manageable and very robust, provided that at the beginning of each analytical session the performance of the system was controlled by checking the retention time of the analytes on the first analytical column with UV detection and by optimising vaporiser temperature of the thermospray by flow injection.     

Inclusive sharing of mass spectrometry imaging data requires a converter for all

With continued efforts towards a single MSI data format, data conversion routines must be made universally available. The benefits of a common imaging format, imzML, are slowly becoming more widely appreciated but the format remains to be used by only a small proportion of imaging groups. Increased awareness amongst researchers and continued support from major MS vendors in providing tools for converting proprietary formats into imzML are likely to result in a rapidly increasing uptake of the format. It is important that this does not lead to the exclusion of researchers using older or unsupported instruments. We describe an open source converter, imzMLConverter, to ensure against this. We propose that proprietary formats should first be converted to mzML using one of the widely available converters, such as msconvert and then use imzMLConverter to convert mzML to imzML. This will allow a wider audience to benefit from the imzML format immediately.