Amaya Albalat

Recent advances in capillary electrophoresis coupled to mass spectrometry for clinical proteomic applications

Proteome analysis using capillary electrophoresis coupled to mass spectrometry (CE‐MS) has been used in a number of clinical applications in the past years. The main focus of CE‐MS‐based studies has been on the investigation of urine, due to the stability of the urinary proteome, ease of collection, and also the low molecular weight range of the urinary proteome, mostly peptides below 30 kDa. The reproducibility of this approach has enabled analysis of over 20u2009000 samples in a comparable way, giving enormous statistical power to any additional study involving this methodological setup. In this article, we review the major technological issues associated with the application of CE‐MS in the routine investigation of the urinary proteome for clinical applications. We pinpoint recent developments that may have a chance to improve on the currently used approach, and highlight obstacles that need to be solved. In the second part of the article, we review the recent clinical applications, aiming to highlight relevant issues, and possible future routine applications in clinical diagnosis. In the end, we provide a short outlook, and indicate future developments to be expected, as well as problems that need to be solved to enable routine application of CE‐MS in a clinical setting.

A comparison between MALDI-MS and CE-MS data for biomarker assessment in chronic kidney diseases

Non-invasive detection of diseases, based on urinary proteomics, is becoming an increasingly important area of research, especially in the area of chronic kidney disease (CKD). Different platforms have been used in independent studies, mostly capillary-electrophoresis coupled ESI-MS (CE-MS), liquid chromatography coupled mass spectrometry, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We have compared the performance of CE-MS with MALDI-MS in detecting CKD, based on a cohort of 137 urine samples (62 cases and 75 controls). Data cross-talk between the two platforms was established for the comparison of detected biomarkers. The results demonstrate superior performance of the CE-MS approach in terms of peptide resolution and obtained disease prediction accuracy rates. However, the data also demonstrate the ability of the MALDI-MS approach to separate CKD patients from controls, at slightly reduced accuracy, but expected reduced cost and time. As a consequence, a practical approach can be foreseen where MALDI-MS is employed as an inexpensive, fast, and robust screening tool to detect probable CKD. In a second step, high resolution CE-MS could be used in those patients only that scored negative for CKD in the MALDI-MS analysis, reducing costs and time of such a program.

Clinical application of urinary proteomics/peptidomics

The technology platforms for proteome analysis have advanced considerably over the last few years. Driven by these advancements in technology, the number of studies on the analysis of the proteome/peptidome, with the aim of defining clinically relevant biomarkers, has substantially risen. Urine has become an increasingly relevant target for clinically oriented proteome analysis; the first clinical trials based on urinary proteomics have been initiated, and studies including several hundred patients have been published. In this article, we summarize the relevant technical aspects in biomarkers discovery and the course from biomarker discovery or ‘potential’ biomarkers to those that have been validated and are clinically important. We discuss experimental design based on the statistics calculated to produce a clinically important end point. We present several examples of proteomic studies that have defined urinary biomarkers for clinical applications, focusing on capillary electrophoresis coupled to mass spectrometry as a technology. Finally, current challenges and considerations for future studies will be discussed.

A peptidomic approach to biomarker discovery for bovine mastitis.

UNLABELLEDnBovine mastitis is usually caused by either Gram positive or Gram negative bacteria, reducing the quantity and quality of milk produced. This investigation using capillary electrophoresis and mass spectroscopy, studied peptides in milk from cows with clinical mastitis in comparison to milk from healthy cows to identify biomarkers for mastitis. In addition, the milk peptidome from udders infected with Gram positive Staphylococcus aureus (S. aureus) or with Gram negative Escherichia coli (E. coli), was examined to assess differential diagnosis between the causative agent. Comparison of the peptidome between healthy (n=10) and mastitic milk (n=27) identified 154 peptides for a biomarker panel which in a model for diagnosis of mastitis showed 100% sensitivity and specificity. β-casein and α(s1) casein provided the majority of peptides identified in this model. The peptidome comparison of milk from mastitis cases caused by S. aureus (n=8) or E. coli (n=11) revealed a biomarker panel of 47 peptides which discriminated between cause of infection with a sensitivity of 75% and a specificity of 100%. β-casein fragments were the most common of the peptides in this model. Peptide biomarkers of milk could be used in the diagnosis of mastitis and can discriminate between these two bacterial causes.nnnBIOLOGICAL SIGNIFICANCEnThe paper describes an innovative approach to the use of gel free proteomics to identify the peptides that are present in milk during clinical mastitis, which is a major cause of loss of production to dairy farmers worldwide. The use of capillary electrophoresis, liquid chromatography and mass spectrometry has been able to identify panels of peptides which can be used for disease diagnosis and for differential diagnosis of the causative bacteria of the infections of the mammary gland. As well as contributing to our knowledge of the pathophysiology of bovine mastitis the results could be the basis of improved detection and differential diagnosis of the disease.

Performance of different separation methods interfaced in the same MS-reflection TOF detector: A comparison of performance between CE versus HPLC for biomarker analysis

One of the aims in the field of proteomics is the identification of a protein or polypeptide, or a range of these compounds, that could provide pre‐symptomatic indication of the onset of a disease. A number of analytical techniques have been employed to try and achieve this end. These techniques have been applied to the complete range of body fluids and tissues that are readily available from clinical studies. Of these sample sources, the urinary low molecular weight peptidome has been shown to reflect changes in the health status of the individual. The alterations that occur in the polypeptide make up of urine, which reflect changes in biological status, are known as biomarkers. To be able to determine these changes no single technique has emerged that can cope with detecting the large number of peptides present and quantifying them over the wide concentration range they exist in. In this investigation, we made use of a single reflectron time of flight (RTOF)‐MS analyser to which we first connected a CE system and then a nanoflow HPLC. Two pooled male and female standard urine samples were compared on these systems. Both techniques had similar results in terms of number of peptides detected and the mass range the peptides were detected over. The major differences in terms of biomarker research were the ability in CE to calibrate the migration time of the peptides to allow comparison between samples. In addition, CE was shown not to suffer from carry over from previous samples as was seen in the LC analysis.

A Pilot Study on the Effect of Short-Term Consumption of a Polyphenol Rich Drink on Biomarkers of Coronary Artery Disease Defined by Urinary Proteomics

Polyphenol rich diets have been associated with a reduced risk of cardiovascular disease. We examined the effect of a polyphenol rich (P-R) drink on biomarkers assessed by urinary proteomics. Thirty nine middle aged and overweight subjects were randomized to P-R drink (n = 20) or placebo (n = 19) in addition to their normal diet. After two weeks urine samples were obtained for assessment of the urinary proteome using capillary electrophoresis coupled to a mass spectrometer. A total of 93 polypeptides were found to be candidates for differential distribution with a nominal p-value <0.05, though these differences did not reach significance when multiple testing was accounted for. Sequences were determined in 19 of these demonstrating that they originate from alpha-1 antitrypsin, collagens, fibrinogen alpha and IgG kappa. Levels of 27 polypeptides were greater than 4-fold different between the two groups. Of these, 7 were previously found to be part of a coronary artery disease (CAD) specific urinary biomarker pattern. Their direction of expression was closer to the healthy state in the P-R drink group and closer to CAD state in the placebo group. Our data suggest that the P-R drink may have beneficial effects on urinary biomarkers of CAD. The data encourage the planning of future prospective studies, aimed at investigating significant effects of polyphenol rich dietary products.

Targeting the live market: Recovery of Norway lobsters Nephrops norvegicus (L.) from trawl-capture as assessed by stress-related parameters and nucleotide breakdown

Abstract The recovery potential of Norway lobsters ( Nephrops norvegicus ) held in on-board seawater tanks after trawl-capture was assessed at two different times of the year (winter and summer). Survival recorded 24xa0h after trawl-capture was 84.83xa0±xa00.93% in the winter compared to 75.35xa0±xa02.92% in the summer. Stress-related parameters in the muscle (arginine phosphate, glycogen and L-lactate) and in the haemolymph (L-lactate) were measured, together with nucleotide breakdown products in the muscle (yielding the “Adenylate Energy Charge” or AEC ratio). All parameters analysed were responsive to the stress of the trawl-capture and subsequently recovered towards resting values, but did so at different rates. The fact that some measures recovered at a faster rate than others should be taken into account when trying to develop an index of metabolic stress for this species. Animals trawled in the winter recovered to AEC values above 0.8 within 4xa0h of placing them in on-board seawater tanks, whereas animals trawled in the summer took 24xa0h to reach these values. Furthermore, at the end of the trials animals trawled in the summer presented significantly higher haemolymph L-lactate and lower muscle glycogen reserves than the animals trawled in the winter, suggesting a faster recovery in the winter compared to the summer. Finally, animals in the winter were better able to endure further stresses (an emersion of 1xa0h while animals were transported to the commercial handling facilities). Therefore, as a code of practice it is advised that trawled N. norvegicus directed to the live trade should be allowed to recover for at least 4–6xa0h in on-board tanks, and extra care should be taken especially in the summer, if further stresses such as additional emersion are to be applied within the first 24xa0h after capture.

The influence of defined ante-mortem stressors on the early post-mortem biochemical processes in the abdominal muscle of the Norway lobster, Nephrops norvegicus (Linnaeus, 1758)

Abstract The effects of four different ante-mortem stressors (exercise, emersion, starvation and a patent infection with the parasite Hematodinium sp.) on post-mortem processes have been investigated in the abdominal muscle of Norway lobster Nephrops norvegicus by measuring changes in the pH, the levels of glycogen, l-lactate, arginine phosphate, ATP, ADP, AMP, IMP, HxR, Hx and the adenylate energy charge (AEC) over a time course of 24 h with samples being taken at 0, 3, 6, 12 and 24 h. The acute stresses of intense exercise and 2 h emersion resulted in a premature onset of anaerobic glycolysis, leading both to an enhanced glycogen depletion rate and an early accumulation of l-lactate. The chronic stressors, starvation and parasite infection, resulted in a complete ante-mortem depletion of muscle glycogen and consequently the failure of post-mortem glycolytic fermentation. Post-mortem pH and ATP inter-conversion were significantly altered in chronically stressed animals. Ante-mortem, a rapid, almost complete depletion of arginine phosphate was observed in all stress groups. The AEC was altered significantly by all stresses, indicating a strong energy demand. The findings suggest that ante-mortem stressors strongly influence the post-mortem biochemical processes. The laboratory-based results are compared to ‘field’ data and effects on post-harvest product quality are discussed.

Effect of capture method on the physiology and nucleotide breakdown products in the Norway lobster (Nephrops norvegicus)

Abstract The effects of capture method (creel and trawl) and trawling time (15, 60 and 150 min) were assessed in Norway lobsters by measuring stress-related metabolites together with nucleotide breakdown products. Furthermore, mechanical damage was scored in the animals captured by trawl. Capture method had a clear impact on the nucleotide profile in the Norway lobster muscle. In rested and creel-caught animals the main nucleotide was ATP, while in trawled animals the main nucleotide was AMP. According to these results the Adenylate Energy Charge (AEC) was lower in trawled animals compared with creel-caught animals, while trawling time did not affect AEC levels significantly. Stress-related and anaerobic metabolites together with muscle pH indicated that trawled animals, even at the shortest time tested, were using anaerobic metabolism. In the haemolymph, l-lactate increased with a delay compared with muscle, suggesting that the concentration of l-lactate in the muscle provides a more immediate measure of capture-stress in this species. Although physiological measures were similar for short and long tows, physical damage increased in long trawls. Further studies should elucidate whether the different physiological stresses and the physical damage that occur during capture could compromise the live transport of trawled animals, and if quality measures could thus be affected.

Improving peptide relative quantification in MALDI-TOF MS for biomarker assessment.

Proteomic profiling by MALDI‐TOF MS presents various advantages (speed of analysis, ease of use, relatively low cost, sensitivity, tolerance against detergents and contaminants, and possibility of automation) and is being currently used in many applications (e.g. peptide/protein identification and quantification, biomarker discovery, and imaging MS). Earlier studies by many groups indicated that moderate reproducibility in relative peptide quantification is a major limitation of MALDI‐TOF MS. In the present work, we examined and demonstrate a clear effect, in cases apparently random, of sample dilution in complex samples (urine) on the relative quantification of peptides by MALDI‐TOF MS. Results indicate that in urine relative abundance of peptides cannot be assessed with confidence based on a single MALDI‐TOF MS spectrum. To account for this issue, we developed and propose a novel method of determining the relative abundance of peptides, taking into account that peptides have individual linear quantification ranges in relation to sample dilution. We developed an algorithm that calculates the range of dilutions at which each peptide responds in a linear manner and normalizes the received peptide intensity values accordingly. This concept was successfully applied to a set of urine samples from patients diagnosed with diabetes presenting normoalbuminuria (controls) and macroalbuminuria (cases).