J.E. Araújo

Novel nanocomposites based on a strawberry-like gold- coated magnetite (Fe@Au) for protein separation in multiple myeloma serum samples

A new process to produce magnetite partially coated with strawberry-like gold nanoparticles in aqueous media is reported. The fast response to magnetic fields and optical properties of gold nanoparticle-based colloidal systems are the two main advantages of this new Fe@Au nanomaterial. These advantages allow for the use of this new colloidal nanomaterial for various purposes in proteomics and biomedicine, as proteins can bind to the surface, and the surface can also be functionalized. As proof-of-concept, the new Fe@Au nanoparticles have been assessed in biomarker discovery as a tool for pre-concentration and separation of proteins from complex proteomes. To this end, sera from healthy people were compared with sera from patients diagnosed with multiple myeloma. The application of this new Fe@Au nanomaterial combined with mass spectrometry has allowed for the identification of 53 proteins, and it has also shown that the heat shock protein HSP75 and the plasma protease C1 inhibitor are potential biomarkers for diagnostics and control of multiple myeloma progression.

Use of MALDI-TOF mass spectrometry fingerprinting to characterize Enterococcus spp. and Escherichia coli isolates ☆

UNLABELLED Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify intact bacteria than conventional microbiology and/or molecular biology methods. The MALDI-TOF MS method is potentially applicable in diagnostic laboratories to characterize commensal bacterial species, some of which are major pathogens, from human or animal gastrointestinal tracts. The aim of this study was to analyze at the cluster and statistical level the capacity of MALDI-TOF MS to distinguish between previously characterized enterococci and Escherichia coli isolated from wild birds of the Azores archipelago. Soluble proteins were extracted from intact cell cultures of 60 isolates of Enterococcus spp. and 60 isolates of E. coli by an expedient method. MALDI-TOF MS was used to obtain 1200 mass spectra that were statistically analyzed and compared. A total of 215 distinct mass-to-charge (m/z) peaks were obtained, including a peak at m/z 4428±3, which is exclusively found in spectra from Enterococcus isolates, and peaks at m/z 5379±3 and m/z 6253±3, which are only detected in spectra from E. coli isolates. By processing mass spectra and analyzing them statistically with MassUp software, including principal component analysis (PCA) and clustering, it was possible to correctly distinguish between isolates of Enterococcus and Escherichia genera. The results of the proteomic analysis confirm that these tools could be used to characterize whole bacterial cells. In the future, with an optimized protocol for obtaining plasmid-associated proteins and the further development of bioinformatics methods, it is likely that mass peak characteristic of antimicrobial resistance will be detected, increasing the potential usefulness of MALDI-TOF in routine clinical assays. BIOLOGICAL SIGNIFICANCE This study highlights the importance of MALDI-TOF MS in the rapid and reliable identification of bacteria by whole-cell analysis. The mass spectrometry approach performed in this study further contributes for the microbial biomarker discovery culminating in a preferable bacteria identification and antimicrobial resistance tool for the future of clinical microbiology. This article is part of a Special Issue entitled: HUPO 2014.

A cost-effective method to get insight into the peritoneal dialysate effluent proteome.

Protein depletion with acetonitrile and protein equalization with dithiothreitol have been assessed with success as proteomics tools for getting insight into the peritoneal dialysate effluent proteome. The methods proposed are cost-effective, fast and easy of handling, and they match the criteria of analytical minimalism: low sample volume and low reagent consumption. Using two-dimensional gel electrophoresis and peptide mass fingerprinting, a total of 72 unique proteins were identified. Acetonitrile depletes de PDE proteome from high-abundance proteins, such as albumin, and enriches the sample in apolipo-like proteins. Dithiothreitol equalizes the PDE proteome by diminishing the levels of albumin and enriching the extract in immunoglobulin-like proteins. The annotation per gene ontology term reveals the same biological paths being affected for patients undergoing peritoneal dialysis, namely that the largest number of proteins lost through peritoneal dialysate are extracellular proteins involved in regulation processes through binding. SIGNIFICANCE Renal failure is a growing problem worldwide, and particularly in Europe where the population is getting older. Up-to-date there is a focus of interest in peritoneal dialysis (PD), as it provides a better quality of life and autonomy of the patients than other renal replacement therapies such as haemodialysis. However, PD can only be used during a short period of years, as the peritoneum lost its permeability through time. Therefore to make a breakthrough in PD and consequently contribute to better healthcare system it is urgent to find a group of biomarkers of peritoneum degradation. Here we report on two cost-effective methods for protein depletion in peritoneal dialysate effluent (PDE). The use of ACN and DTT over PDE to deplete high abundant proteins or to equalize the concentration of proteins, respectively, performs well and with similar protein profiles than when the same chemicals are used in human plasma samples. ACN depletes de PDE proteome from large proteins, such as albumin, and enriches the sample in apolipoproteins. DTT equalizes the PDE proteome by diminishing the levels of large proteins such as albumin and enriching the extract in immunoglobulins. Although the number and type of proteins identified are different, the annotation per gene ontology term reveals the same biological paths being affected for patients undergoing peritoneal dialysate. Thus, the largest number of proteins lost through peritoneal dialysate belongs to the group of extracellular proteins involved in regulation processes through binding. As for the searching of biomarkers, DTT seems to be the most promising of the two methods because acts as an equalizer and it allows interrogating more proteins in the same sample.

Proteomics analysis of the peritoneal dialysate effluent reveals the presence of calcium-regulation proteins and acute inflammatory response

BackgroundPeritoneal dialysis (PD) is a form of renal replacement used for advanced chronic kidney disease. PD effluent holds a great potential for biomarker discovery for diagnosis and prognosis. In this study a novel approach to unravelling the proteome of PD effluent based-on dithiothreitol depletion followed by 2D-SDS-PAGE and protein identification using tandem mass spectrometry is proposed.ResultsA total of 49 spots were analysed revealing 25 proteins differentially expressed, among them many proteins involved in calcium regulation.ConclusionsRemarkably, a group of proteins dealing with calcium metabolism and calcium regulation has been found to be lost through peritoneal dialysate effluent, giving thus a potential explanation to the calcification of soft tissues in patients subjected to peritoneal dialysis and kidney injury. Comparison of literature dealing with PD is difficult due to differences in sample treatment and analytical methodologies.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based profiling as a step forward in the characterization of peritoneal dialysis effluent

The aim of this study was to differentiate patients with glomerulonephritis and diabetic nephropathy using (i) peritoneal dialysate effluent, (ii) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and (iii) bioinformatics tools. Profiles of peritoneal dialysate effluent were obtained using (a) sample preparation consisting of protein concentration through centrifugal concentrators and chemical-assisted protein depletion using DL-dithiothreitol, and (b) MALDI-TOF MS. A free open-source bioinformatics tool, Mass-UP, was used to classify such profiles using principal component analysis and hierarchical clustering. The methodology proposed here allows for classifying two different groups of patients with kidney failure, one with chronic glomerulonephritis and other with diabetic nephropathy.

Classifying patients in peritoneal dialysis by mass spectrometry-based profiling.

Protein equalization with dithiothreitol, protein depletion with acetonitrile and the entire proteome were assessed in conjunction with matrix assisted laser desorption ionization time of flight mass spectrometry-based profiling for a fast and effective classification of patients with renal insufficiency. Two case groups were recruited as proof of concept, patients with chronic glomerulonephritis and diabetic nephropathy. Two key tools were used to develop this approach: protein concentration with centrifugal concentrator tubes with 10 KDa cut-off membranes and chemical assisted protein equalization with dithiothreitol or chemical assisted protein depletion with acetonitrile. In-house developed software was used to apply principal component analysis and hierarchical clustering to the profiles obtained. The results suggest that chemical assisted protein equalization with dithiothreitol is a methodology more robust than the other two ones, as the patients were well grouped by principal component analysis or by hierarchical clustering.

A journey through PROTEOSONICS.

Ultrasonic energy is gaining momentum in Proteomics. It helps to shorten many proteomics workflows in an easy and efficient manner. Ultrasonic energy is nowadays used for protein extraction, solubilisation and cell disruption, to speed protein identification, protein quantification, peptide profiling, metal-protein complexes characterisation and imaging mass spectrometry. The present review gives a perspective of the latest achievements in ultrasonic-based sample treatment for proteomics as well as provides the basic concepts and the tools of the trade to efficiently implement this tool in proteomics labs.

POST-NOAC: Portuguese observational study of intracranial hemorrhage on non-vitamin K antagonist oral anticoagulants.

Background There is a lower reported incidence of intracranial hemorrhage with non-vitamin K antagonist oral anticoagulants compared with vitamin K antagonist. However, the functional outcome and mortality of intracranial hemorrhage patients were not assessed. Aims To compare the outcome of vitamin K antagonists- and non-vitamin K antagonist oral anticoagulants-related intracranial hemorrhage. Methods We included consecutive patients with acute non-traumatic intracranial hemorrhage on oral anticoagulation therapy admitted between January 2013 and June 2015 at four university hospitals. Clinical and demographic data were obtained from individual medical records. Intracranial hemorrhage was classified as intracerebral, extra-axial, or multifocal using brain computed tomography. Three-month functional outcome was assessed using the modified Rankin Scale. Results Among 246 patients included, 24 (9.8%) were anticoagulated with a non-vitamin K antagonist oral anticoagulants and 222 (90.2%) with a vitamin K antagonists. Non-vitamin K antagonist oral anticoagulants patients were older (81.5 vs. 76 years, p = 0.048) and had intracerebral hemorrhage more often (83.3% vs. 63.1%, p = 0.048). We detected a non-significant trend for larger intracerebral hemorrhage volumes in vitamin K antagonists patients (p = 0.368). Survival analysis adjusted for age, CHA2DS2VASc, HAS-BLED, and anticoagulation reversal revealed that non-vitamin K antagonist oral anticoagulants did not influence three-month mortality (hazard ratio (HR) = 0.83, 95% confidence interval (CI) = 0.39–1.80, p = 0.638). Multivariable ordinal regression for three-month functional outcome did not show a significant shift of modified Rankin Scale scores in non-vitamin K antagonist oral anticoagulants patients (odds ratio (OR) 1.26, 95%CI 0.55–2.87, p = 0.585). Conclusions We detected no significant differences in the three-month outcome between non-vitamin K antagonist oral anticoagulants- and vitamin K antagonists-associated intracranial hemorrhage, despite unavailability of non-vitamin K antagonist oral anticoagulants-specific reversal agents.

A comprehensive factorial design study of variables affecting protein extraction from formalin-fixed kidney tissue samples

Formalin-fixed tissues are an important source of biological samples for biomedical research. However, proteomics analysis of formalin-fixed tissues has been set aside by formalin-induced protein modifications, which reduce protein extraction efficiency. In this study, a two level full factorial experimental design (2(4)) was used to determine the effects of the extracting conditions in the efficiency of protein recovery from formalin-fixed kidney samples. The following variables were assessed: temperature of extraction, pH of extraction, composition of the extracting buffer and the use ultrasonic energy applied with probe. It is clearly demonstrated that when hating and ultrasonic energy are used in conjunction, a 7-fold increase (p < 0.05) in protein extraction is obtained if compared to extracting conditions for which neither heating nor ultrasonic energy are used. The optimization study was done following the amount of protein extracted by UV (Nanodrop(®) technology, protein ABS at 280 nm) and by 1D SDS-PAGE. Extracts obtained with the optimized conditions were subjected to LC-MALDI MS/MS. A total of 112 proteins were identified.

S2P: A Desktop Application for Fast and Easy Processing of 2D-Gel and MALDI-Based Mass Spectrometry Protein Data

2D-gel electrophoresis is widely used in combination with MALDI-TOF mass spectrometry in order to analyse the proteome of biological samples. It can be used to discover proteins that are differentially expressed between two groups (e.g. two disease conditions) obtaining thus a set of potential biomarkers. Biomarker discovery requires a lot of data processing in order to prepare data for analysis or in order to merge data from different sources. This kind of work is usually done manually, being highly time consuming and distracting the operator or researcher from other important tasks. Moreover, doing this repetitive process in a non-automated, handling-based manner is error-prone, affecting reliability and reproducibility. To overcome these drawbacks, the S2P, an AIBench based desktop multiplatform application, has been specifically created to process 2D-gel and MALDI-mass spectrometry protein identification-based data in a computer-aided manner. S2P is open source and free to all users at http://www.sing-group.org/s2p.