Hugo M. Santos

Trends in ultrasonic-based equipment for analytical sample treatment

The recent developments achieved in ultrasonic equipment urges the need for the revision of its applications in analytical chemistry. In the present work, the last ultrasonic devices are easily presented and their applications for sample treatment are critically discussed. Comments are given on edge areas of research, such as proteomics or polymer science, which are presently taking advantage of ultrasonic sample treatments. Future applications and trends for ultrasonic-based handling approaches are also given and commented.

Overview on modern approaches to speed up protein identification workflows relying on enzymatic cleavage and mass spectrometry-based techniques.

Recent tools addressed to accelerate the different steps of the sample treatment for protein identification in modern workflows are reviewed and critically commented in this manuscript. Heating, microspin columns, ultrasonic energy, high pressure, infrared energy, microwave energy, alternating electric fields and microreactors are outlined as useful tools that can be used to accelerate all or some of the following steps for in-gel or in-liquid based approaches for protein identification: (i) protein dissolution/denaturation, (ii) protein reduction, (iii) protein alkylation and (iv) protein digestion. The advantages and drawbacks, along with the main differences among the different tools are also commented. Future prospects for hyphenation of methods are also discussed. Researchers are informed also in this work regarding the main problems to be found when implementing any of the above mentioned methods.

A comparison of depletion versus equalization for reducing high-abundance proteins in human serum

In this work three methods to diminish the content of most highly abundant proteins in human serum have been studied and compared. Protein depletion with ACN or DTT and protein equalization with the ProteoMiner™ (PM) have been assessed by 1‐D gel electrophoresis and MS. After treatment 5, 18 and 9 major proteins within the 20 most abundant proteins in serum were identified for the ACN, DTT and PM methods, respectively. The ACN method was efficient for depleting high molecular weight proteins, over 75 KDa, resulting in 10±4% (n=3) of the total protein content remaining in the depleted serum. In addition, 75% of the proteins belonging to the group of the 20 most abundant proteins were not detected, making this depletion strategy a cheap alternative to expensive commercial tools regularly used for removing high abundance proteins from serum. The ACN extract was found rich in apolipoproteins. The dithithreitol method promotes the precipitation of proteins rich in disulfide bonds, mainly albumin, with 73±7% (n=3) of the total protein content remaining in the depleted serum, which was found rich in immunoglobulins. The PM method compresses the dynamic range of the serum proteins, rendering an extract containing 16±2% (n=3) of the total initial protein content. The extract was found to be rich in both apolipoproteins and immunoglobulins. As a general rule the DTT and PM methods provide a compression of the dynamic range of serum protein concentrations while the ACN method allows an effective depletion of the protein fraction above 72 KDa.

Latest developments in sample treatment for 18O-isotopic labeling for proteomics mass spectrometry-based approaches: a critical review.

Nowadays isotopic (18)O-labeling of peptides has recalled the attention of researchers due to its simplicity of application and high versatility for proteomics studies. Protein quantification, differential peptide mass mapping, studies regarding proteins overexpressed or underexpressed, or the searching of biomarkers can be accomplished by using (18)O-labeling. In this critical review we comment on the different ways in which (18)O-labeling can be done, highlighting the key parameters of the different sample treatments to obtain a reliable and reproducible labeling. In addition we describe and compare the latest improvement in terms of sample treatment that allows to reduce the handling and to increase the throughput for this sample treatment. Finally, we hypothesize on the future trends of these methods under the light of the new technological advances to speed protein cleavage.

Mass-Up: an all-in-one open software application for MALDI-TOF mass spectrometry knowledge discovery.

BackgroundMass spectrometry is one of the most important techniques in the field of proteomics. MALDI-TOF mass spectrometry has become popular during the last decade due to its high speed and sensitivity for detecting proteins and peptides. MALDI-TOF-MS can be also used in combination with Machine Learning techniques and statistical methods for knowledge discovery. Although there are many software libraries and tools that can be combined for these kind of analysis, there is still a need for all-in-one solutions with graphical user-friendly interfaces and avoiding the need of programming skills.ResultsMass-Up, an open software multiplatform application for MALDI-TOF-MS knowledge discovery is herein presented. Mass-Up software allows data preprocessing, as well as subsequent analysis including (i) biomarker discovery, (ii) clustering, (iii) biclustering, (iv) three-dimensional PCA visualization and (v) classification of large sets of spectra data.ConclusionsMass-Up brings knowledge discovery within reach of MALDI-TOF-MS researchers. Mass-Up is distributed under license GPLv3 and it is open and free to all users at http://sing.ei.uvigo.es/mass-up.

Development of a fast and efficient ultrasonic-based strategy for DNA fragmentation

Several ultrasound-based platforms for DNA sample preparation were evaluated in terms of effective fragmentation of DNA (plasmid and genomic DNA)-ultrasonic probe, sonoreactor, ultrasonic bath and the newest Vialtweeter device. The sonoreactor showed the best efficiency of DNA fragmentation while simultaneously assuring no cross-contamination of samples, and was considered the best ultrasonic tool to achieve effective fragmentation of DNA at high-throughput and avoid sample overheating. Several operation variables were studied-ultrasonication time and amplitude, DNA concentration, sample volume and sample pre-treatment-that allowed optimisation of a sonoreactor-based strategy for effective DNA fragmentation. Optimal operating conditions to achieve DNA fragmentation were set to 100% ultrasonic amplitude, 100microL sample volume, 8min ultrasonic treatment (2min/sample) for a DNA concentration of 100microgmL(-1). The proposed ultrasonication strategy can be easily implemented in any laboratory setup, providing fast, simple and reliable means for effective DNA sample preparation when fragmentation is critical for downstream molecular detection and diagnostics protocols.

Fast human serum profiling through chemical depletion coupled to gold-nanoparticle-assisted protein separation

The use of chemical protein depletion in conjunction with gold-based nanoparticles for fast matrix assisted laser desoption ionization time of flight mass spectrometry-based human serum profiling was assessed. The following variables influencing the process were optimized: (i) amount of nanoparticles, (ii) sample pH, (iii) amount of protein and (iv) incubation time. pH was found the most important factor to be controlled, with an optimum range comprised between 5.8 and 6.4. The minimum incubation time to obtain an adequate profiling was 30 min. Using this approach, serum from five patients with lymphoma, five patients with myeloma and from two healthy volunteers were correctly classified using Principal component analysis.

Metallothionein responses in the Asiatic clam (Corbicula fluminea) after exposure to trivalent arsenic

Abstract The main objective of this work was to evaluate arsenic effects on metallothionein (MT) induction by exposing a freshwater Asiatic clam (Corbicula fluminea) to different concentrations of this metalloid. The presence of MT-like proteins was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and compared with a standard rabbit MT. In addition, the polarographic response showed good correspondence between standard MT and MT-like curves from C. fluminea, allowing MT quantification. The results show that clams exposed to different concentrations of arsenic are able to induce significant levels of MTs. Although variability was found in MT induction, significant differences in MT levels were found after 28 days of exposure in all treatments in comparison with the controls, suggesting that exposure to arsenic induced MT-like proteins in C. fluminea.

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.