One of the most common adult respiratory diseases worldwide is chronic obstructive pulmonary disease (COPD). In the UK, there are 30,000 deaths annually, meaning that more die of COPD compared with breast, prostate or colon cancer. However, COPD is treatable and preventable, and early diagnosis and treatment can slow down its progression.
COPD is a disease of multiple sets of conditions that includes emphysema and chronic bronchitis, whose sufferers experience breathlessness and coughing.
In emphysema, the walls between the tiny air sacs in the lungs are destroyed, leading to a smaller number of large sacs that are unable to exchange oxygen and carbon dioxide efficiently.
With chronic bronchitis, the airways in the lungs become inflamed and thickened, and excessive mucus is produced, contributing to coughing and breathing difficulties.
The molecular basis of COPD and the mechanisms that govern its initiation and progression are currently poorly understood.
Researchers in Italy and the USA have tried a new approach to assess the suitability of proteomics methods to identify changes in protein expression that are related to the disease pathophysiology.
The scientists’ analyzed sputum from people affected by COPD to different degrees and compared the protein profiles to identify potential biomarkers.
Sputum was collected from 56 subjects using a saline induction method. The subjects were categorized into five groups: healthy never smokers (group A); healthy smokers with no airflow obstruction or mucus hypersecretion (group B); smokers without airflow obstruction but complaining about cough and phlegm (group C); smokers with airflow obstruction but no emphysema (group D); and the COPD group with airflow obstruction and emphysema (group E).
After the sputum was collected, it was treated with dithiothreitol and centrifuged, and the supernatant was mixed with chloroform–methanol. Proteins at the aqueous interface were extracted, reduced, alkylated and digested with trypsin. The digests were then analyzed by HPLC MS/MS with ESI, and the peptides were matched to proteins using standard MS/MS software.
A total of 203 proteins were identified Most proteins were matched with good certainty, using at least three peptide sequences. A total of 118 proteins were detected for the first time and had not been found in earlier studies on sputum and BALF.
The new proteins included those with high isoelectric points, which are difficult to detect on 2D gels, as well as 21 proteins with molecular masses greater than 150 kDa. The novel proteins identified included mucins, low-molecular-weight immune proteins and eosinophils.
The researchers anticipated tthe several proteins that were detected more frequently in some groups of subjects than others. For example, Zn-2 glycoprotein was discovered in 71% of group A samples, but less than 10% in all other groups. This glycoprotein is known to be downregulated in patients with pulmonary injury.
A protein known as Clara cells was observed in 100% of group A, but in smaller proportions in the other subjects. The researchers suggest that Clara cells have an anti-inflammatory function induced by cigarette smoke; thus, loss of this protective effect may contribute to the progression of COPD.
Other proteins, such as histone H4 (found in group E) were discovered in greater quantities in the diseased subjects. The presence of H4 indicates that nuclear acetylation may be disrupted in COPD and emphysema.
Overall, across the five disease groups, 14 proteins displayed differential expressions. The researchers conclude that closer examination of their functions will lead to a clearer understanding of the disease, while a broader study of their frequency may help establish a panel of proteins to serve as biomarkers for the different stages of COPD.
Source: Casado B, Iadarola P, Pannell LK et al. Protein expression in sputum of smokers and chronic obstructive pulmonary disease patients: a pilot study by CapLC-ESI-Q-TOF. J. Proteome Res. 6(12), 4615–4623 (2007).
Protein separation by magnetic attraction
Scientists in China may have discovered a new protein separation technique to rival gel electrophoresis and ion chromatography. The researchers have deciphered a method to isolate specific proteins from a complex mixture by applying a magnetic field, through joining together iron and gold nanoparticles.
Iron and gold nanoparticles individually possess a number of useful properties. For gold particles, these properties include the ability to bind sulfur-containing thiol molecules, such as cysteine, which means that various biomolecules can be attached to them. While for iron particles, these properties include magnetism. Therefore, combining properties from both iron and gold should prove useful.
However, the fabrication process of these nanoparticles tends to be complicated, requiring some variation of coating an iron nanoparticle with gold. Currently, a team of researchers from Tsinghua University (China), led by Yadong Li, has come up with a simpler method, which involves linking together separate iron and gold nanoparticles.
Li and his team created 60 nm iron nanoparticles with amino groups on their surfaces, to which they attached cysteine molecules. The cysteine molecules will naturally bind with 10 nm gold particles, thus producing combined iron and gold nanoparticles. These nanoparticles retain both the magnetic properties of the iron particle and the biomolecule-binding ability of the gold particle. Taking this into consideration, Li and his colleagues chose to carry out protein separation.
The team then decided to take advantage of the remaining thiol binding sites on the gold particles by attaching nickel ion-containing thiol groups. The researchers did this because nickel ions will readily bind with the amino acid histidine. The group then mixed these iron–gold nanoparticles with the full complement of proteins from a strain of Escherichia coli that had been genetically modified to express arginine kinase – a phosphorylation protein.
Li and colleagues expected that the iron–gold nanoparticles would preferentially bind with the arginine kinase, as it would be the only protein in the mixture displaying numerous histidine molecules. The team then easily separated the arginine kinase from the protein mixture by extracting the nanoparticles with a magnet. Finally, in order to release the attached arginine kinase, the nanoparticles were washed in an imidazole buffer solution.
Following this, Li and his team performed gel electrophoresis on both the separated proteins and on the protein mixture prior to separation, in order to assess the ability of the protein separation technique. As expected, the researchers discovered the protein mixture consisted of a wide range of different size proteins. In contrast, the proteins extracted with the nanoparticles formed a neat line in the gel, almost comprising of pure arginine kinase. In addition, the extracted arginine kinase retained much of its catalytic ability.
The methods are hopeful, but still in the early days of development, and Li and colleagues are working on ways to maintain the initial protein loading capacity.
Source: Bao J, Chen W, Liu T et al. Bifunctional Au-Fe3O4 nanoparticles for protein separation. ACS Nano 1(4), 293–298 (2007).
Simplified solid-phase extraction sample preparation
Diagnostic platform: Strata®-X Virtual SPE Kit
Manufacturer: Phenomenex Inc.
Application: Method development software: optimizes solid-phase extraction sample preparation methods
Phenomenex Inc. (CA, USA) is a manufacturer of separation science consumables that has recently introduced a new support and reference tool that will aid chromatographers to develop and optimize solid-phase extraction (SPE) sample preparation methods.
The new tool kit was designed for users of Phenomenex’s Strata® SPE products, which remove contaminants from samples before separation by HPLC or gas chromatography (GC). The Strata-X Virtual SPE Kit contains method development software applicable for samples in tubes, or 96-well plates with user-defined analytes, sample matrix and analysis goals. The kit also provides educational tools and instructions to simplify method development using either robotics or a vacuum manifold.
Included in the kit are SPE methods for reversed and ion-exchange phases, as well as 24 reference cards with tips and information on the range of Strata-X products with an interactive, user-driven troubleshooting CD guide.
Michael Garriques for Phenomenex commented, “The use of SPE is growing rapidly as more applications demand clean samples, SPE is the most selective sample preparation technique, yet many HPLC and GC users do not fully understand the method development. Our new kit makes it simple.”
Sources: www.phenomenex.com; www.strataSPE.com
Biological variation of elderly biomarkers and implication in biomarker discovery
Proteomic methods have been increasingly applied in the hope of finding new biomarkers; suitable compounds that flag the onset of a particular disease or medical condition.
Most often, biomarkers are characterized by their change in concentration, appearance or disappearance compared with the normal healthy state. For example, in prostate cancer, the level of prostate specific antigen (PSA) in blood is regarded as an indicator.
In order for a protein to be considered a biomarker, the normal range of protein expression within the body must be established in order for an abnormal level to be recognized. In the case of PSA, a concentration of 0–2.5 ng/ml is regarded as normal or low, with higher levels raising concern.
However, it is important to stress that no ‘universal level’ of PSA exists. In actual fact, approximately two out of three men with raised levels of PSA will not have prostate cancer; on the contrary, 20% of men with so-called ‘normal levels’ will.
Since variation in concentration of any biomarker may be affected by factors such as gender, age, lifestyle or health and the possibility of analytical processes introducing variability in the measured value; a large panel of subjects should be tested in order to establish baseline normal levels and statistically determine significant variations between individuals.
In Austria, a team of researchers have examined the variations in the blood platelet proteome from a set of 20 healthy individuals aged 56–100 years. This age group was selected because most patients visiting clinics fall into this age range. Platelet proteins were precipitated from protein-free blood samples and labeled with Cye3 or Cye5 fluorescent dyes for separation by 2D differential gel electrophoresis (DIGE).
An internal standard, made up by pooling platelet samples from all individuals and labeled with Cye2 dye, was mixed with the samples. Each sample was then separated by isoelectric focusing 40 times, 20 each within the isoelectric point ranges of pH 4–7 and 6–9, before further separation by SDS-PAGE. Only reproducible spots that were present in the internal standard in at least 90% of the gels were further studied, generating 484 acidic proteins and 424 alkaline proteins to be measured by fluorescence.
The results were then used to calculate standardized abundances and the coefficients of variation for each spot to give values for the total variation between the samples.
The analysis revealed that the abundance of a particular protein spot had varied by approximately 18% between individuals, much lower than reported total variations for other systems that ranged from 24–60% for primary murine cells, porcine mononuclear or cells and plant tissue.
The team credited the reduction in their findings to their use of the DIGE method. The method produced a lower technical variation (mean of 7%) compared with those from the published methods, which used 2D gel electrophoresis without internal standards. Thus, the internal standard mixture improved the statistical confidence of the analysis.
Moderate changes in protein expression are generally observed, with typical ranges of one to two times their coefficients of variation. Taking into account the low abundance variation of 18% found in this study, future clinical proteomics studies using platelets need to be able to employ an analytical method capable of detecting such small differences. Otherwise, potential biomarkers will remain undiscovered or will be missed during screening.
The researchers also estimate that platelet-based proteomic studies for biomarkers should be designed to include at least 20–25 samples per group, in order to ensure that protein expression changes are observed. Analyzing a fewer number of samples may risk increasing the number of false negatives, evidently meaning that an actual change in a biomarker level would not be detected.
To conclude, the team have argued that these criteria could be extended to other biomarker studies, since the total variations are similar in platelets and human tissue types.
Source: Winkler W, Zellner M, Diestinger M et al. Biological variation of the platelet proteome in the elderly population and its implication for biomarker research. Mol. Cell. Proteomics 7(1), 193–203 (Epub ahead of print).
New NMR software is here to help
Diagnostic platform: ACD/1D NMR Assistant
Manufacturer: Advanced Chemistry Development Inc.
Application: Aids synthetic chemists and NMR users in complex data analyses
New NMR software is here to help synthetic chemists and casual users of NMR by reducing the amount of time required to spend on data analysis. The new product provides chemists with a new, quick and easy spectrum of viewing and processing, including interpretation assistance that will promote faster, better and more independent decisions.
A majority of a synthetic chemist’s time is spent on chemical reactions, synthesizing and purifying compounds, as well as preparing reports for publications, patents and presentations. NMR experiments are routinely performed to ensure a reaction has come to completion. In an open-access laboratory, analyses may involve analyzing a paper printout or trying to figure out how to use expert software for simple effects, such as expanding a spectrum region or re-integrating peaks of interest.
ACD/Labs have designed the ACD/1D NMR Assistant, a new NMR software package designed specifically with the chemist in mind. The software helps the chemist by assigning NMR data and automatically evaluates whether a proposed chemical structure is consistent with a 1H NMR spectrum. If the structure does not match, the software will then specifically highlight where the inconsistencies lie, allowing the chemist to focus on the problematic area of interest.
In addition, the new software provides all analysis and processing features that are accessible in an intuitive and logical manner. The software allows chemists to completely process and analyze a spectrum quickly. If a chemist has to prepare a patent submission, the software can create a multiple report in patent format with one click. Moreover, all spectra, tables and structures can be easily copied to common word processing and desktop publishing applications.
Brent Lefebvre of ACD/Labs commented, “We spent the last several years speaking with chemists and spectroscopists who support chemists. In this time, we learned a great deal about their needs and desires with regards to NMR software,” he continued, “Following the first stage of development, we prereleased the software to groups of synthetic chemists in five different pharmaceutical organizations to garner impressions, suggestions and feedback. We then went back to development with this input and built what we have today. This process was a crucial step in the development of this product. We now have a software package that was built by chemists for chemists.”
Source: www.acdlabs.com