Stefano Sbrignadello

High abundance proteins depletion vs low abundance proteins enrichment: comparison of methods to reduce the plasma proteome complexity.

Background To date, the complexity of the plasma proteome exceeds the analytical capacity of conventional approaches to isolate lower abundance proteins that may prove to be informative biomarkers. Only complex multistep separation strategies have been able to detect a substantial number of low abundance proteins (<100 ng/ml). The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity columns or, alternatively, the enrichment of by the use of solid phase hexapeptides ligand libraries. Methodology/Principal Findings Here we present a direct comparison of these two approaches. Following either approach, the plasma sample was further fractionated by SCX chromatography and analyzed by RP-LC-MS/MS with a Q-TOF mass spectrometer. The depletion of the 20 most abundant plasma proteins allowed the identification of about 25% more proteins than those detectable following low abundance proteins enrichment. The two datasets are partially overlapping and the identified proteins belong to the same order of magnitude in terms of plasma concentration. Conclusions/Significance Our results show that the two approaches give complementary results. However, the enrichment of low abundance proteins has the great advantage of obtaining much larger amount of material that can be used for further fractionations and analyses and emerges also as a cheaper and technically simpler approach. Collectively, these data indicate that the enrichment approach seems more suitable as the first stage of a complex multi-step fractionation protocol.

Shape of glucose, insulin, C-peptide curves during a 3-h oral glucose tolerance test: any relationship with the degree of glucose tolerance?

We aimed to analyze the shape of the glucose, insulin, and C-peptide curves during a 3-h oral glucose tolerance test (OGTT). Another aim was defining an index of shape taking into account the whole OGTT pattern. Five-hundred ninety-two OGTT curves were analyzed, mainly from women with former gestational diabetes, with glycemic concentrations characterized by normal glucose tolerance (n = 411), impaired glucose metabolism (n = 134), and Type 2 diabetes (n = 47). Glucose curves were classified according to their shape (monophasic, biphasic, triphasic, and 4/5-phases), and the metabolic condition of the subjects, divided according to the glucose shape stratification, was analyzed. Indices of shape based on the discrete second-order derivative of the curve patterns were also defined. We found that the majority of the glucose curves were monophasic (n = 262). Complex shapes were less frequent but not rare (n = 37 for the 4/5-phases shape, i.e., three peaks). There was a tendency toward the amelioration of the metabolic condition for increasing complexity of the shape, as indicated by lower glucose concentrations, improved insulin sensitivity and β-cell function. The shape index computed on C-peptide, WHOSH(CP) (WHole-Ogtt-SHape-index-C-peptide), showed a progressive increase [monophasic: 0.93 ± 0.04 (dimensionless); 4/5-phases: 1.35 ± 0.14], and it showed properties typical of β-cell function indices. We also found that the type of glucose shape is often associated to similar insulin and C-peptide shape. In conclusion, OGTT curves can be characterized by high variability, and complex OGTT shape is associated with better glucose tolerance. WHOSH(CP) (WHole-Ogtt-SHape-index) may be a powerful index of β-cell function much simpler than model-based indices.

A Low Frequency Electromagnetic Sensor for Indirect Measurement of Glucose Concentration: In Vitro Experiments in Different Conductive Solutions

In recent years there has been considerable interest in the study of glucose-induced dielectric property variations of human tissues as a possible approach for non-invasive glycaemia monitoring. We have developed an electromagnetic sensor, and we tested in vitro its ability to estimate variations in glucose concentration of different solutions with similarities to blood (sodium chloride and Ringer-lactate solutions), differing though in the lack of any cellular components. The sensor was able to detect the effect of glucose variations over a wide range of concentrations (∼78–5,000 mg/dL), with a sensitivity of ∼0.22 mV/(mg/dL). Our proposed system may thus be useful in a new approach for non-invasive and non-contact glucose monitoring.

Influence of Increasing BMI on Insulin Sensitivity and Secretion in Normotolerant Men and Women of a Wide Age Span

The impact of sex and age on glucose metabolism in the development of overweight/obesity is a matter of debate. We hypothesized that insulin sensitivity (IS) and β‐cell function (BF) in a normal white population will differ between males and females and aimed to evaluate the possible effects of BMI and age on metabolic parameters of both sexes. This study is a cross‐sectional analysis of the general community. IS was measured with quantitative insulin sensitivity check index (QUICKI) and oral glucose insulin sensitivity (OGIS) and BF with the insulinogenic index during 75‐g 2‐h oral glucose‐tolerance tests (OGTTs). We studied 611 females and 361 males with normal glycemia according to both fasting and 2‐h glucose (85 ± 0.3 mg/dl (means ± SE) in females and 89 ± 0.4 in males (P < 0.0001), and 93 ± 1 in females and 89 ± 1 in males (P = 0.005), respectively). Females were younger (37 ± 1 years) than males (40 ± 1, P < 0.0001), but no difference was found in mean BMI (BMI = 25.8 ± 0.2 kg/m2 in both). Students two‐sample t‐test was used for simple comparison between and within genders, multiple linear regressions to account for covariates. During the OGTT, females had lower glucose (area under the curve (AUC) 133 ± 1 mg/ml·2 h vs. 148 ± 2; P < 0.00001), while insulinemia was comparable (AUC 5.3 ± 0.1 mU/ml·2 h vs. 5.7 ± 0.2, P = 0.15). IS remained higher in females (473 ± 3 ml/min/m2 vs. 454 ± 3, P < 0.0001) also after having accounted for age and BMI (P = 0.015). No difference was observed in fasting insulin or BF. However, BF increased by 46% with BMI and when accounting for age and BMI, BF of females was significantly higher (P < 0.0001). Because IS and BF are higher in females than in males, sex should be considered in metabolic studies and overweight/obese populations.

Electroimpedance Spectroscopy for the Measurement of the Dielectric Properties of Sodium Chloride Solutions at Different Glucose Concentrations

We investigated possible variations of impedance values in samples of sodium chloride solution (sodium chloride 0.9%) with glucose at different concentrations, ranging from 5000 to around 75 mg/dL. The sodium chloride solution (either saline physiological solution) was chosen since it has similarities to blood but no cell components, which may be confounding factors in this study. Special focus was on the effect of stirring and of temperature variations on the impedance spectrum of samples at different glucose concentrations. We found that variations in glucose concentration directly affect the impedance modulus of the sample both in static conditions and in dynamic conditions due to stirring, as well as at both room temperature and at increased temperature. In fact, even if the impedance variations were often small (around 3-4 mΩ per mg/dL), they were usually clearly measurable. These findings may be the basis for possible development of a new approach, based on impedance technology, for the noninvasive monitoring of glycaemia.

The inter- and intra-operator variability in manual spot segmentation and its effect on spot quantitation in two-dimensional electrophoresis analysis

Separation of complex mixtures of proteins by 2‐DE is a fundamental component of current proteomic technology. Quantitative analysis of the images generated by digitization of such gels is critical for identifying alterations in protein expression within a given biological system. Software packages are designed for this purpose. The accurate definition of protein spot boundaries, using a suitable method of image segmentation, is a key requirement for image analysis. It is often necessary for operators to intervene manually to correct mistakes in spot segmentation; therefore operator subjectivity and differences in ability can weaken the analysis. We estimated the error in spot quantification after manual spot segmentation, which was performed by different operators, using two different software packages. Our results clearly show that this operation was associated with significant inter‐ and intra‐variability and an overestimation of subsequent spot intensity, especially when spots were weak. For comparative studies, we suggest separately analysing spots which have been manually segmented by imposing a requirement for at least a threefold difference in spot intensity in addition to use of statistical tests.

Delta2D and Proteomweaver: Performance evaluation of two different approaches for 2-DE analysis

2‐DE is a fundamental technology used in proteomics research. However, despite its high capacity to simultaneously separate several proteins for subsequent identification and quantitative comparison studies, a drawback for this technique is its limited reproducibility, especially when comparing data from different laboratories. 2‐DE‐related variability can be broadly divided into two categories: experimental and post‐experimental. Experimental variability depends on physical and chemical parameters, whereas post‐experimental variability arises when gels are analyzed by different software packages, particularly when different workflows are followed. In this paper, we compared the analysis performance of two software packages, Delta2D and Proteomweaver, using both standard and experimental gel images. Using standard gel images, the false negative spot count was 50% lower, the false positive count was 77% lower, the true positive count was 19% higher and spot matching was 4% higher in Delta2D when compared to Proteomeweaver. Using experimental gel images, we found that the total amount of time taken to complete the analysis with Delta2D was 30% that of the time needed with Proteomweaver and required fewer user interventions. The differences between ease of use and workflow strategy of these programs is discussed.

Operator- and software-related post-experimental variability and source of error in 2-DE analysis

In the field of proteomics, several approaches have been developed for separating proteins and analyzing their differential relative abundance. One of the oldest, yet still widely used, is 2-DE. Despite the continuous advance of new methods, which are less demanding from a technical standpoint, 2-DE is still compelling and has a lot of potential for improvement. The overall variability which affects 2-DE includes biological, experimental, and post-experimental (software-related) variance. It is important to highlight how much of the total variability of this technique is due to post-experimental variability, which, so far, has been largely neglected. In this short review, we have focused on this topic and explained that post-experimental variability and source of error can be further divided into those which are software-dependent and those which are operator-dependent. We discuss these issues in detail, offering suggestions for reducing errors that may affect the quality of results, summarizing the advantages and drawbacks of each approach.

SDS‐PAGE and two‐dimensional maps in a radial gel format

A novel method for performing 2‐D map analysis is here reported, consisting in a modification of the second dimension run, which is performed not in a conventional square‐ or rectangular‐size gel, but in a radial surface. This has the advantage of permitting resolution of closely adjacent bands, representing strings of isoforms of similar or identical mass but of closely spaced isoelectric points. When used in a mono‐dimensional, SDS‐PAGE format, this system allows the simultaneous running of 62 sample tracks. Examples are given of separation of plasma and urinary proteins.

Improved instrumentation for large‐size two‐dimensional protein maps

Novel instrumentation for performing large‐size (>25 cm) 2‐D maps is reported here. To perform the first dimension, we developed a power supply that can deliver a voltage of up to 15 000 V and allows regulation of current (up to 200 μA) onto each individual focusing IPG strip. The IEF strip tray can accommodate up to 12 IPG strips and the electrodes slide on a ruler, thus permitting running strips of any length up to 45 cm. In addition, this apparatus also includes a second power supply that allows the performance of electrophoresis at high amperage (400 mA) and a Peltier system that allows a 10–80°C temperature control.