Modeling SILAC Data to Assess Protein Turnover in a Cellular Model of Diabetic Nephropathy.

Protein turnover rate is finely regulated through intracellular mechanisms and signals that are still incompletely understood but that are essential for the correct function of cellular processes. Indeed, a dysfunctional proteostasis often impacts the cell's ability to remove unfolded, misfolded, degraded, non-functional, or damaged proteins. Thus, altered cellular mechanisms controlling protein turnover impinge on the pathophysiology of many diseases, making the study of protein synthesis and degradation rates an important step for a more comprehensive understanding of these pathologies. In this manuscript, we describe the application of a dynamic-SILAC approach to study the turnover rate and the abundance of proteins in a cellular model of diabetic nephropathy. We estimated protein half-lives and relative abundance for thousands of proteins, several of which are characterized by either an altered turnover rate or altered abundance between diabetic nephropathic subjects and diabetic controls. Many of these proteins were previously shown to be related to diabetic complications and represent therefore, possible biomarkers or therapeutic targets. Beside the aspects strictly related to the pathological condition, our data also represent a consistent compendium of protein half-lives in human fibroblasts and a rich source of important information related to basic cell biology.

Are there dietary requirements for dispensable amino acids and if so, how do we assess requirements?

PURPOSE OF REVIEW: Nonessential amino acids (NEAAs) represent a relevant portion of dietary protein(s), yet their requirement(s) has not been determined. Despite their nature as dispensable substrates, should either shortage of any NEAA precursor or impaired synthetic reactions occur, NEAA dietary intake may become insufficient. The purpose of this review is to discuss recent hypotheses and data on individual NEAA requirements and metabolism. RECENT FINDINGS: A minimum total NEAA requirement can simply be estimated by subtraction of essential amino acid (EAA) total RDAs, from recommended 'safe' protein intake. By this calculation, NEAA intake would account for two to three times that of the EAAs, under nitrogen-balance conditions. Although the α-amino-nitrogen of the NEAAs is 'not essential', yet it must be furnished by a common pool contributed by both EAAs and NEAAs. Thus, an increased demand for NEAAs may deprive the α-amino-nitrogen body pool(s) possibly limiting the NEAA de novo synthesis itself. Conversely, shortage of NEAAs may require more EAAs to maintain the nitrogen pool. Conditions of increased requirements could those of unbalanced diets, EAA intake below RDA, pregnancy, or else. In addition, the 'obligatory nitrogen losses' may consume NEAAs too. A novel approach to estimate NEAA 'requirements' in humans is proposed. SUMMARY: Methods to estimate NEAA requirements in humans should be the object of further studies.

A High-Fiber Diet Decreases Postabsorptive Protein Turnover but Does Not Alter Insulin Sensitivity in Men with Type 1 Diabetes Mellitus.

BACKGROUND: High-fiber diets (HFDs) are recommended in the diet of persons with diabetes, yet such diets can impair macronutrient digestion and/or absorption, modify insulin sensitivity, and reset metabolism. OBJECTIVES: We studied the effects of a HFD on the kinetics of whole-body protein, a macronutrient that could be affected by dietary fiber, in type 1 diabetes mellitus (T1DM), under both basal-low insulinemic and hyperinsulinemic conditions. METHODS: Eight men with T1DM (body mass index range: 21.8-27.8 kg/m2) were studied twice - before and after the addition of guar gum (∼15 g/d) to their usual diet for ∼4 mo. Whole-body protein degradation (i.e., the rate of appearance [Ra] of endogenous leucine), leucine disposal to protein synthesis (PS), deamination, and reamination, were determined before and after the HFD, both in the postabsorptive state and following a euglycemic, hyperinsulinemic, hyperaminoacidemic clamp, using isotope dilution methods. RESULTS: After the HFD, mean values (± SEs) for postabsorptive leucine Ra decreased by ∼20%: from 2.52 (0.15) to 2.03 (0.16) µmol x kg-1 x min-1, P < 0.049, after vs. before the HFD respectively. PS also decreased, by ∼25%: from 2.03 (0.15) to 1.57 (0.15), P < 0.045. Leucine concentration (P = 0.1) and reamination (P = 0.095) decreased moderately, whereas deamination was unchanged. Following the clamp, plasma amino acid concentrations (P < 0.001), leucine deamination (+ ∼50%, P < 0.00002), reamination (+ ∼30%, P < 0.0007), and PS (+ ∼35%, P < 0.00001) were all increased compared with postabsorptive state values, whereas endogenous leucine Ra was suppressed (by 15%, P < 0.00001, and by 25%, P < 0.001, with the primary or the reciprocal pool models, respectively). No significant differences in these insulin effects before compared with after the HFD were observed. Metabolic control (glycated hemoglobin), daily insulin requirement, and insulin-mediated glucose disposal were unchanged after the HFD. CONCLUSIONS: A HFD downregulates postabsorptive protein turnover in men with T1DM, by decreasing both protein degradation and synthesis, possibly due to a subtle decrease and/or delay in amino acid absorption. It does not significantly affect the insulin (and amino acid sensitivity) to protein turnover, glucose disposal, and metabolic control.

The contribution of muscle, kidney, and splanchnic tissues to leucine transamination in humans.

The first steps of leucine utilization are reversible deamination to α-ketoisocaproic acid (α-KIC) and irreversible oxidation. Recently, the regulatory role of leucine deamination over oxidation was underlined in rodents. Our aim was to measure leucine deamination and reamination in the whole body, in respect to previously determined rates across individual organs, in humans. By leucine and KIC isotope kinetics, we determined whole-body leucine deamination and reamination, and we compared these rates with those already reported across the sampled organs. As an in vivo counterpart of the "metabolon" concept, we analysed ratios between oxidation and either deamination or reamination. Leucine deamination to KIC was greater than KIC reamination to leucine in the whole body (p = 0.005), muscles (p = 0.005), and the splanchnic area (p = 0.025). These rates were not significantly different in the kidneys. Muscle accounted for ≈60% and ≈78%, the splanchnic bed for ≈15% and ≈15%, and the kidney for ≈12% and ≈18%, of whole-body leucine deamination and reamination rates, respectively. In the kidney, percent leucine oxidation over either deamination or reamination was >3-fold greater than muscle and the splanchnic bed. Skeletal muscle contributes by the largest fraction of leucine deamination, reamination, and oxidation. However, in relative terms, the kidney plays a key role in leucine oxidation.

Leucine Transamination Is Lower in Middle-Aged Compared with Younger Adults.

Background: Insulin and age affect leucine (and protein) kinetics in vivo. However, to our knowledge, leucine transamination and the effects of insulin have not been studied in participants of different ages.Objective: The aims of the study were to measure whole-body leucine deamination to α-ketoisocaproate (KIC) and KIC reamination to leucine in middle-aged and younger healthy adults, both in the postabsorptive state and after hyperinsulinemia.Methods: Younger (mean ± SE age: 26 ± 2 y) and middle-aged (54 ± 3 y) healthy men and women were enrolled. Isotope dilution methods with 2 independent leucine and KIC tracers, a dual isotope model and the euglycemic, hyperinsulinemic clamp technique, were used.Results: Leucine deamination [expressed as µmol/(kg × min)] was consistently greater than KIC reamination. In middle-aged adults, postabsorptive leucine deamination (0.77 ± 0.05), reamination (0.49 ± 0.04), and net deamination (0.28 ± 0.04) were ∼30% lower than in the younger group (deamination: 1.12 ± 0.07; reamination: 0.70 ± 0.09; net deamination: 0.42 ± 0.04) (P < 0.002, P < 0.05, and P < 0.015, respectively). After the hyperinsulinemic clamp, plasma leucine and KIC concentrations were reduced by ∼50% in both groups. Deamination and reamination also were suppressed by ∼40-50% in both groups (P < 0.001); however, they remained lower [-35% (P = 0.02) and -25% (P = 0.036), respectively] in the middle-aged than in the younger participants. The leucine rate of appearance and its suppression by insulin were similar in the middle-aged and in the younger subjects. By using both the basal and the clamp data, deamination was directly correlated with the plasma leucine concentration (r = 0.61, P < 0.0025) and reamination to that of plasma KIC (r = 0.79, P < 0.00002). Expressing the data relative to lean body mass did not substantially alter the results.Conclusions: Leucine deamination and reamination are lower in middle-aged than in younger adults, both in the postabsorptive and in the insulin-stimulated state. In middle age, a decreased net leucine transamination may represent a mechanism to spare this essential amino acid.

Effects of CK2 inhibition in cultured fibroblasts from Type 1 Diabetic patients with or without nephropathy.

CK2 is a multifunctional, pleiotropic protein kinase involved in the regulation of cell proliferation and survival. Since fibroblasts from Type 1 Diabetes patients (T1DM) with Nephropathy exhibit increased proliferation, we studied cell viability, basal CK2 expression and activity, and response to specific CK2 inhibitors TBB (4,5,6,7-tetrabenzotriazole) and CX4945, in fibroblasts from T1DM patients either with (T1DM+) or without (T1DM-) Nephropathy, and from healthy controls (N). We tested expression and phosphorylation of CK2-specific molecular targets. In untreated fibroblasts from T1DM+, the cell viability was higher than in both N and T1DM-. CK2 inhibitors significantly reduced cell viability in all groups, but more promptly and with a larger effect in T1DM+. Differences in CK2-dependent phosphorylation sites were detected. In conclusion, our results unveil a higher dependence of T1DM+ cells on CK2 for their survival, despite a similar expression and a lower activity of this kinase compared with those of normal cells.

Circulating myeloid calcifying cells have antiangiogenic activity via thrombospondin-1 overexpression.

Myeloid calcifying cells (MCCs) represent a subpopulation of human monocytes with procalcific potential and are characterized by coexpression of osteocalcin (OC) and bone alkaline phosphatase (BAP). Herein, an in-depth proteomic investigation of MCCs based on fluorescence-activated cell sorting, protein extraction and digestion, isobaric tag for relative and absolute quantitation labeling, fractionation, and analysis on matrix-assisted laser desorption/ionization-time of flight/time of flight and LTQ Orbitrap mass spectrometers identified and quantified more than 700 proteins and revealed pathways activated in OC(+)BAP(+) MCCs compared with those in OC(-)BAP(-) cells. Among proteins referable to angiogenesis, the thrombospondin-1 pathway was markedly up-regulated in MCCs vs. control cells. Up-regulation of the thrombospondin-1 pathway was confirmed by a genome-wide transcriptional analysis. Using in vitro and in vivo angiogenesis assays, we found that freshly isolated MCCs and cultured MCCs display an antiangiogenic function by means of both paracrine activity (conditioned medium) and altered spatial localization in cocultures with endothelial cells. Thrombospondin-1 inhibition by antibody-mediated neutralization or gene knockdown restored the angiogenic activity of OC(+)BAP(+) MCCs toward normal values and abolished the antiangiogenic effects of MCC conditioned medium. These data indicate that circulating MCCs exert antiangiogenic activity by virtue of their overexpression of thrombospondin-1. The study highlights the successful identification and validation of a pathogenic pathway by a gold standard proteomic/transcriptomic analysis of blood cells.

Stepwise Discovery of Insulin Effects on Amino Acid and Protein Metabolism.

A clear effect of insulin deficiency and replacement on body/muscle mass was a landmark observation at the start of the insulin age. Since then, an enormous body of investigations has been produced on the pathophysiology of diabetes mellitus from a hormonal/metabolic point of view. Among them, the study of the effects of insulin on body growth and protein accretion occupies a central place and shows a stepwise, continuous, logical, and creative development. Using a metaphor, insulin may be viewed as a director orchestrating the music (i.e., the metabolic effects) played by the amino acids and proteins. As a hormone, insulin obviously does not provide either energy or substrates by itself. Rather, it tells cells how to produce and utilize them. Although the amino acids can be released and taken up by cells independently of insulin, the latter can powerfully modulate these movements. Insulin regulates (inhibits) protein degradation and, in some instances, stimulates protein synthesis. This review aims to provide a synthetic and historical view of the key steps taken from the discovery of insulin as an "anabolic hormone", to the in-depth analysis of its effects on amino acid metabolism and protein accretions, as well as of its interaction with nutrients.

Beta-Glucans of Cereals: Functional and Technological Properties.

ß-glucans are a polymeric dietary fiber characterized by ß-(1,3) and ß-(1,4) glycosidic bonds between glucose monomers. They are often used as thickeners, stabilizers, and fat substitutes in foods. The functional and technological quality of ß-glucans is attributed to their origin/source, molecular weight, and structural properties. In particular, physical treatments such as drying, cooking, freezing, and refrigeration influence their molecular, morphological, and rheological characteristics. In addition to their useful technical qualities, ß-glucans are recognized for their numerous beneficial impacts on human health. For this reason, the European Food Safety Authority (EFSA) has provided a positive opinion on health claims such as cholesterol lowering and hypoglycemic properties relating to oats and barley ß-glucans. This paper provides insight into the properties of ß-glucans and different treatments affecting their characteristics and then reviews the latest research on ß-glucans as a functional ingredient for people with type 2 diabetes mellitus (T2DM).

Anabolic Resistance in the Pathogenesis of Sarcopenia in the Elderly: Role of Nutrition and Exercise in Young and Old People.

The development of sarcopenia in the elderly is associated with many potential factors and/or processes that impair the renovation and maintenance of skeletal muscle mass and strength as ageing progresses. Among them, a defect by skeletal muscle to respond to anabolic stimuli is to be considered. Common anabolic stimuli/signals in skeletal muscle are hormones (insulin, growth hormones, IGF-1, androgens, and ß-agonists such epinephrine), substrates (amino acids such as protein precursors on top, but also glucose and fat, as source of energy), metabolites (such as ß-agonists and HMB), various biochemical/intracellular mediators), physical exercise, neurogenic and immune-modulating factors, etc. Each of them may exhibit a reduced effect upon skeletal muscle in ageing. In this article, we overview the role of anabolic signals on muscle metabolism, as well as currently available evidence of resistance, at the skeletal muscle level, to anabolic factors, from both in vitro and in vivo studies. Some indications on how to augment the effects of anabolic signals on skeletal muscle are provided.

Skin fibroblasts as a tool for identifying the risk of nephropathy in the type 1 diabetic population.

Human fibroblasts in culture have been employed as an in vitro system to investigate some pathophysiological mechanisms of diabetes mellitus also associated with the development of diabetic nephropathy. In fact, there is increasing evidence that genetic factors either convey the risk of, or protect from, diabetic nephropathy and that the expression profiles and/or the behaviour of the cultured skin fibroblasts from type 1 diabetic patients could reflect these genetic influences. On the other hand, alterations could be attributable not only to changes in DNA sequence, but also to epigenetic factors. Our aim is to make a critical overview of the studies involving primary cultures of skin fibroblasts as tools to investigate the pathophysiology of diabetic nephropathy performed until now in this area. Cultured skin fibroblasts could be useful not only for the identification of patients at risk of developing diabetic renal disease, but also for a better understanding of the complex multifactorial mechanisms leading to the long-term complications in diabetes.

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.

High confidence and sensitivity four-dimensional fractionation for human plasma proteome analysis.

Reducing the complexity of plasma proteome through complex multidimensional fractionation protocols is critical for the detection of low abundance proteins that have the potential to be the most specific disease biomarkers. Therefore, we examined a four dimension profiling method, which includes low abundance protein enrichment, tryptic digestion and peptide fractionation by IEF, SCX and RP-LC. The application of peptide pI filtering as an additional criterion for the validation of the identifications allows to minimize the false discovery rate and to optimize the best settings of the protein identification database search engine. This sequential approach allows for the identification of low abundance proteins, such as angiogenin (10(-9) g/L), pigment epithelium growth factor (10(-8) g/L), hepatocyte growth factor activator (10(-7) g/L) and thrombospondin-1 (10(-6) g/L), having concentrations similar to those of many other growth factors and cytokines involved in disease pathophysiology.

Neither Incretin or Amino Acid Responses, nor Casein Content, Account for the Equal Insulin Response Following Iso-Lactose Loads of Natural Human and Cow Milk in Healthy Young Adults.

Human milk contains <50% less protein (casein) than cow milk, but is equally effective in insulin secretion despite lower postingestion hyperaminoacidemia. Such potency of human milk might be modulated either by incretins (glucagon-like polypeptide-1,GLP-1); glucose-inhibitory-polypeptide, GIP), and/or by milk casein content. Healthy volunteers of both sexes were fed iso-lactose loads of two low-protein milks, i.e., human [Hum] (n = 8) and casein-deprived cow milk (Cow [↓Cas]) (n = 10), as well as loads of two high-protein milks, i.e., cow (n = 7), and casein-added human-milk (Hum [↑Cas]) (n = 7). Plasma glucose, insulin, C-peptide, incretins and amino acid concentrations were measured for 240'. All milks induced the same transient hyperglycemia. The early [20'−30'] insulin and C-peptide responses were comparable among all milk types apart from the low-protein (Cow [↓Cas]) milk, where they were reduced by <50% (p < 0.05 vs. others). When comparing the two high-protein milks, GLP-1 and GIP [5'−20'] responses with the (Hum [↑Cas]) milk were lower (by ≈2−3 fold, p < 0.007 and p < 0.03 respectively) than those with cow milk, whereas incretin secretion was substantially similar. Plasma amino acid increments largely reflected the milk protein content. Thus, neither casein milk content, nor incretin or amino acid concentrations, can account for the specific potency of human milk on insulin secretion, which remains as yet unresolved.

Effects of chronic metabolic acidosis on splanchnic protein turnover and oxygen consumption in human beings.

BACKGROUND & AIMS: Although metabolic acidosis stimulates protein catabolism, its effects on splanchnic protein turnover and energy expenditure have not been measured in human beings. We investigated the effects of chronic metabolic acidosis (CMA) on splanchnic protein dynamics and oxygen consumption in human beings by using a leucine tracer and mass-balance techniques. METHODS: Five subjects were studied after 6 days of HCl-, CaCl(2)-, and NH(4)Cl-induced acidosis; 8 subjects served as controls. Blood samples were collected from the radial artery and the hepatic veins. Measurements were performed on plasma and whole-blood samples. RESULTS: Based on plasma measurements, subjects who had undergone CMA had lower rates of splanchnic proteolysis (-35%) and protein synthesis (-50%; P < .05) than controls, as well as a negative leucine kinetic balance (-6.81 +/- 2.48 micromol/kg/min/1.73 m(2) body surface [BS](-1)), compared with the neutral balance in control plasma samples (0.76 +/- 2.11 micromol/kg/min/1.73; P < .05 between groups). Based on measurements from whole blood, splanchnic proteolysis and protein synthesis did not differ significantly between CMA and control samples, and the net leucine kinetic balance was neutral in both groups (CMA, -0.69 +/- 1.57; controls, -0.74 +/- 3.45 micromol/kg/min/1.73). In CMA whole-blood measurements, splanchnic oxygen consumption (44.8 +/- 4.3 mL/min/1.73 m(2) BS) was slightly lower than in controls (57.5 +/- 8.4 mL/min/1.73 m(2) BS; P = NS). Splanchnic protein synthesis correlated with oxygen consumption (r = 0.82; P < .001). CONCLUSIONS: CMA reduces splanchnic protein turnover and results in a negative leucine balance--an effect that apparently is offset by the contribution of blood cells to organ leucine (and protein) dynamics. Protein synthesis is a major contributor (about 67%) to energy expenditure in splanchnic organs.

Effect of Reversal of Whey-Protein to Casein Ratio of Cow Milk, on Insulin, Incretin, and Amino Acid Responses in Humans.

SCOPE: Milk-proteins, besides lactose, stimulate insulin and incretin secretion. Although whey-proteins (WP) are more efficient than casein (Cas) in hormone secretion, the effects of reversal of the (WP/Cas) ratio in whole-milk are poorly known. METHODS AND RESULTS: Healthy volunteers received two different cow-milk drinks, at identical lactose (0.36 g × kg-1 BW) and total-protein (0.18 g × kg1 BW) loads, but at reversed WP/Cas ratio. One is cow-whole milk with a ≈20/80 [WP/Cas] ratio, the other an experimental cow-milk with a ≈70/30 [WP/Cas] ratio ([↑WP↓Cas]-milk). Both milk-types induced the same mild hyperglycemic response. Following [↑WP↓Cas]-milk, the [20'-90'] insulin incremental area (iAUC) (+ ≈44%, p < 0.035), and the [20'-120'] C-peptide iAUC (+ ≈47%, p < 0.015) are greater than those with cow-milk. Similarly, following [↑WP↓Cas]-milk, the GLP-1 [20'-90'] iAUC (+96%, p < 0.025), and the GIP [30'-60'] iAUC (+140%, p < 0.006), were greater than those with cow-milk. Plasma total and branched-chain amino acids are also greater following the [↑WP↓Cas] than cow-milk. CONCLUSIONS: Reversal of the (WP/Cas) ratio in cow-milk enhanced the insulin response, an effect possibly mediated by incretins and/or amino acids(s). These data may be useful in designing specific milk formulas with different effects on insulin and incretin response(s).

The effects of rosiglitazone and high glucose on protein expression in endothelial cells.

Rosiglitazone is a thiazolidinedione used to treat insulin resistance in diabetes. Although thiazolidinediones may also exert cardiovascular effects, contrasting results were reported. Favorable effects were shown for pioglitazone, whereas adverse reactions were suspected for rosiglitazone. Therefore, a reassessment of the molecular effects of rosiglitazone on vascular cells is required. We tested the effects of rosiglitazone on the proteome of human endothelial cells grown under either normal or high glucose levels. Protein profiles were analyzed in both membrane and cytosolic fractions. About 150 cytosolic proteins, and approximately 100 membrane proteins, were detected. Two-thirds of the proteins significantly altered by high glucose were also modulated by rosiglitazone in an antagonistic way. Half of these proteins are involved in apoptosis. Using an independent assay of apoptosis based on nucleosome quantification, an approximately 20% stimulation by high versus normal glucose was shown (p < 0.05). Conversely, rosiglitazone reduced apoptosis by approximately 30-50% in cells exposed to either glucose conditions (p < 0.001). In addition, rosiglitazone differently modulated cytoskeleton and energy metabolism-related proteins. Our data show novel, potential sites of action of rosiglitazone through protein expression of endothelial cells. These mechanisms may foster new investigations on the overall vascular effects of this compound, and help to discriminate between desired and adverse effects.

Proteomic analysis of clonal interstitial aortic valve cells acquiring a pro-calcific profile.

Calcific degeneration represents the most frequent aortic valve disease observed in industrialized countries. Our aim is to study modifications in the cytosolic and membrane protein profile of aortic interstitial valve cells (VIC) acquiring a pro-calcific phenotype. We studied a clonal population of bovine VIC that expresses bone-related proteins (such as alkaline phosphatase [ALP]) and calcifies a collagen matrix in response to endotoxin (LPS) treatment. A proteomic analysis was performed on proteins extracted from cells treated for 12 days with LPS (100 ng/mL) versus control. We identified 34 unique cytosolic and 10 unique membrane-associated proteins showing significant changes after treatment. These proteins are involved in several cellular functions, such as chaperone-mediated protein folding, protein metabolism and transport, cell redox/nitric oxide homeostasis, and cytoskeletal organization. Reduced expression of proteins involved in NOS bioactivity (such as DDAH-1 and -2) suggested a role for the l-arginine/ADMA ratio in controlling VIC phenotypic profile. In accordance with this hypothesis, we observed that exposure of clonal cells to l-arginine prevented LPS-induced ALP expression and collagen calcification. In conclusion, we identified several proteins involved in structural, metabolic, and signaling functions that are significantly altered in aortic VIC acquiring a pro-calcific profile, thus giving new insights into the pathogenesis of aortic valve degeneration.

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.

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.