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.

Decreased Homocysteine Trans-Sulfuration in Hypertension With Hyperhomocysteinemia: Relationship With Insulin Resistance.

Context: Homocysteine is an independent cardiovascular risk factor and is elevated in essential hypertension. Insulin stimulates homocysteine catabolism in healthy individuals. However, the mechanisms of hyperhomocysteinemia and its relationship with insulin resistance in essential hypertension are unknown. Objective: To investigate whole body methionine and homocysteine kinetics and the effects of insulin in essential hypertension. Design and Setting: Eight hypertensive male subjects and six male normotensive controls were infused with l-[methyl-2H3,1-13C]methionine for 6 hours. In the last 3 hours a euglycemic, hyperinsulinemic clamp was performed. Steady-state methionine and homocysteine kinetics were determined in postabsorptive and hyperinsulinemic conditions. Results: Postabsorptive hypertensive subjects had elevated homocysteine concentrations (+30%, P = 0.035) and slightly (by 15% to 20%) but insignificantly lower methionine rates of appearance (Ras) (P = 0.07 to P = 0.05) and utilization for protein synthesis (P = 0.06) than postabsorptive normotensive controls. Hyperinsulinemia suppressed methionine Ra and protein synthesis, whereas it increased homocysteine trans-sulfuration, clearance, and methionine transmethylation (the latter only in the normotensive subjects). However, in the hypertensive subjects trans-sulfuration was significantly lower (P < 0.05) and increased ~50% less [by +1.59 ± 0.34 vs +3.45 ± 0.52 µmol/kg lean body mass (LBM) per hour, P < 0.005] than in normotensive controls. Homocysteine clearance through trans-sulfuration was ~50% lower in hypertensive than in normotensive subjects (P < 0.005). In the hypertensive subjects, insulin-mediated glucose disposal was ~45% lower (460 ± 44 vs 792 ± 67 mg/kg LBM per hour, P < 0.0005) than in normotensive controls and was positively correlated with the increase of trans-sulfuration (P < 0.0015). Conclusions: In subjects with essential hypertension, hyperhomocysteinemia is associated with decreased homocysteine trans-sulfuration and probably represents a feature of insulin resistance.

Decreased VLDL-Apo B 100 Fractional Synthesis Rate Despite Hypertriglyceridemia in Subjects With Type 2 Diabetes and Nephropathy.

CONTEXT: Subjects with type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN) often exhibit hypertriglyceridemia. The mechanism(s) of such an increase are poorly known. OBJECTIVE: We investigated very low-density lipoprotein (VLDL)-Apo B 100 kinetics in T2DM subjects with and without DN, and in healthy controls. DESIGN: Stable isotope (13)C-leucine infusion and modeling analysis of tracer-to-tracee ratio dynamics in the protein product pool in the 6-8-h period following tracer infusion were employed. SETTING: Male subjects affected by T2DM, either with (n = 9) or without (n = 5) DN, and healthy male controls (n = 6), were studied under spontaneous glycemic levels in the post-absorptive state. RESULTS: In the T2DM patients with DN, plasma triglyceride (TG) (mean ± SD; 2.2 ± 0.8 mmol/L) and VLDL-Apo B 100 (17.4 ± 10.4 mg/dL) concentrations, and VLDL-Apo B 100 pool (0.56 ± 0.29 g), were ∼60-80% greater (P < .05 or less) than those of the T2DM subjects without DN (TG, 1.4 ± 0.5 mmol/L; VLDL-Apo B 100, 9.9 ± 2.5 mg/dL; VLDL-Apo B 100 pool, 0.36 ± 0.09 g), and ∼80-110% greater (P < .04 or less) than those of nondiabetic controls (TG, 1.2 ± 0.4 mmol/L; VLDL-Apo B 100, 8.2 ± 1.7 mg/dL; VLDL-Apo B 100, 0.32 ± 0.09 g). In sharp contrast however, in the subjects with T2DM and DN, VLDL-Apo B 100 fractional synthesis rate was ≥50% lower (4.8 ± 2.2 pools/d) than that of either the T2DM subjects without DN (9.9 ± 4.3 pools/d; P < .025) or the control subjects (12.5 ± 9.1 pools/d; P < .04). CONCLUSIONS: The hypertriglyceridemia of T2DM patients with DN is not due to hepatic VLDL-Apo B 100 overproduction, which is decreased, but it should be attributed to decreased apolipoprotein removal.

Roles of insulin, age, and asymmetric dimethylarginine on nitric oxide synthesis in vivo.

We tested the effects of insulin on production of nitrous oxide (NO)-related substances (nitrites and nitrates [NOx]) after (15)N-arginine intravenous infusion and on asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) concentrations in conditions reportedly associated with altered NO availability, i.e., aging, hypertension, hypercholesterolemia, and type 2 diabetes mellitus (T2DM). A total of 26 male subjects (age 23-71 years, BMI 23-33 kg/m(2)), some of whom were affected by mixed pathologic features, were enrolled. NOx fractional synthesis rate (FSR) was lower in elderly (P < 0.015) and T2DM subjects (P < 0.03) than in matched control subjects. Hyperinsulinemia generally increased both NOx FSR and absolute synthesis rate (ASR) and reduced NOx, ADMA, and SDMA concentrations. Insulin sensitivity was impaired only in T2DM. With use of simple linear regression analysis across all subjects, age was inversely correlated with both NOx FSR (R(2) = 0.23, P < 0.015) and ASR (R(2) = 0.21, P < 0.02). NOx FSR inversely correlated with both ADMA and SDMA. With use of multiple regression analysis and various models, NOx FSR remained inversely associated with age and ADMA, whereas ASR was inversely associated with age and diabetes. No association with insulin sensitivity was found. We conclude that whole-body NOx production is decreased in aging and T2DM. Age, ADMA concentration, and T2DM, but not insulin resistance, appear as negative regulators of whole-body NOx production.

Molecular targets of antimicrobial photodynamic therapy identified by a proteomic approach.

Antimicrobial photodynamic therapy (PDT) is a promising tool to combat antibiotic-resistant bacterial infections. During PDT, bacteria are killed by reactive oxygen species generated by a visible light absorbing photosensitizer (PS). We used a classical proteomic approach that included two-dimensional gel electrophoresis and mass spectrometry analysis, to identify some proteins of Staphylococcus aureus that are damaged during PDT with the cationic PS meso-tetra-4-N-methyl pyridyl porphine (T4). Suspensions of S. aureus cells were incubated with selected T4 concentrations and irradiated with doses of blue light that reduced the survival to about 60% or 1%. Proteomics analyses of a membrane proteins enriched fraction revealed that these sub-lethal PDT treatments affected the expression of several functional classes of proteins, and that this damage is selective. Most of these proteins were found to be involved in metabolic activities, in oxidative stress response, in cell division and in the uptake of sugar. Subsequent analyses revealed that PDT treatments delayed the growth and considerably reduced the glucose consumption capacity of S. aureus cells. This investigation provides new insights towards the characterization of PDT induced damage and mechanism of bacterial killing using, for the first time, a proteomic approach.

Polar electrophoresis: shape of two-dimensional maps is as important as size.

The performance of two-dimensional electrophoresis in conventional gels in Cartesian coordinates (2-DE) vs. polar coordinates (2-PE) is here evaluated. Although 2-DE is performed in much longer Immobiline gels in the first dimension (17 cm) vs. barely 7-cm in 2-PE, an equivalent resolving power is found. Moreover, due to the possibility of running up to seven Immobiline strips in the radial gel format, the reproducibility of spot position is seen to be higher, this resulting in a 20% higher matching efficiency. As an extra bonus, strings of "isobaric" spots (i.e. polypeptides of identical mass with different pI values) are more resolved in the radial gel format, especially in the 10 to 30 kDa region, where the gel area fans out leaving extra space for spot resolution. In conclusion, this novel gel format in the second dimension of 2D gels is seen as an important improvement of this technique, still one of the most popular in proteome analysis.

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 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.

Insulin resistance of amino acid and protein metabolism in type 2 diabetes.

Although insulin resistance in T2DM (type 2 diabetes mellitus) is usually referred to glucose and lipid metabolism, the question whether such a resistance affects also amino acid and protein metabolism is both relevant and not easy to be answered. Available data indicate a reduced response to insulin in the inhibition of proteolysis at low, near basal hormone levels, whereas such a response appears to be normal at high physiological doses. In most studies in T2DM subjects the stimulation of whole-body protein synthesis in the presence of hyperinsulinemia and euaminoacidemia appears to be normal, although one single study reported lower rates in male T2DM subjects with obesity. The response to insulin of plasma protein synthesis (albumin and fibrinogen) is also normal. However, some metabolic steps of amino acids related to vascular complications (methionine and arginine) exhibit a defective response to insulin in T2DM subjects with nephropathy. In summary, although gross alterations in the response of whole-body protein turnover are not evident in T2DM, specific investigations reveal subtle abnormalities in metabolic steps of selected amino acids. Furthermore, the effects of interaction between diabetes (with the associated insulin resistance) and older age in the pathogenesis of sarcopenia in the elderly deserve more specific studies.

Caldesmon over-expression in type 1 diabetic nephropathy.

Substantial evidence supports a genetic susceptibility to develop nephropathy in type 1 diabetes and a key pathogenic role of actin cytoskeleton dysfunction in this complication. We previously reported that many cytoskeletal proteins were either up- or down-regulated in fibroblast cells from type 1 diabetic (T1DM) patients with nephropathy. The gene of one of these proteins, caldesmon, lies in a chromosomal region linked to nephropathy and its promoter region contains a single nucleotide polymorphism that is associated with nephropathy. Hence, we analyzed caldesmon gene and protein expression in cultured fibroblasts from T1DM patients with and without nephropathy and from control subjects. Caldesmon gene was studied in cells cultured under normal glucose levels by quantitative real-time RT-PCR. Caldesmon protein isoforms were quantified both under normal and high glucose conditions by two-dimensional electrophoresis. Caldesmon gene was over-expressed in fibroblasts from diabetic patients with nephropathy, in comparison to both those from diabetic patients without nephropathy and those from controls. We quantified six caldesmon protein isoforms, two of them were increased whereas another one was decreased only in fibroblasts from diabetic patients with nephropathy. None of these isoforms showed any difference in their relative abundance in response to high glucose. Variable results in response to high glucose were observed in the expression of other proteins in the three experimental groups. Our data lend further support to an involvement of caldesmon in the susceptibility to diabetic nephropathy in type 1 diabetes, independently from environmental glucose levels.

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.

Nitric oxide synthesis is reduced in subjects with type 2 diabetes and nephropathy.

OBJECTIVE: Nitric oxide (NO) is a key metabolic and vascular regulator. Its production is stimulated by insulin. A reduced urinary excretion of NO products (NOx) is frequently found in type 2 diabetes, particularly in association with nephropathy. However, whether the decreased NOx excretion in type 2 diabetes is caused by a defective NOx production from arginine in response to hyperinsulinemia has never been studied. RESEARCH DESIGN AND METHODS: We measured NOx fractional (FSR) and absolute (ASR) synthesis rates in type 2 diabetic patients with diabetic nephropathy and in control subjects, after l-[(15)N(2)-guanidino]-arginine infusion, and use of precursor-product relationships. The study was conducted both before and after an euglycemic hyperinsulinemic ( approximately 1,000-1,200 pmol/l) clamp. RESULTS: In type 2 diabetes, NOx FSR was reduced both under basal (19.3 +/- 3.9% per day, vs. 22.9 +/- 4.5% per day in control subjects) and hyperinsulinemic states (24.0 +/- 5.6% per day, vs. 37.9 +/- 6.4% per day in control subjects; P < 0.03 by ANOVA). Similarly, in type 2 diabetes, NOx ASR was lower than in control subjects under both conditions (basal, 0.32 +/- 0.06 vs. 0.89 +/- 0.34 mol per day; hyperinsulinemia, 0.35 +/- 0.07 vs. 1.15 +/- 0.38 mol per day; P = 0.01 by ANOVA). In type 2 diabetes, the ability of insulin to stimulate both the FSR (4.7 +/- 3.2% per day) and the ASR (0.03 +/- 0.04 mol per day) of NOx was several-fold lower than that in control subjects (15.0 +/- 2.9% per day and 0.25 +/- 0.07 mol per day, P < 0.03 and P < 0.02, respectively). Also the fraction of arginine flux converted to NOx (basal, 0.22 +/- 0.05% vs. 0.65 +/- 0.25%; hyperinsulinemia, 0.32 +/- 0.06% vs. 1.03 +/- 0.33%) was sharply reduced in the patients (P < 0.01 by ANOVA). CONCLUSIONS: In type 2 diabetic patients with nephropathy, intravascular NOx synthesis from arginine is decreased under both basal and hyperinsulinemic states. This defect extends the concept of insulin resistance to NO metabolism.

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.

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.

Effect of liver cirrhosis on phenylalanine and tyrosine metabolism.

PURPOSE OF REVIEW: Phenylalanine conversion to tyrosine (i.e., 'hydroxylation') is the first irreversible step in phenylalanine catabolism and a source of circulating tyrosine. The purpose of the present review is both to examine hydroxylation from a biochemical standpoint and to report data measured in vivo under physiological conditions, as well as in liver and kidney disease. RECENT FINDINGS: The simultaneous infusion of phenylalanine and tyrosine tracers in humans allows us to determine the hydroxylation rate in vivo. Hydroxylation accounts for a minor ( approximately 10-20%) although significant portion of tyrosine flux. The liver and the kidney are the key organs accounting for virtually the whole-body hydroxylation rates. It is regulated by substrate availability, being acutely stimulated by mixed meal ingestion and by dietary adaptation to high phenylalanine intakes. Theoretically, it may be impaired in advanced liver and kidney disease. Nevertheless, in compensated liver cirrhosis, hydroxylation as well as tyrosine flux are not decreased but rather increased. Only in end stage liver disease hydroxylation may be impaired and is corrected by transplantation. Hydroxylation is also reduced in end stage renal disease. SUMMARY: Phenylalanine hydroxylation in vivo appears to represent a regulatory step of phenylalanine disposal and tyrosine production under acute and/or extreme conditions.

Middle age is not associated with altered fibrinogen concentration and production in males.

Whether ageing is associated with increased fibrinogen concentration and production remains unclear. We measured fibrinogen fractional (FSR) and absolute synthesis (ASR) rates in male volunteers, of either young (mean age: 28 years, range: 22-34) or middle age (mean age: 57 years, range: 38-72), using a leucine-tracer isotope dilution technique. In the middle-age group, neither fibrinogen FSR (20.8 +/- 1.6%/day) nor ASR (1.8 +/- 0.1 g/day), or concentration (274 +/- 15 mg/dl), were different from those of the younger group (FSR: 20.2 +/- 1.4; ASR: 1.7 +/- 0.2; concentration: 265 +/- 8, respectively). Leucine Ra, an index of endogenous proteolysis, was approximately 20% lower in the older than in the younger group (P < 0.02). Thus, middle age in males is not associated with increased fibrinogen concentration and turnover, whereas endogenous protein breakdown in decreased. Factor(s) different from age per se are likely to be involved in the dysfibrinogenemia possibly occurring with ageing. Protein turnover is already reduced in middle-age males.

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.

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.

Kinetics of albumin homocysteinylation measured with matrix-assisted laser/desorption ionization mass spectrometry versus with a radioactive tracer.

Homocysteinylation is a post-translational protein modification which involves homocysteine-thiolactone and may be responsible for many pathophysiological changes secondary to hyperhomocysteinemia. Therefore, methods to measure protein homocysteinylation in intact biological samples are required. We tested whether matrix assisted-laser/desorption ionization mass spectrometry (MALDI-MS) can detect time- and dose-dependent changes in in vitro homocysteine-thiolactone binding to human serum albumin. We have compared this method with a 35S-thiolactone radioactive binding assay. Incubations with and without dithiothreitol allowed measurement of the amide-linked and disulfide-linked thiolactone-protein adducts, respectively. A good correspondence in time- and dose-dependent protein-thiolactone formation was observed between the two methods. A maximum of 9 to 12 thiolactone residues were bound to each albumin molecule. The 35S-thiolactone bound albumin tightly, particularly at the lowest concentrations, with approximately 70% of the binding amide-linked. Although the results of the two methods were rather similar, the radioactive method appears to be more sensitive than the MALDI-MS technique.