Mass Spectrometry Investigation of Some ATP-Binding Cassette (ABC) Proteins.

Drug resistance remains one of the main causes of poor outcome in cancer therapy. It is also becoming evident that drug resistance to both chemotherapy and to antibiotics is driven by more than one mechanism. So far, there are at least eight recognized mechanisms behind such resistance. In this review, we choose to discuss one of these mechanisms, which is known to be partially driven by a class of transmembrane proteins known as ATP-binding cassette (ABC) transporters. In normal tissues, ABC transporters protect the cells from the toxic effects of xenobiotics, whereas in tumor cells, they reduce the intracellular concentrations of anticancer drugs, which ultimately leads to the emergence of multidrug resistance (MDR). A deeper understanding of the structures and the biology of these proteins is central to current efforts to circumvent resistance to both chemotherapy, targeted therapy, and antibiotics. Understanding the biology and the function of these proteins requires detailed structural and conformational information for this class of membrane proteins. For many years, such structural information has been mainly provided by X-ray crystallography and cryo-electron microscopy. More recently, mass spectrometry-based methods assumed an important role in the area of structural and conformational characterization of this class of proteins. The contribution of this technique to structural biology has been enhanced by its combination with liquid chromatography and ion mobility, as well as more refined labelling protocols and the use of more efficient fragmentation methods, which allow the detection and localization of labile post-translational modifications. In this review, we discuss the contribution of mass spectrometry to efforts to characterize some members of the ATP-binding cassette (ABC) proteins and why such a contribution is relevant to efforts to clarify the link between the overexpression of these proteins and the most widespread mechanism of chemoresistance.

Mass Spectrometry Contribution to Pediatric Cancers Research.

For over four decades, mass spectrometry-based methods have provided a wealth of information relevant to various challenges in the field of cancers research. These challenges included identification and validation of novel biomarkers for various diseases, in particular for various forms of cancer. These biomarkers serve various objectives including monitoring patient response to the various forms of therapy, differentiating subgroups of the same type of cancer, and providing proteomic data to complement datasets generated by genomic, epigenetic, and transcriptomic methods. The same proteomic data can be used to provide prognostic information and could guide scientists and medics to new and innovative targeted therapies The past decade has seen a rapid emergence of epigenetics as a major contributor to carcinogenesis. This development has given a fresh momentum to MS-based proteomics, which demonstrated to be an unrivalled tool for the analyses of protein post-translational modifications associated with chromatin modifications. In particular, high-resolution mass spectrometry has been recently used for systematic quantification of chromatin modifications. Data generated by this approach are central in the search for new therapies for various forms of cancer and will help in attempts to decipher antitumor drug resistance. To appreciate the contribution of mass spectrometry-based proteomics to biomarkers discovery and to our understanding of mechanisms behind the initiation and progression of various forms of cancer, a number of recent investigations are discussed. These investigations also include results provided by two-dimensional gel electrophoresis combined with mass spectrometry.

Mass Spectrometry-Based Proteomics: Analyses Related to Drug-Resistance and Disease Biomarkers.

Mass spectrometry-based proteomics is a key player in research efforts to characterize aberrant epigenetic alterations, including histone post-translational modifications and DNA methylation. Data generated by this approach complements and enrich datasets generated by genomic, epigenetic and transcriptomics approaches. These combined datasets can provide much-needed information on various mechanisms responsible for drug resistance, the discovery and validation of potential biomarkers for different diseases, the identification of signaling pathways, and genes and enzymes to be targeted by future therapies. The increasing use of high-resolution, high-accuracy mass spectrometers combined with more refined protein labeling and enrichment procedures enhanced the role of this approach in the investigation of these epigenetic modifications. In this review, we discuss recent MS-based studies, which are contributing to current research efforts to understand certain mechanisms behind drug resistance to therapy. We also discuss how these MS-based analyses are contributing to biomarkers discovery and validation.

Mean blood glucose-independent HbA1c racial disparity and iron status in youth with Type 1 DM.

INTRODUCTION: Black patients have higher HbA1c than Whites even after adjustment for mean blood glucose (MBG). Decreased iron status has been associated with increased HbA1c independently of glucose. We hypothesized that decreased iron status might account for higher HbA1c in Black patients. METHODS: Pediatric patients with T1D in the Diabetes Center at Children's Hospital of New Orleans who self-identified as either Black or White were recruited for the study. At the time of their clinic visit labs were obtained for ferritin (Fer), soluble transferrin receptor (sTfR), HbA1c, and CBC. MBG was derived from patient's home glucose meter records over the last 30 days. Total body iron (TBI) and sTfr/log10 Fer (R/lFer) were calculated. RESULTS: A total of 80 (35 Blacks/45 Whites; 41 female/39 male) patients were recruited. Unadjusted levels of HbA1c, MBG, sTfR, Fer, RDW-CV, and RDW-SD were all higher in Blacks than Whites. TBI and R/lFer were not different between groups. Fer was correlated with Hb, MBG but not HbA1c. sTfR was correlated with HbA1c, MCV, MCH, and RDW-SD. In multiple variable analysis with HbA1c as the dependent variable, race and MBG were statistically significant in the model. However, measures of iron status: Fer, sTfR, R/lFer and TBI were not statistically influential. CONCLUSION: After adjustment for race, MBG and RDW-CV, iron indices were not statistically significant independent predictors of HbA1c levels. These observations indicate that factors besides iron status and CBC indices contribute to MBG-independent racial disparity in HbA1c.

Racial disparity in HbA1c persists when fructosamine is used as a surrogate for mean blood glucose in youth with type 1 diabetes.

BACKGROUND: Blacks have been reported to have higher hemoglobin A1c (HbA1c) than Whites even after adjustment for differences in blood glucose levels. Potentially glucose-independent racial disparity in HbA1c is an artifact of glucose ascertainment methods. In order to test this possibility, we examined the relationship of HbA1c with race after adjustment for concurrent fructosamine level as a surrogate for mean blood glucose (MBG). METHODS: Youth with type 1 diabetes self-identified as either Black or White had blood drawn for HbA1c, fructosamine complete blood count, ferritin, and soluble transferrin receptor (sTfR) at a clinic visit. MBG was calculated as the average of self-monitored capillary glucoses over the preceding 30 days. The effect of race on HbA1c was evaluated in a general linear model adjusting for either MBG or fructosamine, along with other covariates. RESULTS: Fructosamine was correlated with both HbA1c (r = 0.73, P < .0001), MBG (r = 0.46, P < .0001), red cell distribution width coefficient of variation (RDW-CV) (r = 0.31, P = .0045), Fe (r = 0.27, P = .017), and sTfR (r = 0.32, P = .0042). HbA1c was approximately 0.7% higher in Blacks than Whites after adjustment for fructosamine along with age, gender, RDW-CV, Fe, sTfR. CONCLUSIONS: Blacks tend to have higher HbA1c than Whites even after statistical adjustment for fructosamine levels as a surrogate for MBG. Thus, HbA1c tends to overestimate corresponding MBG or fructosamine levels in Black patients. Racial differences should be taken into consideration when using HbA1c as a guide to diagnosis and therapy of diabetes in mixed-race populations.

Palliative Care Issues and Challenges in Saudi Arabia: Knowledge Assessment Among Nursing Students.

BACKGROUND: Nurses are the heart of the palliative care team who provide high standards of care since they spend the longest time with patients. However, lack of adequate knowledge of palliative care is considered as one of the main obstacles to palliative care improvement. AIM: A survey was conducted to assess Saudi nursing students' knowledge about palliative care. METHODS: Using a descriptive design, 154 students who enrolled in first year and who will graduate within 1 year were surveyed in a nursing college located in Riyadh, the capital of Saudi Arabia. The Palliative Care Quiz for Nursing (PCQN) was used to assess students' knowledge. RESULTS: The sample mean age was 22.1 (standard deviation (SD): 2.2), and most of the students were predominately female (93; 60.4%) and the majority were senior students who will be graduated within the current academic year (105; 68.2%). The total mean score of palliative care knowledge was low at 7.30 (SD: 0.56; range: 0-13), and the number of correctly answered statements ranged from 16 (10.4%, statement no. 7) to 91 (59.1%, statement no. 18). Eleven misconceptions about palliative care among participants are conceptually categorized into 3categories: principle and philosophy of palliative care, pain and symptom management, and psychological and spiritual care. CONCLUSION: Integrating palliative care education within the nursing programs is the backbone to improve nursing students' knowledge, namely, in principles of palliative care and symptom management.

Differences in Red Blood Cell Indices Do Not Explain Racial Disparity in Hemoglobin A1c in Children with Type 1 Diabetes.

We assessed the association of erythrocyte indices on mean blood glucose-independent racial disparity in hemoglobin A1c (HbA1c) in youth with type 1 diabetes. Blacks still had higher HbA1c after adjustment for mean blood glucose, red blood cell indices, age, and sex. Such differences need to be taken into account when interpreting HbA1c in Black patients.

Prevalence of traumatic dental injuries and associated factors among preschool children in Amman, Jordan.

BACKGROUND/AIMS: Dental trauma is a major public health problem. However, baseline data regarding traumatic injuries to primary teeth in Jordan are lacking. The study aimed at evaluating the prevalence of traumatic dental injuries to primary anterior teeth among preschool children in Amman (Jordan), investigating the relationship between dental trauma and associated factors, and assessing the treatment provided and treatment need. MATERIALS AND METHODS: After obtaining ethical approval and parental consent, a cross-sectional population-based study examined a total of 1198 children attending 39 preschools randomly selected from different areas of Amman. Chi-square test and stepwise logistic regression modeling were used for statistical analysis. RESULTS: The prevalence of traumatic dental injuries was 26.4%. The upper incisors were more likely to sustain dental trauma (91.7%). No statistically significant association was established between dental trauma and any of the socio-demographic variables. The most common type of dental trauma was enamel fracture (43.1%) followed by pulp injury (39.7%). The odds ratio suggested that the risk of dental trauma was 1.89 times greater if the overjet was >3 mm, 1.93 times greater if the child had an anterior open bite, and 2.56 times greater if the child had inadequate lip coverage. Only 25.3% of children diagnosed with a TDI visited a dentist following their trauma. CONCLUSIONS: The prevalence of dental trauma among preschool children in Amman (Jordan) was high; therefore, it is highly recommended to plan campaigns targeting parents, children, and medical/dental care providers that stress the importance of preventing dental trauma and treating it promptly.

Dataset on the impact of implementing a shared governance model on the level of professional governance among nurses in Saudi Arabia: Insights from experimental data.

Shared governance is a concept that has been gaining popularity in the nursing field. It is a framework that allows nurses to have a greater role in clinical decision-making. This approach recognizes the expertise and knowledge that nurses possess and allows them to be active participants in the decision-making process. It is a way to empower nurses and to ensure that the best possible care is being provided to patients. By promoting shared governance, nurses are able to work collaboratively with other healthcare professionals and provide high-quality care that is evidence-based and patient-centered. This article presents data that was collected in an empirical study to investigate the impact of implementing a shared governance model on the perceptions of professional governance among nurses working in a tertiary hospital in Saudi Arabia by measuring the level of shared governance from the lowest level, the traditional governance level (management and administration only), to the highest level, the self-governance level (staff only), through six dimensions of nursing professional governance, including personnel, information, resources, participation, practice, and goals. The study was conducted over 8 months between July 2022 to February 2023 with the involvement of a random sample of 200 clinical nurses who completed a structured questionnaire before and after the study interventions as part of quasi-research. The interventions included designing and implementing a shared governance model, and providing a shared governance training to clinical to nurse participants. The pretest-posttest experimental group showed that there were improvements in the level of shared governance (shared governance level - primarily management/administration with some staff input), which denotes the effectiveness of nursing professionals governance training among nurses working in a tertiary hospital in Saudi Arabia. The data used in this study can be utilized by future studies for benchmarking purposes.

The Association of Transformational Leadership on Safety Practices Among Nurses: The Mediating Role of Patient Safety Culture.

Background: To ensure best possible patient outcomes, patient safety is a major component of healthcare delivery system that needs to be prioritized. Safety practices among nurses are essential to maintain patient safety, especially the practices of medication administration, handover, patient falls and unplanned extubations prevention. Purpose: To investigate the mediating effect of patient safety culture between the relationship of transformational leadership and safety practices among nurses. Methods: The data in this cross-sectional study were gathered from a survey targeted clinical nurses using a random sampling technique. The study was conducted in a medical city in Saudi Arabia, and two hundred nurses were surveyed. The Multifactor Leadership, Hospital Survey on Patient Safety Culture, and Nursing Safety Practice questionnaires were used in the study. Results: The results revealed significant positive associations between transformational leadership, patient safety culture, and nursing safety practices. Moreover, patient safety culture mediates the association between transformational leadership and safety practices among nurses. Conclusion: Enhancing transformational leadership capabilities among nurse managers should be considered in order to improve nursing safety practices. Additionally, patient safety culture should be measured and improved periodically to ensure better nursing safety practices.

The epidemiology of gestational diabetes, gestation hypertension, and anemia in North Palestine from 2018 to 2020: A retrospective study.

BACKGROUND: In Palestine, women face a challenging environment and a demanding lifestyle, which puts them at a higher risk of experiencing complications during pregnancy. This study aimed to examine the risk factors linked with abnormal hemoglobin (Hb) levels, gestational diabetes (GD), and gestational hypertension (GH) among pregnant women. The results was compared between women residing in cities or villages areas and those in refugee camps. METHODS: Medical records (N = 7889) for pregnant women at primary healthcare centers in the North West Bank were reviewed for sociodemographic and medical data along with the reported fasting blood sugar, hemoglobin, and blood pressure in the first and second trimesters from July 2018 to July 2020. However, only 6640 were included in the analysis as 1249 were excluded for having multiple pregnancy or lost to follow up. Complications and risk factors were defined according to the available global guidelines. Then, descriptive analysis was used to show the percentages of different risk factors and complications among them. The correlation between the several characteristics and variables with these complications was assessed by calculating the odds ratios (OR) using logistic regression. P-values of <0.05 were considered significant. RESULTS: The prevalence of adolescent pregnancy was the highest among women living in rural areas (9.8%) and grand multigravidity among refugee women (22%). The overall prevalence of anemia was higher in the second-trimester (16.2%) than in the first-trimester (11.2%), with anemic women in the first-trimester being more likely to be anemic in the second-trimester (OR = 8.223, P-value<0.001). Although anemia was less common in the first-trimester among refugees than among women living in urban areas (OR = 0.768, P-value = 0.006), it was more prevalent in the second-trimester (OR = 1.352, P-value<0.001). Moreover, refugee women were at lower risk than women living in urban areas of having GD (OR = 0.687, P-value<0.001) and diabetes mellitus (OR = 0.472, P-value<0.001) in the second-trimester. GH was associated with GD (OR = 1.401, P-value = 0.003) and DM (OR = 1.966, P-value<0.001). CONCLUSION: The findings of this study provide evidence-based data on the considerable prevalence of pregnancy complications, anemia, GD, and GH among Palestinian pregnant women living in the north of the West Bank. Multi gravida, gestational age, Hb levels, and the type of settings were strong predictors of pregnancy complications. Therefore, a national plan is needed to ensure adequate maternal care for all, especially disadvantaged women, those in rural areas and refugee camps.

Traumatic dental injuries among 12-year-old schoolchildren in Jordan: prevalence, risk factors and treatment need.

PURPOSE: To assess the prevalence and related factors of traumatic dental injuries (TDI) among 12-year-old Jordanian schoolchildren. MATERIALS AND METHODS: For this cross-sectional study among schoolchildren, a sample of 2560 schoolchildren (1209 boys, 1351 girls) aged 12 years were randomly selected. The epidemiological classification adopted by the World Health Organization (WHO) and modified by Andreasen et al was used to record TDI on incisors. RESULTS: Of the children examined, 5.5% showed TDI. The difference in prevalence between boys and girls was statistically significant (P < 0.01). Maxillary central incisors were the most affected and the most common type of crown injury was enamel/dentin fracture. The relationship between dental injuries and geographic location, area, and socioeconomic indicators was not statistically significant. Most of the TDI occurred at home, followed by school; the most frequent cause was falls. No statistically significant association was found between TDI and dental caries experience (P > 0.05). There was a tendency for boys (P < 0.01), children with an incisal overjet > 3 mm (P < 0.01) and incompetent lip closure (P < 0.001) to have experienced dental injuries. Treatment need due to dental injuries was very high. CONCLUSION: The prevalence of traumatic injuries to permanent incisors in 12-year-old Jordanian schoolchildren was relatively low. TDI was associated with gender, overjet and lip competence, but was not influenced by dental caries experience. There was a great unmet treatment need.

Postoperative Morbidity of Dental Paediatric Patients Treated under General Anaesthesia at a University Hospital: An Observational Study.

Aims: To assess prevalence of postoperative morbidity signs and symptoms in children treated under GA, and to investigate the association between pre- and intra-operative factors with postoperative morbidity. Study design and. Methods: Prospective, observational study supported by pre-operative, intra-operative, and postoperative questionnaire conducted on paediatric patients treated for dental reasons under general anaesthesia at a university hospital. Results: Hundred and fifty patients were included with average age 5.5 years. The majority (92%) experienced at least one postoperative morbidity sign and symptom on the day of treatment under GA and the majority of symptoms subside by the third day. Dental pain (81.3%), sleepiness (70%), and poor appetite (46.7%) were the most frequently reported. Logistic regression analysis showed that age, gender, type of treatment provided, use of LA, and duration of procedure were significantly associated with postoperative morbidity. Conclusions: Most patients experienced one or more morbidity sign/s or symptom/s during first postoperative day and the majority subsides by the third day. Dental pain and poor appetite were the most and long lasting reported. Factors that would predict the occurrence of postoperative morbidity were gender, patient's age, and duration of procedure.

Traditional vs digital communication channels for improving compliance with fixed orthodontic treatment.

OBJECTIVES: To compare the efficacy of traditional and digital communication strategies in improving compliance with fixed orthodontic therapy and to investigate the effect of gender, baseline oral hygiene habits, socioeconomics, and parents' education on orthodontic compliance. MATERIALS AND METHODS: Orthodontic patients were randomly allocated to three groups. Group 1 received traditional communication including verbal and written instructions, whereas group 2 and group 3 received, in addition to traditional communication, weekly text messages or e-mails with audiovisual links, respectively. Baseline demographics (age, gender, baseline oral hygiene habits, socioeconomics, and parents' education) as well as compliance indicators (treatment duration, failed appointments, incidence and total number of appliance breakages) were recorded. For statistical analysis, Pearson chi-square, independent t-test, and one-way analysis of variance were used (P < .05). RESULTS: Of 120 patients (aged 12 to 18 years) recruited, 108 completed the trial (G1 = 37, G2 = 35, G3 = 36). Weekly text messages failed to improve patient compliance. On the other hand, sending weekly e-mails with audiovisual links significantly (P = .014) reduced the incidence of appliance breakage as compared with the control group. Females had a significantly lower incidence of breakage (P = .041) and a fewer total number of breakages (P = .021). Patients from households with high income had significantly better compliance (P < .05). A higher level of parents' education was significantly associated with a lower incidence and total number of breakages (P < .01). CONCLUSIONS: Communication with patients using link-rich e-mails and reminders could improve patient compliance with fixed orthodontic treatment. Female patients, high household income, and high parent education are associated with better compliance with fixed orthodontic treatment.

Metabolic Syndrome Associated with Tobacco and Caffeine Products Use Among Refugee Adolescents: Risk of Dyslipidemia.

INTRODUCTION: Due to their stressful lives, Palestinian refugees are prone to use cognitive enhancers (CE) and psychostimulant substances (PS). Recognising health problems associated with CE/PS use can indicate how preventive programs should be directed towards adolescents. Unfortunately, research has not thoroughly investigated the health problems related to tobacco and caffeine products, the most socially acceptable CE/PS among Palestinian refugee adolescents. METHODOLOGY: In 2021, schoolchildren from five Palestinian refugee camps (N=271) aged 12-<16 years agreed to give blood samples and to be interviewed to address the association between metabolic syndrome (MetS) and tobacco smoking, coffee, energy drinks (ED), black tea, and chocolate consumption. MetS was diagnosed based on International Diabetic Federation Criteria. To address the association, we used binary logistic regression models adjusted to age, gender, BMI, and central obesity. RESULTS: The prevalence of MetS was [17 (6.3%)]; (7.4%) in males, (5.1%) in females. The prevalence of substance use was as follows: ED [163 (48.9%)], coffee [255 (76.6%)], black tea [295 (89.1%)], cigarette [35 (10.5%)], waterpipe [59 (17.7%)], and chocolate [309 (93.6%)], with male predominance. Cigarette smokers have increased central obesity (p-value=0.024), and decreased HDL-cholesterol (p-value=0.015) than non-smokers. Similar results were observed for waterpipe smokers besides the increased levels of triglycerides (p-value=0.01). ED consumers have increased central obesity (p-value=0.03) and fast blood sugar (p-value=0.003) than non-consumers. Chocolate consumers have decreased central obesity (p-value=0.008) and increased HDL levels (p-value=0.04) than non-consumers. ED consumers were significantly at higher risk of MetS (OR=9.97, p-value=0.019) than non-consumers and chocolate consumers were at lower risk of MetS (OR=0.14, p-value=0.046) than non-consumers. Waterpipe smokers were at a higher risk of having increased plasma triglycerides than non-consumers (OR=3.4, p-value=0.03). CONCLUSION: The high prevalence of ED consumption and waterpipe smoking in Palestinian refugee adolescents is problematic. Their use was associated with higher odds of MetS, dyslipidemia, and central obesity, which justifies greater attention.

Shared governance: a children's hospital journey to clinical nursing excellence.

BACKGROUND: Shared governance is examined through a framework for developing independent decision making in professional nursing practice and improving patient care outcomes. AIMS: This study is designed to obtain a baseline measurement of the degree of shared governance in a selected children's hospital in Saudi Arabia. METHODS: The study was guided by the Donabedian model. The Professional Nursing Governance Index was used. A total of 400 questionnaires were distributed to nurses working at the hospital, with a response rate of 77% (n = 307). Descriptive and inferential statistics were used for analysis. RESULTS: The results corresponded with those from nurses and managers in most subscales of the Index of Professional Nursing Governance (information, goals, resources, participation and practice). However, nurses working in the operating theatre and surgical unit have a perceived higher level of shared governance than those in critical care units and medical wards. CONCLUSIONS: The results could encourage shareholders and leaders in the nursing field to develop the perception of shared governance by adopting a shared governance model, which in turn might improve the quality of nursing care.

Alu-repeat polymorphism in the tissue plasminogen activator (t-PA) gene, seminal t-PA concentration, and male fertility impairment: A case-control study.

BACKGROUND: Tissue plasminogen activator (t-PA) is a protein involved in the fibrinolytic system that catalyzes the conversion of plasminogen into the active plasmin. The activity of t-PA is controlled by plasminogen activator inhibitor-1. t-PA has crucial functions during spermatogenesis. One polymorphism was reported for t-PA gene, either the presence of a 300-bp Alu-repeat (Alu + ) or its absence (Alu - ). OBJECTIVE: The current work aimed at studying the association between Alu polymorphism in the t-PA gene and male infertility. MATERIALS AND METHODS: Using polymerase chain reaction on genomic DNA isolated from the blood of 79 participants, a region polymorphic for Alu element insertion in t-PA gene was amplified. In addition, total t-PA concentration, plasminogen activator inhibitor-1 /t-PA complex concentration, and t-PA activity in seminal plasma were measured by enzyme-linked immunosorbent assay. RESULTS: The results indicate that the percentage of infertile participants (n = 50) who were homozygous for t-PA Alu insertion (Alu + / + ), heterozygous Alu + / - or homozygous for t-PA Alu deletion (Alu - / - ) did not change significantly (p = 0.43, 0.81, and 0.85, respectively) when compared with the control participants (n = 29). On the other hand, a significant decrease (p = 0.0001) of t-PA total concentration in seminal plasma was observed in the infertile group in comparison with the control group. However, the results indicate that there is no association between the t-PA Alu different genotypes and the total t-PA seminal concentration in the infertile group when compared to the control group (p = 0.63). CONCLUSION: Data obtained from the current study does not support an association between t-PA Alu polymorphism and t-PA seminal concentration or male infertility.

Study of proteomic changes associated with healthy and tumoral murine samples in neuroblastoma by principal component analysis and classification methods.

BACKGROUND: The adrenal gland is the election organ forming primary neuroblastoma (NB) tumours, the most common extracranial solid tumours of infancy and childhood. METHODS: Samples of adrenal gland belonging to healthy and diseased nude mouse were analysed by 2D gel-electrophoresis. The resulting 2D-PAGE maps were digitized by PDQuest and investigated by principal component analysis (PCA). RESULTS: The analysis of the loadings of the first principal component (PC) permitted the evaluation of the spots characterising each class of samples. Moreover, the soft-independent model of class analogy (SIMCA) method confirmed the separation of the samples in the two classes and allowed the identification of the modelling and discriminating spots. Very good correlation was found between the data obtained by analysis of 2D maps via the commercial software PDQuest and the present PCA analysis. In both cases, the comparison between such maps showed up- and down-regulation of 84 polypeptide chains, out of a total of 700 spots detected by a fluorescent stain, Sypro Ruby. Spots that were differentially expressed between the two groups were analysed by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry and 14 of these spots were identified so far.

Structural and conformational variants of human beta2-microglobulin characterized by capillary electrophoresis and complementary separation methods.

The small (Mr = 11729) serum protein beta2-microglobulin is prone to precipitate as amyloid in a protein conformational disorder (PCD) that occurs in a significant number of patients on chronic hemodialysis. Analyses by capillary electrophoresis (CE) were undertaken to study beta2-microglobulin conformations under native separation conditions and showed an apparent heterogeneity of purified preparations when the sample matrix included organic solvents such as acetonitrile, trifluoroethanol and ethanol. We here present LC-MS, CE-MS, and CE studies of changes of separation profiles as a function of capillary temperature, organic solvent concentration, and analysis time. The results suggest that the apparent beta2-microglobulin heterogeneity observed by CE is caused by two distinct protein conformations that are present in beta2-microglobulin under partly denaturing conditions and that Met99-oxidized and normal (i.e. nonoxidized) beta2-microglobulin behave similarly with respect to the potential to attain this alternative conformation. CE is an attractive method to study early and intermediate soluble folding variants that may be involved in PCDs and CE thus may have an important role as a tool for understanding other PCDs characterized by amyloid deposition.

Critical survey of quantitative proteomics in two-dimensional electrophoretic approaches.

The present review attempts to cover a number of methods that appeared in the last few years for performing quantitative proteome analysis. However, due to the large number of methods described for both electrophoretic and chromatographic approaches, we have limited this excursus only to conventional two-dimensional (2D) map analysis, coupling orthogonally a charge-based step (isoelectric focusing) to a size-based separation (sodium dodecyl sulfate (SDS)-electrophoresis). The first and oldest method applied in 2D mapping is based on statistical analysis performed on sets of gels via powerful software packages, such as the Melanie, PDQuest, Z3 and Z4000, Phoretix and Progenesis. This method calls for separately-running a number of replicas for control and treated samples, the merging and comparing between these two sets of data being accomplished via the softwares just mentioned. Recent developments permit analyses on a single gel containing mixed samples differentially labelled and resolved by either fluorescence or isotopic means. In one approach, a set of fluorophors, called Cy3 and Cy5, are selected for differentially tagging Lys residues, via a "minimal labelling" protocol. A variant of this, adopts a newer set of fluorophors, also of the Cy3 and Cy5 type, reacting on Cys residues, via a strategy of "saturation labelling". There are at present two methods for quantitative proteomics in a 2D gel format exploiting stable isotopes: one utilizes tagging Cys residues with [2H0]/[2H3]-acrylamide; the other one, also based on a Cys reactive compound, exploits [2H0]/[2H4] 2-vinylpyridine. The latter reagent achieves 100% efficiency coupled to 100% specificity. The advantages and limitations of the various protocols are discussed.