An Altered Metabolism in Leukocytes Showing in vitro igG Memory From SARS-CoV-2-Infected Patients.

Coronavirus disease 2019 (COVID 19) is a systemic infection that exerts a significant impact on cell metabolism. In this study we performed metabolomic profiling of 41 in vitro cultures of peripheral blood mononuclear cells (PBMC), 17 of which displayed IgG memory for spike-S1 antigen 60-90 days after infection. By using mass spectrometry analysis, a significant up-regulation of S-adenosyl-Homocysteine, Sarcosine and Arginine was found in leukocytes showing IgG memory. These metabolites are known to be involved in physiological recovery from viral infections and immune activities, and our findings might represent a novel and easy measure that could be of help in understanding SARS-Cov-2 effects on leukocytes.

The role of energy intake and energy misreporting in the associations between eating patterns and adiposity.

BACKGROUND/OBJECTIVES: Research examining associations between eating occasion (EO) frequency and adiposity is inconclusive; studies examining the impact of energy misreporting are rare. This study examined associations between eating patterns and adiposity, with adjustment for energy misreporting, in a nationally representative sample of Australian adults. SUBJECTS/METHODS: Dietary intake was assessed via two 24-h recalls collected during the 2011-12 National Nutrition and Physical Activity Survey (n=4050 adults, aged ⩾19 years). Frequencies of all EOs, meals and snacks were calculated. Height, weight and waist circumference (WC) were measured. Energy misreporting was assessed as the ratio of energy intake to predicted energy expenditure (EI:EE). Energy misreporters were identified by EI:EE ratios, <0.68 or >1.32. Multivariate regression models assessed associations between eating patterns and body mass index (BMI), WC, overweight/obesity (BMI ⩾25 kg m-2) and central overweight/obesity (WC ⩾94 cm in men and ⩾80 cm in women). RESULTS: After adjustment for covariates and EI:EE, frequency of all EOs, meals (women only) and snacks was positively associated with WC and BMI (all P<0.01). Snack, but not meal frequency, was also associated with overweight/obesity (men: OR=1.22, 95% CI 1.07-1.39; women: OR=1.26, 95% CI 1.10-1.43) and central overweight/obesity (men: OR=1.17, 95% CI 1.04-1.32; women: OR=1.21, 95% CI 1.06-1.37). Multivariate analysis that excluded energy misreporters and adjusted for EI yielded either null or inverse associations (P<0.05). CONCLUSIONS: These findings suggest that the associations between eating patterns and adiposity are complicated by the role of EI and energy misreporting. Longitudinal research that considers the impact of EI and energy misreporting is needed to better understand the relationship between eating patterns and obesity.

Effectiveness of interventions to promote physical activity and/or decrease sedentary behaviour among rural adults: a systematic review and meta-analysis.

Physical inactivity and overweight and obesity are more prevalent among rural than urban populations. This study aimed to review published evidence of the effectiveness of interventions to increase physical activity (PA) and/or decrease sedentary behaviour (SB) among rural adults and to identify factors associated with effectiveness. Seven electronic databases were searched for controlled trials of a PA or SB intervention. Meta-analysis was conducted using random effects models and meta-regression. Thirteen studies were included in the qualitative synthesis (n = 4,848 participants) and 12 in the meta-analysis (n = 4,820). All studies were interventions to increase PA. Overall, there was no effect on PA (standardized mean difference [SMD] 0.11; 95% confidence interval [CI] -0.04, 0.25) or SB (SMD 0.07; 95% CI -0.33, 0.20). In PA subgroup analyses, studies employing objective outcome measures demonstrated effects in favour of the intervention (SMD 0.65, 95% CI 0.30, 1.00), while those using self-reported measures did not (SMD 0.00; 95% CI -0.11, 0.10). This review highlights significant gaps in our understanding of how best to promote PA and reduce SB among rural adults. Future studies should use objective measures of PA as study outcomes. The absence of interventions to decrease SB is of concern, with immediate action required to address this large knowledge gap.

What predicts children's active transport and independent mobility in disadvantaged neighborhoods?

This study examined two year changes in children's active transport and independent mobility and prospective associations between individual, social and physical environmental predictors of interest and these behaviors two years later. Overall, 43.5% of children (12.0±2.1 years) used active transport on the school journey at T1 and at T2 (p=0.77), and 35.3% engaged in independent mobility on the school journey at T1 and 29.6% at T2 (p=0.07). Enjoyment, parental safety concerns, and proximity to walking tracks were associated with independent mobility on the school journey. Road safety and social norms were associated with active transport and independent mobility to local destinations. These factors provide potential targets for interventions.

Clustering of diet, physical activity and sedentary behaviour among Australian children: cross-sectional and longitudinal associations with overweight and obesity.

BACKGROUND/OBJECTIVES: Evidence suggests diet, physical activity (PA) and sedentary behaviour cluster together in children, but research supporting an association with overweight/obesity is equivocal. Furthermore, the stability of clusters over time is unknown. The aim of this study was to examine the clustering of diet, PA and sedentary behaviour in Australian children and cross-sectional and longitudinal associations with overweight/obesity. Stability of obesity-related clusters over 3 years was also examined. SUBJECTS/METHODS: Data were drawn from the baseline (T1: 2002/2003) and follow-up waves (T2: 2005/2006) of the Health Eating and Play Study. Parents of Australian children aged 5-6 (n=87) and 10-12 years (n=123) completed questionnaires. Children wore accelerometers and height and weight were measured. Obesity-related clusters were determined using K-medians cluster analysis. Multivariate regression models assessed cross-sectional and longitudinal associations between cluster membership, and body mass index (BMI) Z-score and weight status. Kappa statistics assessed cluster stability over time. RESULTS: Three clusters, labelled 'most healthy', 'energy-dense (ED) consumers who watch TV' and 'high sedentary behaviour/low moderate-to-vigorous PA' were identified at baseline and at follow-up. No cross-sectional associations were found between cluster membership, and BMI Z-score or weight status at baseline. Longitudinally, children in the 'ED consumers who watch TV' cluster had a higher odds of being overweight/obese at follow-up (odds ratio=2.8; 95% confidence interval: 1.1, 6.9; P<0.05). Tracking of cluster membership was fair to moderate in younger (K=0.24; P=0.0001) and older children (K=0.46; P<0.0001). CONCLUSIONS: This study identified an unhealthy cluster of TV viewing with ED food/drink consumption, which predicted overweight/obesity in a small longitudinal sample of Australian children. Cluster stability was fair to moderate over 3 years and is a novel finding. Prospective research in larger samples is needed to examine how obesity-related clusters track over time and influence the development of overweight and obesity.

Clustering of children's obesity-related behaviours: associations with sociodemographic indicators.

BACKGROUND/OBJECTIVES: Research suggests obesity-related behaviours cluster together in children and adolescents, but how these cluster patterns differ by sociodemographic indicators remains unclear. Furthermore, few studies examining clustering of behaviours have included younger children or an objective measure of physical activity (PA) and sedentary behaviour. Therefore, the aim of this study was to examine clustering patterns of diet, PA and sedentary behaviour in 5- to 6- and 10- to 12-year-old children, and their cross-sectional associations with sociodemographic indicators. SUBJECTS/METHODS: In this cross-sectional study, data from the baseline wave (2002/2003) of the Health Eating and Play study (HEAPS) were used. Questionnaires were completed by parents of Australian children aged 5-6 (n=362) and 10-12 years (n=610). Children wore accelerometers for up to 7 days. K-medians cluster analysis identified groups of children with similar diet, PA and sedentary behaviours. Chi-square tests assessed cluster differences by gender, maternal education and marital status. RESULTS: For each age group, three reliable and meaningful clusters were identified and labelled 'most healthy', 'energy-dense (ED) consumers who watch TV' and 'high sedentary behaviour/low moderate-to-vigorous PA (MVPA)'. Clusters varied by sociodemographic indicators. For example, a higher proportion of older girls comprised the 'high sedentary behaviour/low MVPA' cluster (χ(2)=22.4, P<0.001). Among both age groups, the 'ED consumers who watch TV' cluster comprised more children with lower educated mothers (younger children: χ(2)=34.9, P<0.001; older children: χ(2)=27.3, P<0.001). CONCLUSIONS: Identification of cluster patterns of obesity-related risk factors in children, and across sociodemographic groups may assist the targeting of public health initiatives, to those most in need.

Resilience to obesity among socioeconomically disadvantaged women: the READI study.

OBJECTIVE: This cross-sectional study aimed to identify sociodemographic and behavioural characteristics of 'overweight-resilient' women, that is, women who were in a healthy body weight range, despite living in socioeconomically disadvantaged neighbourhoods that place them at increased risk of obesity. The study also aimed to test a comprehensive theoretically derived model of the associations between intrapersonal, social and environmental factors and obesity among this target group. PARTICIPANTS: A total of 3235 women aged 18-45 years from 80 urban and rural neighbourhoods throughout Victoria, Australia, participated in the Resilience for Eating and Activity Despite Inequality study. MEASUREMENTS: Women reported height, weight, sociodemographic characteristics, leisure-time physical activity, dietary behaviours and a range of theoretically derived cognitive, social and neighbourhood environmental characteristics hypothesized to influence obesity risk. A theoretical model predicting body mass index (BMI) was tested using structural equation models. RESULTS: Women classified as 'resilient' to obesity tended to be younger, born overseas, more highly educated, unmarried and to have higher or undisclosed household incomes. They engaged in more leisure-time physical activity and consumed less fast foods and soft drinks than overweight/obese women. Neighbourhood characteristics, social characteristics and cognitive characteristics all contributed to explaining variation in BMI in the hypothesized directions. CONCLUSIONS: These results demonstrate several characteristics of women appearing 'resilient' to obesity, despite their increased risk conferred by residing in socioeconomically disadvantaged neighbourhoods. Acknowledging the cross-sectional study design, the results advance theoretical frameworks aimed at investigating obesity risk by providing evidence in support of a comprehensive model of direct and indirect effects on obesity of neighbourhood, as well as social, cognitive and behavioural characteristics.

Comparative proteomic analysis of hemocyanins in Dinocras cephalotes and Perla marginata (Plecoptera).

Hemocyanins are large oligomeric respiratory proteins found in many arthropods and mollusks. The overall expression of hemocyanin mRNA, revealed by studies on Plecoptera hemocyanin sequencing, has raised the question of whether the protein is expressed or not. In fact, the presence of expressed hemocyanin has only been reported in the literature for one species, Perla marginata (Panzer, 1799). In this paper, we report the presence of hemocyanin and hexamerin proteins in Dinocras cephalotes (Curtis, 1827), a species closely related to P. marginata. To assess the presence of hemocyanin, we used a reproducible and highly sensitive method based on liquid chromatography tandem mass spectrometry. We conclude that regardless of its putative function (respiratory, immune defense, storage protein), the hemocyanin is actually expressed in species in which its mRNA is present.

The association between home environmental variables and soft drink consumption among adolescents. Exploration of mediation by individual cognitions and habit strength.

Soft-drink consumption is one of the important target behaviours for the prevention of excessive weight gain among adolescents. To be able to modify these behaviours in obesity prevention interventions, further understanding of the underlying factors and mediational pathways is required. The present study aimed to explore associations between home environment variables and adolescent soft drink consumption and potential mediation of these associations by individual cognitions derived from the Theory of Planned Behaviour and habit strength. The ENDORSE study (N=1005) provided data on soft drink consumption and on home environment variables related to soft drink consumption (availability, accessibility, parental modelling, and parental rules), cognitive variables (intention, attitude, perceived behaviour control, and parental norm) and habit strength. Multiple mediation analyses were conducted using regression analyses according to the steps described by MacKinnon to assess the association between home environment variables and soft drink consumption and mediation of these associations by cognitive variables and habit strength. The bootstrapping method was used to calculate the confidence intervals. There were significant associations between the home environment variables and soft drink consumption. After inclusion of the mediators the strength of these associations was reduced. In the multiple mediator models, habit strength (39.4-62.6%) and intention (19.1-36.6%) were the strongest mediators. Intention and habit strength partly mediate the associations between home environment factors and soft drink consumption, suggesting that home environment variables influence soft drink consumption both indirectly and directly.

The longitudinal influence of home and neighbourhood environments on children's body mass index and physical activity over 5 years: the CLAN study.

OBJECTIVE: To determine the independent contributions of family and neighbourhood environments to changes in youth physical activity and body mass index (BMI) z-score over 5 years. METHODS: In 2001, 2004 and 2006, 301 children (10-12 years at baseline) had their height and weight measured (BMI was converted to z-scores using Centers for Disease Control and Prevention reference charts; see http://www.cdc.gov/growthcharts) and moderate-to-vigorous physical activity (MVPA) assessed using accelerometers. In 2001, parents reported on the home environment (social support, role modelling, rules and restrictions, physical environment) and perceived neighbourhood environment (local traffic, road safety, sporting venues, public transport), and Geographic Information Systems were used to map features of the neighbourhood environment (destinations, road connectivity, traffic exposure). Generalized estimating equations were used to predict average BMI z-score and MVPA over time from baseline home and perceived and objective neighbourhood environment factors. RESULTS: Among boys, maternal education and heavy traffic were inversely associated, and sibling physical activity, maternal role modelling of MVPA and the presence of dead-end roads were positively associated with MVPA. Having unmarried parents, maternal MVPA role modelling and number of home sedentary items were positively associated with BMI z-score among boys. Among girls, having siblings, paternal MVPA role modelling, physical activity rules and parental physical activity co-participation were positively associated with MVPA. Having unmarried parents and maternal sedentary behaviour role modelling were positively associated, and number of sedentary behaviour rules and physical activity items were inversely associated with BMI z-score among girls. CONCLUSION: The home environment seems more important than the neighbourhood environment in influencing children's physical activity and BMI z-score over 5 years. Physical activity and weight gain programmes among youth should focus on parental role modelling, rules around sedentary and active pursuits, and parental support for physical activity. Intervention studies to investigate these strategies are warranted.

Predictors of time spent outdoors among children: 5-year longitudinal findings.

BACKGROUND: Given the importance of physical activity for health and age-related declines in physical activity, understanding influences on related behaviours, such as time outdoors, is crucial. This study aimed to understand individual, social and physical environmental influences on longitudinal changes in urban children's time outdoors. METHODS: The time children spent outdoors in 2001, 2004 and 2006 (aged 5-6 and 10-12 years at baseline) was reported by their parents (n=421). In 2001, individual, social and physical environmental factors were self-reported by parents. Generalized estimating equations examined longitudinal relationships between baseline predictors and average change in time outdoors over 5 years. RESULTS: Children's time outdoors significantly declined over time. "Indoor tendencies" inversely predicted time outdoors among younger and older boys, and younger girls. Social opportunities positively predicted time outdoors among younger boys, while "outdoor tendencies" positively predicted time outdoors among older boys. Parental encouragement for activity positively predicted time outdoors among younger and older girls, while lack of adult supervision for active play outdoors after school inversely predicted time outdoors among older girls and older boys. CONCLUSION: Individual (indoor and outdoor tendencies) and social factors (social opportunities, parental encouragement and parental supervision) predicted children's time outdoors over 5 years. Interventions targeting reduced indoor tendencies, increased outdoor play with others, and increased parental encouragement and supervision are warranted.

A prospective examination of children's time spent outdoors, objectively measured physical activity and overweight.

OBJECTIVE: This study aimed to determine whether time spent outdoors was associated with objectively measured physical activity, body mass index (BMI) z-score and overweight in elementary-school aged children, cross-sectionally and prospectively over 3 years. METHODS: Three-year cohort study with data collected during 2001 and 2004. Nineteen randomly selected state elementary schools across Melbourne, Australia. One hundred and eighty eight 5-6-year-old and 360 10-12-year-old children. Baseline parent reports of children's time spent outdoors during warmer and cooler months, on weekdays and weekends. At baseline and follow-up, children's moderate and vigorous physical activity (MVPA) was objectively assessed by accelerometry, and BMI z-score and overweight was calculated from measured height and weight. RESULTS: Cross-sectionally, each additional hour outdoors on weekdays and weekend days during the cooler months was associated with an extra 27 min week(-1) MVPA among older girls, and with an extra 20 min week(-1) MVPA among older boys. Longitudinally, more time outdoors on weekends predicted higher MVPA on weekends among older girls and boys (5 min week(-1)). The prevalence of overweight among older children at follow-up was 27-41% lower among those spending more time outdoors at baseline. CONCLUSION: Encouraging 10-12-year-old children to spend more time outdoors may be an effective strategy for increasing physical activity and preventing increases in overweight and obesity. Intervention research investigating the effect of increasing time outdoors on children's physical activity and overweight is warranted.

Is availability of public open space equitable across areas?

This study examined whether availability of public open spaces that can be used for recreation varies according to neighbourhood socio-economic disadvantage. Density and area of public open spaces were examined using a geographic information system and postal boundaries were used to define neighbourhoods. Neighbourhood socio-economic status (SES) was stratified into quintiles. Once neighbourhood population and geographic area were considered there were no differences in the number or total area of free-access, restricted access or sporting/recreation open spaces across quintiles of neighbourhood SES. Future research should examine whether the quality of public open spaces differ by neighbourhood SES.

Perceptions of local neighbourhood environments and their relationship to childhood overweight and obesity.

OBJECTIVES: To examine associations between parent and child perceptions of the local neighbourhood and overweight/obesity among children aged 5-6 and 10-12 y. DESIGN: Cross-sectional survey. SUBJECTS: In total, 291 families of 5-6-y-old and 919 families of 10-12-y-old children. MEASURES: Parent's perceptions of local neighbourhood and perceived child access to eight local destinations within walking distance of home; 10-12-y-old children's perception of local neighbourhood; socio-demographic characteristics (survey). Children's height and weight (measured). RESULTS: No perceptions of the local neighbourhood were associated with weight status among 5-6-y-old children. Among 10-12-y-old children, those whose parents agreed that there was heavy traffic in their local streets were more likely to be overweight or obese (OR=1.4, 95% CI=1.0-1.8), and those whose parents agreed that road safety was a concern were more likely to be obese (OR=3.9, 95% CI=1.0-15.2), compared to those whose parents disagreed with these statements. CONCLUSIONS: This study suggests that parental perceptions of heavy traffic on local streets and concern about road safety may be indirect influences on overweight and obesity among 10-12-y-old children. Future work should also consider perceptions of the neighbourhood related to food choice.

Evidence-based strategies to promote physical activity among children, adolescents and young adults: review and update.

This paper updates evidence reviewed in the first edition of Getting Australia Active on effective physical activity (PA) intervention strategies among children, adolescents and young adults. Intervention studies published between 1999 and September 2003 were identified using electronic databases and hand searching. A total of 28 discrete studies were identified (31 papers). Six of nine studies reported significant effects on child or youth PA in school settings. Those that incorporated whole-of-school approaches including curriculum, policy and environmental strategies appeared to be more effective than those that incorporated curriculum-only approaches. Five of 10 studies with children and two of five studies with adolescents reported increased PA or decreased sedentary behaviour in other settings. Interventions that included contact with families generally appeared to be most effective. One study with adolescents provided some evidence of the potential effectiveness of interventions based in primary care. Two of four papers reported modest short-term results among young adults, including increased PA stage of change or a higher likelihood of being adequately physically active, but none showed any evidence of sustained increases in PA. There is an urgent need for additional studies examining interventions aimed at young adults. Across the three life stages, future studies should include objective PA measures, longer-term follow-up, larger sample sizes, a specific focus on PA (rather than weight) and culturally-specific strategies that build evidence in Australian populations. Future studies should target high risk groups and a broad range of settings and strategies focusing on reducing sedentary behaviours as well as increasing PA.

Identification of a furazolidone metabolite responsible for the inhibition of amino oxidases.

1 Furazolidone, a drug widely used in human and veterinary medicine, exhibits inhibition of monoamine oxidase activity, as observed in the tissues of a number of different animal species, including man. The aim of the current study was to determine which of the two possible metabolites, 3-amino-2-oxazolidone (AOZ) or beta-hydroxyethylhydrazine (HEH), a well-known carcinogenic compound, is involved in the toxicological effects reported. 2 A new spectrometric method was set up to differentiate intracellular HEH from AOZ inside cells. This method works well at low pH where both AOZ and HEH are free in solution and available to react with the chemical chromophore (DAB). 3 The results confirm that furazolidone has to be metabolized in the intact cell in order to exhibit mitochondrial monoamine oxidase inhibition, whereas AOZ itself is able to exert a reversible monoamine oxidase inhibition. AOZ also inhibits bovine serum amino oxidase. On the contrary, HEH gives irreversible inhibition of both enzymes. However, the reversible nature of the AOZ inhibition with respect to HEH suggests that the two metabolites act by different mechanisms which do not require the biotransformation of AOZ to HEH. 4 Cell lysates, previously incubated with AOZ, were directly analysed and the formation of HEH from AOZ was not detected, supporting the conclusion that the amino oxidase inhibition observed on treatment with furazolidone was attributable to AOZ and not to HEH.

Validity and reliability of a physical activity recall instrument among overweight and non-overweight men and women.

This study examined the validity and reliability of survey items measuring one-week recall of physical activity (PA) and examined differences by weight status. A sub-aim of the study was to objectively assess the intensity of activity that most closely matches self-report. A questionnaire was administered to adults twice, three days apart. It was again administered after subjects wore a MTI/CSA accelerometer for seven days (n = 118). Several metabolic equivalent (MET) thresholds were applied to the accelerometer data. Agreement between test and re-test estimates of sufficient physical activity for health benefits (150 min/week) was high (% agreement > 90%). Correlations (rho) between total reported PA (mins/day) and accelerometer data were 0.29 (p < 0.05) among men and 0.25 (p < 0.05) among women. Among men, self-reported duration of moderate PA (3-5.9 METS) and accelerometer data were significantly correlated (rho = 0.40, p < 0.01), with no differences by weight status. Among women, a significant relationship was found only for those who were not overweight (rho = 0.52, p < 0.001). A significant correlation between self-reported duration of vigorous PA (6+ METS) and accelerometer data was only found for overweight men (rho = 0.40, p < 0.05). When lower MET thresholds were applied to the accelerometer data, women's reported duration of moderate-intensity PA was most strongly correlated with moderate PA (accelerometer) defined as 2.0-5.9 METS (rho = 0.39, p < 0.01). The recall instrument provides a consistent measure of physical activity and validation coefficients were similar to those obtained for other physical activity recall questionnaires. However, the ability to measure PA by self-report may vary by weight status.

Isoforms of photosystem II antenna proteins in different plant species revealed by liquid chromatography-electrospray ionization mass spectrometry.

The high selectivity offered by reversed-phase high-performance liquid chromatography on-line coupled to electrospray ionization mass spectrometry has been utilized to characterize the major and minor light-harvesting proteins of photosystem II (Lhcb). Isomeric forms of the proteins, revealed either on the basis of different hydrophobicity enabling their chromatographic separation or on the basis of different molecular masses identified within one single chromatographic peak, were readily identified in a number of monocot and dicot species. The presence of several Lhcb1 isoforms (preferably in dicots) can explain the tendency of dicot Lhcb1 to form trimeric aggregates. The Lhcb1 molecular masses ranged from 24,680 to 25,014 among different species, whereas within the same species, the isoforms differed by 14-280 mass units. All Lhcb1 proteins appear to be highly conserved among different species such that they belong to a single gene group that has several different gene family members. In all species examined, the number of isoforms corresponded more or less to the genes cloned previously. Two isoforms of Lhcb3 were found in petunia and tomato. For Lhcb6, the most divergent of all light-harvesting proteins, the greatest number of isoforms was found in petunia, tobacco, tomato, and rice. Lhcb2, Lhcb4, and Lhcb5 were present in only one form. The isoforms are assumed to play an important role in the adaptation of plants to environmental changes.

High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies.

The use of tetrahydrofuran/decanol as porogens for the fabrication of micropellicular poly(styrene/divinylbenzene) monoliths enabled the rapid and highly efficient separation of peptides and proteins by reversed-phase high-performance liquid chromatography (RP-HPLC). In contrast to conventional, granular, porous stationary phases, in which the loading capacity is a function of molecular mass, the loadability of the monoliths both for small peptides and large proteins was within the 0.40.9-pmol range for a 60- x 0.2-mm capillary column. Lower limits of detection obtained by measuring UV-absorbance at 214 nm with a 3-nl capillary detection cell were 500 amol for an octapeptide and 200 amol for ribonuclease A. Upon reduction of the concentration of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ionization mass spectrometry (ESI-MS) at the cost of only a small decrease in separation efficiency. Detection limits for proteins with ESI-MS were in the lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides and proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferrin and by the analysis of membrane proteins related to the photosystem II antenna complex of higher plants.

High performance liquid chromatography-electrospray mass spectrometry for the simultaneous resolution and identification of intrinsic thylakoid membrane proteins.

In higher plants, both photosystem I (PSI) and II (PSII) consist of membrane-embedded proteins that contain more than one transmembrane alpha helix. PSI is a multiprotein complex consisting of a core complex of thirteen proteins surrounded by four different types of light harvesting antenna proteins. Up to now, the protein components of both photosystems have been characterized by SDS-PAGE and/or immunoblotting and, therefore, identification made only on the basis of electrophoretic mobility, which is sometimes not sufficient to discriminate between individual membrane proteins. This is also complicated by the fact that some proteins, such as the antenna proteins, have almost identical molecular mass and amino acid sequence, making it difficult to identify and ascertain the relative stoichiometry of the proteins. In this paper, we report the complete resolution of the antenna proteins and most of the core components of PSI from spinach, together with the identification of proteins by molecular mass, successfully deduced by the combined use of HPLC coupled on-line with a mass spectrometer equipped with an electrospray ion source (ESI-MS). The proposed RP-HPLC-ESI-MS method holds several advantages over SDS-PAGE, including better protein separation, especially for antenna proteins, mass accuracy, speed, efficiency, and the potential to reveal isomeric forms. Moreover, the molecular masses determined by HPLC-ESI-MS are in good agreement with the molecular masses of the individual components calculated on the basis of their nucleotide-derived amino acid sequences, indicating an absence of post-translational modifications in these proteins. It follows that if the method proposed is useful for these highly hydrophobic proteins, it may be of general use for any membrane proteins, where the presence of detergent for solubilization may compromise their characterization.