Article pubs.acs.org/jpr
Whole Serum 3D LC-nESI-FTMS Quantitative Proteomics Reveals Sexual Dimorphism in the Milieu Inteŕ ieur of Overweight and Obese Adults Nasser M. Al-Daghri,*,†,‡,§,○ Omar S. Al-Attas,†,‡,○ Harvey E. Johnston,∥,⊥ Akul Singhania,∥ Majed S. Alokail,†,‡ Khalid M. Alkharfy,†,# Sherif H. Abd-Alrahman,†,‡ Shaun l. Sabico,†,‡ Theodoros I. Roumeliotis,⊥ Antigoni Manousopoulou-Garbis,∥,⊥ Paul A. Townsend,△,○ Christopher H. Woelk,∥,○ George. P. Chrousos,‡,□,○ and Spiros D. Garbis*,∥,⊥,○ †
Biomarkers Research Program, Biochemistry Department, College of Science, ‡Prince Mutaib Chair for Biomarkers of Osteoporosis, Biochemistry Department, College of Science, §Center of Excellence in Biotechnology Research, and #Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh 12372, Kingdom of Saudi Arabia ∥ Cancer Sciences and Clinical Experimental Sciences (CES) Units, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton SO17 1BJ, United Kingdom ⊥ Institute for Life Sciences, Centre for Proteomic Research, University of Southampton, Highfield Campus, Southampton SO17 1BJ, United Kingdom △ Institute of Cancer Sciences, Faculty of Medical and Human Sciences, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, United Kingdom □ First Department of Pediatrics, University of Athens, Athens 106 79, Greece S Supporting Information *
ABSTRACT: Linking gender-specific differences to the molecular etiology of obesity has been largely based on genomic and transcriptomic evidence lacking endophenotypic insight and is not applicable to the extracellular fluid compartments, or the milieu intérieur, of the human body. To address this need, this study profiled the whole serum proteomes of age-matched nondiabetic overweight and obese females (n = 28) and males (n = 31) using a multiplex design with pooled biological and technical replicates. To bypass basic limitations of immunodepletion-based strategies, subproteome enrichment by size-exclusion chromatography (SuPrE-SEC) followed by iTRAQ 2D-LC-nESI-FTMS analysis was used. The study resulted in the reproducible analysis of 2472 proteins (peptide FDR < 5%, q < 0.05). A total of 248 proteins exhibited significant modulation between men and women (p < 0.05) that mapped to pathways associated with β-estradiol, lipid and prostanoid metabolism, vitamin D function, immunity/inflammation, and the complement and coagulation cascades. This novel endophenotypic signature of gender-specific differences in whole serum confirmed and expanded the results of previous physiologic and pharmacologic studies exploring sexual dimorphism at the genomic and transcriptomic level in tissues and cells. Conclusively, the multifactorial and pleiotropic nature of human obesity exhibits sexual dimorphism in the circulating proteome of importance to clinical study design. KEYWORDS: whole serum milieu intérieur, sexual dimorphism, subproteome enrichment, size exclusion chromatography, depletion-free, multiplex quantitative proteomics, iTRAQ, FT-MS, cardiovascular physiology
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index (BMI) is >25 or ≥30 kg/m2, respectively.7 The increasingly high rates of obesity have reached levels of a global epidemic with major concurrent and increased rates of morbidity and mortality that impact long-term negative
INTRODUCTION
Gender-specific differences can affect how obesity modulates the physiologic status of various hormones beyond insulin, such as vitamin D,1−3 glucagon,4 glucocorticoids,5 estrogens,6 and androgens.5,6 As a well-established pathological condition, obesity occurs when excess adipose tissue has accumulated in the visceral and subcutaneous compartments of the human body. The World Health Organization has defined the overweight and obese status in humans when the body mass © XXXX American Chemical Society
Special Issue: Proteomics of Human Diseases: Pathogenesis, Diagnosis, Prognosis, and Treatment Received: April 2, 2014
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dx.doi.org/10.1021/pr5003406 | J. Proteome Res. XXXX, XXX, XXX−XXX
Journal of Proteome Research
Article
Table 1. Clinical Profile of Subjects Studieda group 1 clinical parameters N age (years) BMI (kg/m2) systolic blood pressure (mm Hg) diastolic blood pressure (mm Hg) fasting glucose (mmol/L) triglycerides (mmol/L) total cholesterol (mmol/L) HDL cholesterol (mmol/L) LDL cholesterol (mmol/L) a
females 14 38.5 ± 33.5 ± 113.5 ± 73.6 ± 5.0 ± 1.8 ± 5.1 ± 0.7 ± 0.56 ±
10.3 6.8 15.0 12.1 0.9 0.86 1.3 0.15 0.23
group 2 p value
males 15 40.4 ± 32.2 ± 115.4 ± 75.6 ± 5.9 ± 2.01 ± 4.9 ± 0.58 ± 0.62 ±
11.9 6.9 12.3 7.26 0.59 1.7 0.93 0.25 0.09
0.84 0.83 0.87 0.82 0.23 0.86 0.84 0.48 0.71
females 14 37.7 ± 32.3 ± 113.7 ± 75.4 ± 5.6 ± 2.1 ± 5.6 ± 0.83 ± 0.54 ±
12 8.9 5.1 5.2 1.1 1.4 2.2 0.32 0.26
males 16 41.3 ± 31.5 ± 112.8 ± 70.0 ± 5.8 ± 1.77 ± 4.8 ± 0.75 ± 0.51 ±
12.4 5.9 12.5 5.77 0.59 1.23 0.63 0.27 0.09
p value 0.74 0.90 0.92 0.29 0.80 0.77 0.89 0.76 0.86
Data presented as mean ± standard deviation.
by the authors.36 These findings conjectured that the albumin and IgG proteins, comprising over 90% of the total protein mass in plasma or serum, have an intrinsic propensity as carrier substrates to interact with a wide array of lower abundant proteins. This study demonstrated that immunodepletion of albumin and IgG proteins also coremoved lower abundance proteins of potential clinical utility. Analogous trends on the basic limitations of immunodepletion strategies to serum or plasma analysis were also observed by other groups.34,37−42 An aim of this study was to extend the utility of the author’s reported method described above to relatively quantify the protein profiles of whole sera derived from age-matched, nondiabetic male and female patients that were overweight or obese. This study tested the hypothesis that the in-depth quantitative proteomic assessment of whole serum as a nondepleted milieu intérieur can effectively capture systemswide protein expression patterns with their encoded networks and pathways consequent to the sexually dimorphic nature of obesity.
implications to healthcare costs. Obesity has been associated with the metabolic syndrome and its components, including carbohydrate intolerance, insulin resistance and an increased risk of type-2 diabetes,8,9 hypertension and blood hypercoagulation,9 cardiovascular disease,10 and cancer.11 Additionally, obesity disrupts several metabolic pathways, including those of carbohydrates and lipids.12−14 Obesity has also been suggested to play a role in the response to stress and has been associated with smoldering systemic inflammation.15,16 The obesity-mediated disruption of such biological processes may exhibit intrinsic synergistic or antagonistic interdependence between the altered reactant concentrations, in which sexual dimorphism might play a pivotal role.17−23 This association has been largely based on genomic and transcriptomic evidence that lacks endophenotypic insight and is not applicable to the extracellular fluid compartments, or milieu intérieur, of the human body. First coined by Claude Bernard, the milieu intérieur is deemed an ideal study matrix through which the internal physiology can be understood on a system-wide basis, thus capturing the complex dynamics of homeostasis and its perturbations.24−28 As an integral body milieu intérieur, the whole blood serum or plasma constitutes its circulating compartment that encompasses secreted or shed proteins from all organs and tissues under a given physiological or pathological state. Therefore, a systems-wide capture of molecular perturbations innate to sexual dimorphism and its effects on obesity largely depends on the comprehensive, nontargeted and unbiased interrogation of the whole serum or plasma milieu intérieur. The in-depth and reproducible proteome analysis of whole serum or plasma remains a challenge, principally stemming from the complexity and dynamic range of its protein content.29−32 Advancements made in mass spectrometry based proteomics by various groups, including the authors, have now made it possible to effectively profile an unprecedented large range of proteins in whole serum with a high-degree of sensitivity, specificity, and selectivity, without recourse to the common depletion strategies of the high abundance proteins, such as albumins and the immunoglobulins.29,33−35 The proposed quantitative proteomics method described here constitutes an extension to the authors’ prior nonquantitative method. From only 100 μL of whole serum, this method captured a diverse range of proteins and phosphoproteins, not observed by depletion-based strategies.29 Key serum proteins identified were verified with immunohistochemistry in their respective prostate tissue specimens from a separate study
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MATERIALS AND METHODS
Patient Cohort
A total of 59 nondiabetic, apparently healthy consenting adults (28 women and 31 men) aged 28−53 years, with BMIs in the range of overweight (>25) and obesity (>30 and
