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• W2536066790 abstract "Each year over 90 million units of blood are transfused worldwide. Our dependence on this blood supply mandates optimized blood management and storage. During storage, red blood cells undergo degenerative processes resulting in altered metabolic characteristics which may make blood less viable for transfusion. However, not all stored blood spoils at the same rate, a difference that has been attributed to variable rates of energy usage and metabolism in red blood cells. Specific metabolite abundances are heritable traits; however, the link between heritability of energy metabolism and red blood cell storage profiles is unclear. Herein we performed a comprehensive metabolomics and proteomics study of red blood cells from 18 mono- and di-zygotic twin pairs to measure heritability and identify correlations with ATP and other molecular indices of energy metabolism. Without using affinity-based hemoglobin depletion, our work afforded the deepest multi-omic characterization of red blood cell membranes to date (1280 membrane proteins and 330 metabolites), with 119 membrane protein and 148 metabolite concentrations found to be over 30% heritable. We demonstrate a high degree of heritability in the concentration of energy metabolism metabolites, especially glycolytic metabolites. In addition to being heritable, proteins and metabolites involved in glycolysis and redox metabolism are highly correlated, suggesting that crucial energy metabolism pathways are inherited en bloc at distinct levels. We conclude that individuals can inherit a phenotype composed of higher or lower concentrations of these proteins together. This can result in vastly different red blood cells storage profiles which may need to be considered to develop precise and individualized storage options. Beyond guiding proper blood storage, this intimate link in heritability between energy and redox metabolism pathways may someday prove useful in determining the predisposition of an individual toward metabolic diseases. Each year over 90 million units of blood are transfused worldwide. Our dependence on this blood supply mandates optimized blood management and storage. During storage, red blood cells undergo degenerative processes resulting in altered metabolic characteristics which may make blood less viable for transfusion. However, not all stored blood spoils at the same rate, a difference that has been attributed to variable rates of energy usage and metabolism in red blood cells. Specific metabolite abundances are heritable traits; however, the link between heritability of energy metabolism and red blood cell storage profiles is unclear. Herein we performed a comprehensive metabolomics and proteomics study of red blood cells from 18 mono- and di-zygotic twin pairs to measure heritability and identify correlations with ATP and other molecular indices of energy metabolism. Without using affinity-based hemoglobin depletion, our work afforded the deepest multi-omic characterization of red blood cell membranes to date (1280 membrane proteins and 330 metabolites), with 119 membrane protein and 148 metabolite concentrations found to be over 30% heritable. We demonstrate a high degree of heritability in the concentration of energy metabolism metabolites, especially glycolytic metabolites. In addition to being heritable, proteins and metabolites involved in glycolysis and redox metabolism are highly correlated, suggesting that crucial energy metabolism pathways are inherited en bloc at distinct levels. We conclude that individuals can inherit a phenotype composed of higher or lower concentrations of these proteins together. This can result in vastly different red blood cells storage profiles which may need to be considered to develop precise and individualized storage options. Beyond guiding proper blood storage, this intimate link in heritability between energy and redox metabolism pathways may someday prove useful in determining the predisposition of an individual toward metabolic diseases. The potency of harvested red blood cells (RBCs) 1The abbreviations used are:RBCred blood cells 2,3-DPG, 2,3-disphosphoglycerate3PG3-phosphoglycerate6PGD6-phosphogluconate dehydrogenaseADPadenosine diphosphateALDOAfructose bisphosphate aldolaseAMPadenosine monophosphateATPadenosine triphosphateBPGMbisphosphoglycerate mutaseCA1carbonic anhydraseDHAPdihydroxyacetone phosphateENO1Alpha-enolaseESIelectrospray ionizationFDPfructose diphosphateGAPDHglyceraldehyde 3-phosphate dehydrogenaseG6Pglucose 6 phosphateGCLCglutamate-cysteine ligaseGPx1glutathione peroxidase 1GPx4glutathione peroxidase 4GPIglucose phosphate isomeraseGSHglutathione reducedGSSGglutathione oxidizedGSTglutathione S transferaseLDHlactate dehydrogenaseMSmass spectrometryMS/MStandem mass spectrometrynLC-MS/MSnanoflow liquid chromatography-tandem mass spectrometryPEPphosphoenolpyruvatePFKphosphofructokinasePGAM1phosphoglycerate mutasePGK1phosphoglycerate kinasePKLRpyruvate kinasePGM2phosphoglucomutase-2. depends on their ability to survive and maintain function during storage. RBC viability primarily depends on their ability to resist programmed cell death-related fragmentation and phagocytosis by maintaining proper energetics and avoiding hemolysis, in which they break down into microvesicles and toxic byproducts including iron, heme, hemoglobin, and oxidized lipids. The released iron can feed bacterial infections and free hemoglobin can interfere with nitric oxide signaling (1.Hod E.A. Spitalnik S.L. Stored red blood cell transfusions: Iron, inflammation, immunity, and infection.Transfus. Clin. Biol. 2012; 19: 84-89Crossref PubMed Scopus (73) Google Scholar, 2.Alexander J.T. El-Ali A.M. Newman J.L. Karatela S. Predmore B.L. Lefer D.J. Sutliff R.L. Roback J.D. Red blood cells stored for increasing periods produce progressive impairments in nitric oxide–mediated vasodilation.Transfusion. 2013; 53: 2619-2628Crossref PubMed Scopus (39) Google Scholar). A number of small and retrospective studies have suggested that prolonged RBC storage is associated with negative clinical outcomes; however, three larger randomized clinical trials showed no negative effects of longer-stored RBCs (3.Offner P.J. Moore E.E. Biffl W.L. Johnson J.L. Silliman C.C. INcreased rate of infection associated with transfusion of old blood after severe injury.Arch. Surg. 2002; 137: 711-717Crossref PubMed Scopus (294) Google Scholar, 4.Sparrow R.L. Red Blood Cell Storage Duration and Trauma.Transfus. Med. Rev. 2015; 29: 120-126Crossref PubMed Scopus (39) Google Scholar, 5.Zallen G. Offner P.J. Moore E.E. Blackwell J. Ciesla D.J. Gabriel J. Denny C. Silliman C.C. Age of transfused blood is an independent risk factor for postinjury multiple organ failure.Am. J. Surg. 1999; 178: 570-572Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 6.Mary Keller Jean Raymond Wayne LaMorte Frederick Millham and Erwin Hirsch Effects of age of transfused blood on length of stay in trauma patients: a preliminary report.J. Trauma Inj. Infect. Crit. Care. 2002; 53: 1023-1025Crossref PubMed Scopus (80) Google Scholar). In short, the viability of stored RBCs is variable and not fully understood, but the accumulation of biophysical and metabolic changes known as storage lesions are linked to the ability to maintain flux through metabolic pathways during storage (7.Hess J.R. Red cell changes during storage.Transfus. Apher. Sci. 2010; 43: 51-59Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 8.Hess J.R. for the Biomedical Excellence for Safer Transfusion (BEST) Collaborative Scientific problems in the regulation of red blood cell products.Transfusion. 2012; 52: 1827-1835Crossref PubMed Scopus (64) Google Scholar). red blood cells 2,3-DPG, 2,3-disphosphoglycerate 3-phosphoglycerate 6-phosphogluconate dehydrogenase adenosine diphosphate fructose bisphosphate aldolase adenosine monophosphate adenosine triphosphate bisphosphoglycerate mutase carbonic anhydrase dihydroxyacetone phosphate Alpha-enolase electrospray ionization fructose diphosphate glyceraldehyde 3-phosphate dehydrogenase glucose 6 phosphate glutamate-cysteine ligase glutathione peroxidase 1 glutathione peroxidase 4 glucose phosphate isomerase glutathione reduced glutathione oxidized glutathione S transferase lactate dehydrogenase mass spectrometry tandem mass spectrometry nanoflow liquid chromatography-tandem mass spectrometry phosphoenolpyruvate phosphofructokinase phosphoglycerate mutase phosphoglycerate kinase pyruvate kinase phosphoglucomutase-2. Poststorage RBC adenosine triphosphate (ATP) concentration is the single best predictor of RBC in vivo recovery in autologous blood transfusions (9.Reid T.J. Babcock J.G. Derse-Anthony C.P. Hill H.R. Lippert L.E. Hess J.R. The viability of autologous human red cells stored in additive solution 5 and exposed to 25°C for 24 hours.Transfusion. 1999; 39: 991-997Crossref PubMed Scopus (32) Google Scholar, 10.Hess J.R. Measures of stored red blood cell quality.Vox Sang. 2014; 107: 1-9Crossref PubMed Scopus (100) Google Scholar, 11.Luten M. Roerdinkholder-Stoelwinder B. Schaap N.P.M. De Grip W.J. Bos H.J. Bosman G.J.C.G.M. Survival of red blood cells after transfusion: a comparison between red cells concentrates of different storage periods.Transfusion. 2008; 48: 1478-1485Crossref PubMed Scopus (185) Google Scholar, 12.Nakao K. Wada T. Kamiyama T. Nakao M. Nagano K. A direct relationship between adenosine triphosphate-level and in vivo viability of erythrocytes.Nature. 1962; 194: 877-878Crossref PubMed Scopus (104) Google Scholar). Specifically, high ATP concentrations are correlated with low levels of hemolysis and other storage lesions in RBCs. Interestingly, poststorage ATP levels vary greatly between individuals but are consistent on repeat measure within an individual. This observation suggests that poststorage ATP, and thus stored RBC viability, may be influenced and/or determined by inheritance (13.Gilroy T.E. Brewer G.J. Sing C.F. Genetic control of glycolysis in human erythrocytes.Genetics. 1980; 94: 719-732Crossref PubMed Google Scholar, 14.van ′t Erve T.J. Wagner B.A. Martin S.M. Knudson C.M. Blendowski R. Keaton M. Holt T. Hess J.R. Buettner G.R. Ryckman K.K. Darbro B.W. Murray J.C. Raife T.J. The heritability of metabolite concentrations in stored human red blood cells.Transfusion. 2014; 54: 2055-2063Crossref PubMed Scopus (44) Google Scholar, 15.Chakraborty S. Chaudhuri A.B.D. Heritability of Some Important Parameters of the Antioxidant Defense System Like Glucose-6-Phosphate Dehydrogenase,.Catalase, Glutathione Peroxidase and Lipid Peroxidation in Red Blood Cells by Twin Study. 2001; 1: 1-4Google Scholar). In prior analyses of these samples and in additional studies of mono- and di-zygotic twins, some metabolite concentrations including glucose 6-phosphate, fructose 1,6-bisphosphate, glutathione, and glutathione disulfide were determined to be heritable in stored RBCs (13.Gilroy T.E. Brewer G.J. Sing C.F. Genetic control of glycolysis in human erythrocytes.Genetics. 1980; 94: 719-732Crossref PubMed Google Scholar, 14.van ′t Erve T.J. Wagner B.A. Martin S.M. Knudson C.M. Blendowski R. Keaton M. Holt T. Hess J.R. Buettner G.R. Ryckman K.K. Darbro B.W. Murray J.C. Raife T.J. The heritability of metabolite concentrations in stored human red blood cells.Transfusion. 2014; 54: 2055-2063Crossref PubMed Scopus (44) Google Scholar, 15.Chakraborty S. Chaudhuri A.B.D. Heritability of Some Important Parameters of the Antioxidant Defense System Like Glucose-6-Phosphate Dehydrogenase,.Catalase, Glutathione Peroxidase and Lipid Peroxidation in Red Blood Cells by Twin Study. 2001; 1: 1-4Google Scholar, 16.van ′t Erve T.J. Doskey C.M. Wagner B.A. Hess J.R. Darbro B.W. Ryckman K.K. Murray J.C. Raife T.J. Buettner G.R. Heritability of glutathione and related metabolites in stored red blood cells.Free Radic. Biol. Med. 2014; 76: 107-113Crossref PubMed Scopus (44) Google Scholar). The metabolite concentrations of ribulose 5-phosphate, sorbitol, and xylulose 5-phosphate are heritable suggesting a genetic control of glucose metabolism (14.van ′t Erve T.J. Wagner B.A. Martin S.M. Knudson C.M. Blendowski R. Keaton M. Holt T. Hess J.R. Buettner G.R. Ryckman K.K. Darbro B.W. Murray J.C. Raife T.J. The heritability of metabolite concentrations in stored human red blood cells.Transfusion. 2014; 54: 2055-2063Crossref PubMed Scopus (44) Google Scholar, 15.Chakraborty S. Chaudhuri A.B.D. Heritability of Some Important Parameters of the Antioxidant Defense System Like Glucose-6-Phosphate Dehydrogenase,.Catalase, Glutathione Peroxidase and Lipid Peroxidation in Red Blood Cells by Twin Study. 2001; 1: 1-4Google Scholar, 16.van ′t Erve T.J. Doskey C.M. Wagner B.A. Hess J.R. Darbro B.W. Ryckman K.K. Murray J.C. Raife T.J. Buettner G.R. Heritability of glutathione and related metabolites in stored red blood cells.Free Radic. Biol. Med. 2014; 76: 107-113Crossref PubMed Scopus (44) Google Scholar). Because RBCs eject all organelles, including the nucleus and mitochondria upon maturing, they have no ability to synthesize proteins in response to environmental stimuli. The lack of mitochondria in mature RBCs also leaves these cells unable to rely on oxidative phosphorylation; instead, RBCs are reliant on glycolysis for energy production. These unique metabolic attributes of RBCs provide a highly instructive model for unraveling how genetic regulation of metabolic pathways can impact blood storage viability. Herein, we describe a multi-omics analysis of genetic and environmental factors dictating RBC variability. Our approach involved an extensive proteomic and metabolomics analysis of RBCs derived from a cohort of 18 mono- and di-zygotic twin-pairs. The primary challenge of performing proteomic analyses on red blood cells is the wide dynamic range characterized by an abundance of hemoglobin. This was surmounted by focusing our analysis on the membrane fraction of red blood cells. Although other studies have relied on time intensive affinity enrichment, utilizing the membrane fraction granted us the second greatest proteomic depth achieved in red blood cells which allowed us to process a multitude of clinical samples. Furthermore, much of the complexity and diversity in red blood cells is associated with the membrane including many energy metabolism components (17.Mohandas N. Gallagher P.G. Red cell membrane: past, present, and future.Blood. 2008; 112: 3939-3948Crossref PubMed Scopus (725) Google Scholar, 18.Almizraq R. Tchir J.D.R. Holovati J.L. Acker J.P. Storage of red blood cells affects membrane composition, microvesiculation, and in vitro quality.Transfusion. 2013; 53: 2258-2267PubMed Google Scholar, 19.Lewis I.A. Campanella M.E. Markley J.L. Low P.S. Role of band 3 in regulating metabolic flux of red blood cells.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 18515-18520Crossref PubMed Scopus (92) Google Scholar). Despite the simplified composition of mature RBCs, i.e. no nucleus or mitochondria, detection and quantification of the RBC proteome presents a few challenges. First, RBCs must be purified from other blood cells. During this process, typically differential centrifugation, care must be taken to limit contamination, especially from the abundant plasma proteins. The next, and most significant, obstacle is the large dynamic range of protein abundance within the RBC (20.Pasini E.M. Kirkegaard M. Salerno D. Mortensen P. Mann M. Thomas A.W. Deep coverage mouse red blood cell proteome a first comparison with the human red blood cell.Mol. Cell. Proteomics. 2008; 7: 1317-1330Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 21.Pasini E.M. Lutz H.U. Mann M. Thomas A.W. Red blood cell (RBC) membrane proteomics — Part II: Comparative proteomics and RBC patho-physiology.J. Proteomics. 2010; 73: 421-435Crossref PubMed Scopus (33) Google Scholar). Although the actual dynamic range of the RBC proteome is not yet known, the technical challenges are analogous to measuring the proteome of plasma, which has a dynamic range approaching twelve orders of magnitude (22.Anderson N.L. Anderson N.G. The human plasma proteome: history, character, and diagnostic prospects.Mol. Cell. Proteomics MCP. 2002; 1: 845-867Abstract Full Text Full Text PDF PubMed Scopus (3551) Google Scholar). Also, similar to the plasma proteome, in which a single protein (albumin) constitutes 55% of the total protein content, hemoglobin comprises 97%, by mass, of the RBC proteome, making protein depletion a necessary consideration (23.Barasa B. Slijper M. Challenges for red blood cell biomarker discovery through proteomics.Biochim. Biophys. Acta. 2014; 1844: 1003-1010Crossref PubMed Scopus (16) Google Scholar). Of the remaining 3%, carbonic anhydrase accounts for 1/3, so that the remaining 2% of total protein mass is made up of several thousand different proteins. Identifying these low abundance proteins from the background presented by hemoglobin and carbonic anhydrase, is challenging (24.Zubarev R.A. The challenge of the proteome dynamic range and its implications for in-depth proteomics.Proteomics. 2013; 13: 723-726Crossref PubMed Scopus (120) Google Scholar, 25.Goodman S.R. Daescu O. Kakhniashvili D.G. Zivanic M. The proteomics and interactomics of human erythrocytes.Exp. Biol. Med. 2013; 238: 509-518Crossref Scopus (54) Google Scholar). Several methods attempt to counter this obstacle by use of various types of affinity or ion exchange separation techniques (26.Ringrose J.H. van Solinge W.W. Mohammed S. O'Flaherty M.C. van Wijk R. Heck A.J.R. Slijper M. Highly efficient depletion strategy for the two most abundant erythrocyte soluble proteins improves proteome coverage dramatically.J. Proteome Res. 2008; 7: 3060-3063Crossref PubMed Scopus (65) Google Scholar, 27.D'Amici G.M. Rinalducci S. Zolla L. Depletion of hemoglobin and carbonic anhydrase from erythrocyte cytosolic samples by preparative clear native electrophoresis.Nat. Protoc. 2012; 7: 36-44Crossref Scopus (19) Google Scholar, 28.Walpurgis K. Kohler M. Thomas A. Wenzel F. Geyer H. Schänzer W. Thevis M. Validated hemoglobin-depletion approach for red blood cell lysate proteome analysis by means of 2D PAGE and Orbitrap MS.Electrophoresis. 2012; 33: 2537-2545Crossref PubMed Scopus (19) Google Scholar). Even when employing these methods, most RBC proteome analyses yield detection of less than 1000 proteins, with the exception of one which identified 1,578 (29.Roux-Dalvai F. Peredo A.G. de Simó C. Guerrier L. Bouyssié D. Zanella A. Citterio A. Burlet-Schiltz O. Boschetti E. Righetti P.G. Monsarrat B. Extensive analysis of the cytoplasmic proteome of human erythrocytes using the peptide ligand library technology and advanced mass spectrometry.Mol. Cell. Proteomics. 2008; 7: 2254-2269Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Most of these studies, especially those with the deepest coverage, require extensive protein and/or peptide fractionation which, in turn, yields considerable increases in analysis time—both sample preparation and instrument acquisition. Recent years have ushered in an era of proteomics where advances in peptide separation and mass spectrometer performance has accelerated the rate and depth of proteome analysis (30.Riley N.M. Hebert A.S. Coon J.J. Proteomics moves into the fast lane.Cell Syst. 2016; 2: 142-143Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). We reasoned that application of this technology, combined with straightforward reversed-phase proteome fractionation, could expedite sample preparation and afford reasonably deep RBC proteomic analysis in short order, thus, affording the throughput for quantitative comparison of clinical RBC samples. Using our method, we show that in RBCs the concentrations of components in crucial energy metabolism pathways are inherited en bloc at distinct levels. This results in different RBC storage phenotypes which can be used to further understanding of changes during storage and develop improved storage guidelines and methods. Furthermore, this rich data set will provide a valuable resource for continuing studies of RBCs and the heritability of disease. This report is a continuation of twin studies reported previously and utilized the same study subjects (14.van ′t Erve T.J. Wagner B.A. Martin S.M. Knudson C.M. Blendowski R. Keaton M. Holt T. Hess J.R. Buettner G.R. Ryckman K.K. Darbro B.W. Murray J.C. Raife T.J. The heritability of metabolite concentrations in stored human red blood cells.Transfusion. 2014; 54: 2055-2063Crossref PubMed Scopus (44) Google Scholar, 16.van ′t Erve T.J. Doskey C.M. Wagner B.A. Hess J.R. Darbro B.W. Ryckman K.K. Murray J.C. Raife T.J. Buettner G.R. Heritability of glutathione and related metabolites in stored red blood cells.Free Radic. Biol. Med. 2014; 76: 107-113Crossref PubMed Scopus (44) Google Scholar, 31.van ′t Erve T.J. Wagner B.A. Ryckman K.K. Raife T.J. Buettner G.R. The concentration of glutathione in human erythrocytes is a heritable trait.Free Radic. Biol. Med. 2013; 65Crossref PubMed Google Scholar, 32.Van ′t Erve T.J. Wagner B.A. Martin S.M. Knudson C.M. Blendowski R. Keaton M. Holt T. Hess J.R. Buettner G.R. Ryckman K.K. Darbro B.W. Murray J.C. Raife T.J. The heritability of hemolysis in stored human red blood cells.Transfusion. 2015; 55: 1178-1185Crossref PubMed Scopus (51) Google Scholar). The study was approved by the Human Subjects office of The University of Iowa Carver College of Medicine. Written informed consent was obtained from all participating subjects. Subjects were qualified for participation by meeting criteria for autologous blood donation according to standard operating procedures of The University of Iowa DeGowin Blood Center. Twin pairs were not required to donate samples at the same time and each individual donated a single blood unit. Standard health history and demographic information were obtained at the time of enrollment and informed consent. Reported height and weight were used to calculate body mass index (BMI). BMI was derived from the formula: BMI = weight (kg)/(height (m))2. Each subject donated one unit of whole blood which were processed according to standard operating procedures into a leukocyte-reduced RBC unit in CP2D/AS-3 extended storage media (Hemonetics Corp, Braintree, MA). During processing, integral leukocyte reduction filters were retained for extraction of DNA. Samples of AS-3 preserved RBC units were prepared from the main unit on each day of sampling. The AS-3 preserved RBCs were sampled by sterile docking of tubing to the RBC unit, back-filling the tubing with RBCs and sectioning into segments. This procedure was performed on the first day after donation (day 0), and every 14 days thereafter until day 56. This resulted in 5 time points at day 0, 14, 28, 42, and 56. Segments were drained into 5 ml Eppendorf tubes; after mixing an aliquot is removed for complete blood count (CBC) testing using a hematology analyzer (Sysmex XE-2100™ Automated Hematology System, Sysmex Corp, Kobe, Japan). The remaining sample was centrifuged at 500 × g for 5 min, after which the storage media (AS-3) was removed. Samples were further processed and used for measurement of ATP, GSH, and GSSG in RBCs as previously described (14.van ′t Erve T.J. Wagner B.A. Martin S.M. Knudson C.M. Blendowski R. Keaton M. Holt T. Hess J.R. Buettner G.R. Ryckman K.K. Darbro B.W. Murray J.C. Raife T.J. The heritability of metabolite concentrations in stored human red blood cells.Transfusion. 2014; 54: 2055-2063Crossref PubMed Scopus (44) Google Scholar, 16.van ′t Erve T.J. Doskey C.M. Wagner B.A. Hess J.R. Darbro B.W. Ryckman K.K. Murray J.C. Raife T.J. Buettner G.R. Heritability of glutathione and related metabolites in stored red blood cells.Free Radic. Biol. Med. 2014; 76: 107-113Crossref PubMed Scopus (44) Google Scholar). Whole venous blood (EDTA, Vacutainer® purple top blood collection tube, 8 ml) collected from participants prior to blood donation was centrifuged at 500 × g for 5 min, followed by removal of the plasma and buffy coat. RBCs were washed twice with cold isotonic saline solution. After washing, a 30 μl aliquot of the packed red blood cells (pRBCs) was removed for complete blood count (CBC) analysis (Sysmex XE-2100™ Automated Hematology System, Sysmex Corp). A 100 μl aliquot of pRBCs was lysed with 900 μl of nanopure water. Samples were thoroughly mixed and stored at −80 °C prior to proteomic and metabolomic analyses. DNA for zygosity testing was obtained from leukocyte reduction filters by rinsing filters with 15 ml DPBS. The rinse volume was centrifuged at 500 × g for 10 min and the cell pellet was resuspended in 2 ml of DPBS. DNA was extracted from the cell pellet using a nucleic acid extraction instrument (AutoGen QuickGene 610L, AutoGen, Holliston, MA) and kit (Fuji QuickGene DNA Whole Blood Kit, AutoGen). Genotyping was performed using a previously developed panel of 24 single nucleotide polymorphisms (SNPs) (10.Hess J.R. Measures of stored red blood cell quality.Vox Sang. 2014; 107: 1-9Crossref PubMed Scopus (100) Google Scholar). SNP genotyping was performed using PCR assays (TaqMan, Applied Biosystems, Foster City, CA) on a Genotyping System (EP1 SNP, Fluidigm, San Francisco, CA) with a Dynamic Array Integrated Fluidic Circuit (GT48.48, Fluidigm). Monozygotic (MZ) twins were identified by 90% or greater genotype concordance; all other twin pairs were identified as dizygotic (DZ). The untargeted metabolic profiling method employed for this analysis combined three independent platforms: ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) optimized for basic species, UHPLC/MS/MS optimized for acidic species, and gas chromatography/mass spectrometry (GC/MS). Samples were analyzed using procedures described in van ′t Erve et al. (14.van ′t Erve T.J. Wagner B.A. Martin S.M. Knudson C.M. Blendowski R. Keaton M. Holt T. Hess J.R. Buettner G.R. Ryckman K.K. Darbro B.W. Murray J.C. Raife T.J. The heritability of metabolite concentrations in stored human red blood cells.Transfusion. 2014; 54: 2055-2063Crossref PubMed Scopus (44) Google Scholar). A 50 μl aliquot of red blood cells lysed in 500 μl DI water was centrifuged at 4 °C for 30 min at 5 G. The supernatant was discarded and the pellet was resuspended in 100 μl lysis buffer (8 m Urea, 100 mm Tris, 10 mm TCEP, 40 mm chloroacetamide). The samples were then diluted with 50 mm Tris pH 7.5 until the pH reached 7.5 (∼ 1 ml). Trypsin digestion was performed overnight at room temperature with trypsin (Promega, Madison, WI) added at a 1:50 (w/w) enzyme to protein ratio with an estimated protein quantity of 500 μg. A second trypsin digestion was performed the following morning at 1:200 (w/w) enzyme to protein ratio for 1 h. Each digest was quenched by the addition of TFA and desalted over tC18 Sep-Pak cartridges (Waters, Milford, MA). Samples were fractionated using high pH reverse phase separation to increase proteomic depth. The solvent system consisted of mobile phase A (20 mm ammonium bicarbonate) and mobile phase B (20 mm ammonium bicarbonate 80% acetonitrile) which was run on an Ultimate 3000 UPLC system (Dionex Sunnyvale, CA) with a reverse phase C18 column. Gradient elution was performed at 400 μl min−1 with the gradient increased from 0 to 6% B over 5 min followed by an increase to 80% B until 24 min and a wash at 100% B for 3 min. Eight fractions were collected from each sample which were subsequently pooled resulting in four total fractions per sample. Samples were analyzed using a LC/MS instrument comprising an Orbitrap Elite hybrid mass spectrometer (Thermo Fisher Scientific). Reverse phase columns were prepared in house using a 75–360 μm inner-outer diameter bare-fused silica capillary with laser pulled tip. The column was packed with 1.7 μm diameter, 130 Å pore size, Bridged Ethylene Hybrid C18 particles (Waters) to a final length of 35 cm. The column was installed on a Dionex Ultimate 3000 UPLC system and heated to 60 °C using an in house designed column heater for all runs (33.Hebert A.S. Richards A.L. Bailey D.J. Ulbrich A. Coughlin E.E. Westphall M.S. Coon J.J. The one hour yeast proteome.Mol. Cell. Proteomics MCP. 2014; 13: 339-347Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 34.Richards A.L. Hebert A.S. Ulbrich A. Bailey D.J. Coughlin E.E. Westphall M.S. Coon J.J. One-hour proteome analysis in yeast.Nat. Protoc. 2015; 10: 701-714Crossref PubMed Scopus (85) Google Scholar). Mobile phase buffer A was composed of water, 0.2% formic acid, and 5% DMSO. Mobile phase B was composed of acetonitrile, 0.2% formic acid, and 5% DMSO. 1 μg of sample was injected as determined by quantitative colorimetric peptide assay (Pierce, Rockford, IL). Gradient elution was performed at 300 nL min−1 with the gradient increased linearly from 0 to 60% B over 103 min followed by a linear increase to 100% B until 106 min and a wash at 100% B for 4 min. Survey scans of peptide precursors were collected from 300–1500 Th with an AGC target of 1,000,000 and a resolution of 60,000 followed by data dependent CID MS/MS scans of the 20 most intense peaks in the quadrupole linear ion trap mass analyzer. Precursors with charge states equal to 1 or unassigned were rejected and a 45 s dynamic exclusion was set to expedite identifications. Label free quantification was performed using Maxquant software version 1.5.2.8 (35.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9150) Google Scholar) and the Andromeda search engine (36.Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment.J. Proteome Res. 2011; 10: 1794-1805Crossref PubMed Scopus (3448) Goog" @default.
• W2536066790 created "2016-10-28" @default.
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• W2536066790 date "2016-12-01" @default.
• W2536066790 modified "2023-10-16" @default.
• W2536066790 title "Multi-omics Evidence for Inheritance of Energy Pathways in Red Blood Cells" @default.
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