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  • * CHAPTER 3Red blood cells: proteomics,physiology and metabolism

    Erica M. Pasini, Alan W. Thomas, Matthias Mann

    IRON2009_CAP.3(88-107):EBMT2008 4-12-2009 16:04 Pagina 88

  • 1. IntroductionThe discovery of the pulmonary circulation by Ibn al-Nafis and Servetus in the 13thcentury went largely unnoticed due to systematic destruction of Servetus manuscripts,but the subsequent description of the systemic circulation by William Harvey wasfollowed by the first successful blood transfusions between animals and attemptedtransfusions from animal to man, which were repeatedly fatal and which wereultimately banned at the end of the 17th century. Around the time that suchtransfusions were being banned the red blood cell (RBC) became a focus of scientificinterest after being described almost simultaneously by the Dutch biologists JanSwammerdam and Anton van Leeuwenhoek, made possible by the invention of themicroscope (1).Since those early days, developments in the RBC field, such as the establishmentof safe transfusion technology after the discovery of A, B, O and Rh blood groupantigens on the RBC membrane in the early 20th century, have been closelyassociated with the development of new technologies that have changed scienceand had a huge impact on the quality of life.During the 20th century in-depth biochemical, immunological and molecularcharacterisation of single cellular proteins led to significantly increased understandingof RBC structure and function. At the end of the century new technologies emergedthat allowed determination of the entire m-RNA (micro-array, transcriptomics) andprotein (mass-spectrometry, proteomics) make-up of cells. Although in principle bothtranscriptomics and proteomics provide information about protein expression,micro-array measurements may be skewed where cellular m-RNA species are nottranslated to proteins. Furthermore, mature RBCs lack nuclei, organelles andtranscription/translation apparatus so that transcriptomics cannot be applied,leaving proteomics as the method of choice for global RBC protein determinations(Figure 1).

    2. Proteomics and the RBCProteomics, a general term describing mass-spectrometric protein expression studies(2), is best defined as a pipeline involving sample isolation/purification, samplepreparation and optional fractionation, mass spectrometric analyses, databasequery and validation of the resulting protein list (Figure 2).

    Isolation/purification. As mass-spectrometry (MS) has become increasingly sensitive,the initial steps of sample isolation and purification have become ever morecritical to the reliability of data generated. This is because components of minorcontaminating populations may be detected, leading to protein misattribution

    DISORDERS OF ERYTHROPOIESIS, ERYTHROCYTES AND IRON METABOLISM89

    CHAPTER 3 RBC proteomics

    IRON2009_CAP.3(88-107):EBMT2008 4-12-2009 16:04 Pagina 89

  • THE HANDBOOK 2009 EDITION90

    DNA

    m-RNA protein

    Genomics

    StaticTranscriptomics

    Dynamic

    Proteomics

    protein

    Ribosom

    rRNA

    mRNA

    tRNAAmino acid

    Figure 1: Omics approaches

    Blood sample(isolation)

    Protein identification

    Purification steps

    Database queryProtein listvalidation

    IPI00022361

    Sample fractionation

    Sample digestion

    Sample analysis

    200nL/min

    Plasma (+EDTA)

    White blood cells,the "buffy coat"

    Red blood cells

    Figure 2: Typical ESI-MS proteomics pipeline

    IRON2009_CAP.3(88-107):EBMT2008 4-12-2009 16:04 Pagina 90

  • DISORDERS OF ERYTHROPOIESIS, ERYTHROCYTES AND IRON METABOLISM91

    (e.g. attribution of platelet proteins to the RBC). False positives can also arise fromdatabase misidentification. However the use of high resolution, high accuracydata in combination with proper statistical techniques (e.g. decoy databases) hasvery effectively reduced the chances of misidentification.For RBCs isolated for proteomic analysis, low level contaminants are most likely toderive from other blood cells and plasma. To date, isolation/purification hasinvolved some or all of the following: repeated washes with isotonic medium(removal of plasma and platelets), elimination of the buffy coat (upper layer) ateach washing step, the use of filters (leucocyte removal), density gradients e.g.percol, renografin-percol (granulocyte, reticulocyte removal) and customised nets(granulocyte removal) (Figure 3). Each method has advantages and disadvantagesand the choice of isolation/purification procedures should be guided by the aimsof the proteomics study.

    Preparation/fractionation. Typically, to generate peptides suitable for MS, a purifiedsample is then either enzymatically cleaved directly in solution or is fractionatedprior to enzymatic cleavage. Fractionation step(s) help reduce sample complexity,thereby facilitating MS, and can help in subsequent protein allocation as they provide

    CHAPTER 3 RBC proteomics

    Whole blood collectedfrom mouse

    passed on filters(Plasmodipur)

    Lympholitecentrifugation

    Aliquoted blood Washed RBCs

    RBCs afternylon nets1

    passage

    -Plasma/serum

    -Retics &Platelets

    -WBC& PMN

    -WBC

    -WBC& PMN Optional in human,

    necessary in mice

    Renografin-Percol1

    centrifugation

    -PMN &PlateletsPurity check by manual and automatic counting:

    Normally: 1 WBC every 1000 RBCAfter purification: ~ 1WBC :1,000,000 RBC

    ~ 0-2 Reticulocytes :1,000,000 RBC~ 0-5 Platelets :1,000,000 RBC

    RBC Purification step

    Figure 3: Purification of the RBC sample

    1Vettore et al. Am J Hemat 1980; 8: 291-297.

    IRON2009_CAP.3(88-107):EBMT2008 4-12-2009 16:04 Pagina 91

  • Mass analyser Q-STAR LTQ-FT

    Quantity measured Time of flight Cyclotron frequency

    Mass accuracy 10 ppm 1 ppm

    Resolution at m/z 1000 10,000 1,000,000

    Sensitivity 1 femtomol 1 attomol

    Dynamic range 104 106

    further information regarding the proteins from which peptides were derived. e.g.molecular weight (MW) in the case of SDS-PAGE, or location when using membranespecific regimes. For SDS/PAGE fractionation, gels are typically sliced at known MWboundaries prior to in-gel proteolytic digestion and subsequent peptide elution. Insome cases fractionation steps may involve procedures isolating specific protein typessuch as membrane proteins, and as such may contribute both to purification andfractionation. RBC membrane and soluble fractions are both characterised by largenumbers of proteins with a wide range of expression e.g. Band 3 is 2000 times moreabundant than the complement receptor 1. To obtain the most sensitive outcomes,such complex cells require a reduction in complexity to levels compatible with thedynamic range of the MS instrument being used (Table 1).The use of different extraction methodologies (e.g. using various solvents,detergents, ion-chelators and ionic solutions to extract proteins from RBCmembranes) allows the detection of proteins with different biochemicalcharacteristics. These methodologies can be applied independently to aliquots ofthe same sample prior to MS analysis, furnishing a very broad picture of the sampleat hand. For the RBC membrane samples have been prepared by hypotonic bufferlysis - a procedure ensuring the preservation of RBC membrane characteristics -followed by extraction procedures including delipidation (e.g. EtOH), differentialdisruption of ionic bonds (using calcium carbonate) and disanchoring of thecytoskeleton (e.g, EDTA) (Table 2). Strategies to improve the detection of lowabundance RBC membrane proteins by selective solubilisation of Band 3, a veryabundant, highly hydrophobic membrane component, using fluorinated solvents havebeen recently proposed.The MS ionisation method has often guided the choice between one and twodimensional (1D- and 2D) -SDS/PAGE for sample fractionation. 1D has traditionallybeen associated with electrospray ionisation (ESI) and 2D with matrix-assisted laserdesorption ionisation (MALDI) as this requires a more extensive sample deconvolutiondue to the low specificity of peptide-mass fingerprinting.

    THE HANDBOOK 2009 EDITION92

    Table 1: Comparison of the characteristics of MS-analysers

    IRON2009_CAP.3(88-107):EBMT2008 4-12-2009 16:04 Pagina 92

  • MS approaches. MALDI has generally been coupled to time of flight (TOF)-analysersand ESI with ion trap, quadrupole and Fourier transform ion cyclotrone (FT-MS)analysers. More recently, different peptide ionisation methods have been coupledwith different detectors. New MALDI instruments coupled with TOF/TOF analysersor quadrupole ion traps have made protein identification of complex samplessignificantly easier and may in some cases have supplanted binomial 2D-MALDI-MS,but automation of MALDI-coupled liquid chromatography (LC) remains a challenge(2D-gel electrophoresis has limitations in reproducibility, dynamic range and depthof analysis).In ESI-LC (Figure 4), peptides are analysed via a high-pressure liquid chromatography

    Extractionmethod

    IPI00025257Semaphorin 7A

    IPI00423570Transformingprotein p21

    IPI00044556Erythroidmembrane-associatedprotein

    IPI00220194Solute carrierfamily 2,member 1

    IPI00218319Tropomyosinalpha 3 chain

    GPI anchored Looselymembraneassociated

    Membranebound

    Integralmembraneprotein

    Cytoskeletalprotein

    Before sampletreatment

    1

    EtOH FT-Q_STAR 3-2 1 3-1 11-17 5-6

    Carbonate & EtOHsaturated-100mM

    0-3 0-8 28-3 4

    Carbonate x 2saturated-100mM

    2-1 9-5 26-28 0-3

    Carbonate x 2 &EtOH saturated-100mM

    1-4 3-7 26-29 0-3

    Gel FT-Q_STAR 0-3 2-7 12-72 14-12

    NO_cytoskeleton(EDTA) lowthreshold-normal

    5-0 5-0 6-0 29-11

    Exclusion list ofEDTA

    5-3 4-3 4-3

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