Human Pituitary Growth Hormone (hGH) . ¢  Human Pituitary Growth Hormone (hGH) Mass...

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  • Human Pituitary

    Growth Hormone (hGH)

    Mass Spectrometry, Quantification, Isoforms, PTM’s, Future Trends

    World Anti-Doping Agency

    Rome, Italy June 15-16, 2011

    Dominic M. Desiderio, Ph.D.

    Department of Neurology The University of Tennessee Health Science Center

    Memphis, Tennessee, USA

  • Anterior Pituitary: Rich neurochemistry. Master regulatory gland in the body. Five different cell types: Each cell type is under close hypothalamic control, and secretes a specific hormone. Somatotrophs: 35-45%.

    Thyrotropin-Releasing Hormone (TRH). Burgus, Dunn, Desiderio, Ward, Vale, Guillemin. Nature 226, 321-325 (1970).

  • Long-Term Goals of this Research Program

     Elucidate molecular mechanisms in the anterior human pituitary: – Controls. – Adenomas (microadenomas and macroadenomas):

    Need accurate molecular classification. Need effective pharmacology. Model system for more-aggressive tumors*.

    Collaborating neurosurgeons: Jim Robertson, M.D. Zhu, et al. Peptides 16, 1097-1107 (1995). Nelson Oyesiku, M.D. Moreno, et al. Cancer Res. 65, 1-8 (2005). *Dworakowska, D. and Grossman, A.B. The pathophysiology of pituitary adenomas. Best Pract. Res. Clin. Endocrin. Metabol. 23, 525-541 (2009).

  • Functional categories of 56 differentially expressed proteins (DEP’s) in human pituitary adenomas. Controls (n = 8); NF- (3); LH+ (3); FSH+ (3); FSH+ + LH+ (3); unknown (3); prolactinomas (4). Zhan and Desiderio. Mass Spectrometry Reviews 23, 1-35 (2004). A. Down-regulated proteins (34): I, neuro-endocrine and hormones; II, cytokine and cellular signal-related proteins; III, cellular defense and stress resistance; IV, mRNA splicing, transport or translation-related enzyme; V, DNA-binding proteins; VI, Metabolic enzymes; VII, Immunologic regulation proteins and tumor-related antigen; VIII, Transport proteins; IX, cell proliferation, differentiation, apoptosis-related proteins; X, others. B. Up-regulated proteins (22): I, metabolic enzyme-related proteins; II, energy metabolism; III, cellular signal proteins; IV, cell cycle, cell growth and proliferation proteins; V, cellular defense response; VI, protein folding-related protein; VII, others.

    I 14%

    I I 15%

    I I I 9%

    I V 3%V

    3%VI 6%

    VI I 15%

    VI I I 17%

    I X 6%

    X 12%

    I 35%

    II 5%

    III 14%

    IV 9%

    V 9%

    VI 5%

    VII 23%

    A B

  • Growth Hormone (hGH)  Pituitary (acidophilic somatotrophs):

    – hGH-N gene. Chromosome 17q24.2.  22 kDa. 191 amino acids.

    – 20 kDa. 176 amino acids. – 18 kDa. 153 amino acids. – 17 kDa. 145 amino acids.

    Oligomers. Protein-bound (GHBP). PTM’s: phosphorylation, glycosylation, deamidation,

    acetylation, oxidation, other.

     Placenta (villous syncytiotrophoblasts): – hGH-V genes: Chromosome 17q22-24.

     chorionic somatomammotropin-like gene;  chorionic somatomammotropin gene A;  somatomammotropin gene B;  GH-variant gene.

  • GH Axis Cone, et al. in Endocrinology, Larsen, et al. eds. 2003

  • Growth Hormone Signaling

    G H R

    G H R

    DAG

    PKC

    C a2 +

    PIP2

    PIP3

    JAK2 JA K2

    STAT1

    STAT1

    STAT5

    STAT5

    STAT3

    STAT3

    STAT3 STAT1

    P

    P

    P

    P

    P

    P P

    P

    P

    P

    P

    P SHP1

    PI3K

    MAPK

    p90RSK

    IGF1 IGF2

    IGF2 IGF1

    P

    CEBP

    PI3K PCL-γ

    Growth Hormone

    Cell Survival

    GLE SPi2.1 CYP2/3

    SIE SIE SIE

    Gene Expression

    Cell Surviv

    Protein Synthesis

    Glucose Uptake

    Glucose

    GLUT4 Vesicle

    STAT5 P

    STAT5 P 2009 ProteinLounge.com C

  • Pituitary hGH Isoforms 1 (88%); 2 (8%); 3 (3%); 4 (1%)

  • Zhan, Giorgianni, Desiderio. Proteomics, 1228-1241 (2005). See also: Kohler, Thomas, Puschel, Schanzer, Thevis. J. Proteome Research 8, 1071-1076 (2009). Thevis. Mass spectrometry in sports drug testing. Wiley (2010) 360 pp.

    Spot No.

    Swiss-Prot No.

    GH splice isoform

    1** P01241 Isoform 1/2 2 P01241 Isoform 3; Isoform 1/2 3 P01241 Isoform 1 4* P01241 Isoform 1 5 P01241 Isoform 1 6** P01241 Isoform 1 7 P01241 Isoform 1 8 P01241 Isoform 1 9 P01241 Isoform 2 10** P01241 Isoform 1 11 P01241 Isoform 1 12 P01241 Isoform 1 13 P01241 Isoform 4; isoform 3 14** P01241 Isoform 1 15** P01241 Isoform 1 16 P01241 Isoform 1 17 P01241 Isoform 1 18 P01241 Isoform 1 19** P01241 Isoform 1 20 P01241 Isoform 1 21 P01241 Isoform 1 22 P01241 Isoform 1 23 P01241 Isoform 3; isoform 1 24 P01241 Isoform 1

  • LC-MS/MS identified two phosphopeptides from hGH in spot 5: FDTNpSHNDDALLK SLVYGApSDSNVYDLLK

    1 2

    3 4 5 55

    44

    44.87

    0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 Time (min)

    100

    R el

    at iv

    e A

    bu nd

    an ce

    31.61

    3.05 6.46 39.01 42.62 5.53 38.08 10.31 48.91 35.83 30.54 58.42 12.14 55.56 65.53 50.43 14.17 28.60 60.80 66.05 19.68

    200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z

    0

    5

    10

    15

    20

    25

    30

    35

    40

    45

    50

    55

    60

    65

    70

    75

    80

    85

    90

    95

    100

    R el

    at iv

    e A

    bu nd

    an ce

    736.57

    654.51 735.65 785.13

    727.23 605.51 444.08 676.90

    1423.07 1028.14 1178.96 894.00 792.78 569.01 259.87 1310.17 445.02 384.99 1528.25

    200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z

    0

    5

    10

    15

    20

    25

    30

    35

    40

    45

    50

    55

    60

    65

    70

    75

    80

    85

    90

    95

    100

    R el

    at iv

    e A

    bu nd

    an ce

    762.92 863.24

    651.25

    812.52

    1361.16 1173.02 713.93 912.85

    1362.34 1075.21 488.23 1233.02 1676.96

    1524.28 1067.27 1076.17 299.98 1450.97 1264.04 591.06 434.91 1679.01 1563.52 960.05 260.23 418.06 1680.10

    Neutral-fragment chromatogram: Only peptides that generated a signal In the MS/MS spectrum at (M+2H-98)2+ are displayed. Beranova-Giorgianni, et al. Pituitary 9, 109-120 (2006).

    MS/MS spectrum MS/MS spectrum

  • Endogenous Phosphorylated hGH (phGH)

    73 phosphopeptides  26 pituitary hormones. Beranova-Giorgianni, et al. Pituitary 9, 109-120 (2006).

     phGH: – 132pSer and 176pSer.

     132pSer was also studied in Giorgianni, et al. Proteomics 4, 587-598 (2004).

     Endogenous phGH: – Unknown biological role.

     in vitro-synthesized pGH. – Liberti and Joshi, BBRC 137, 806-812 (1986).

     No other endogenous phGH references found in the literature.

  • MS/MS Quantification of Peptides

     Synthetic opioid peptide analog: µ opioid receptor agonist: – Dmt-D-Arg-Phe-Lys-NH2 = Dmt1DALDA. (2’,6’dimethyl-Tyr)

    • 2H5-Dmt1DALDA internal standard. • Ovine plasma. Pharmacokinetics. • LC-nanospray-ion trap MS/MS.

    • Wan, et al. J. Chromatog. B 803, 83-90 (2003).

    • LC-Q-TOF-MS/MS. • Wan and Desiderio. Rapid Commun. Mass Spectrom. 17, 538-546, (2003).

     Endogenous β-endorphin (BE1-31) in human pituitaries: – via NAIIK  NAI transition. NAIIK = tryptic peptide = BE20-24

     2H4-NAIIK internal standard.

     Endogenous methionine enkephalin (ME) in human pituitaries: – via YGGFM  YGGF transition.

     2H5-ME internal standard. – Zhu and Desiderio. Mass Spectrometry Reviews 15, 213-240 (1996).

  • Heterogeneity Analysis of Human Pituitary Proteomes

     Pituitary control tissue (n=8 tissues).  2DGE (triplicate); PDQuest. M 27 y.o. B F 40 W M 45 W M 36 W F 34 B F … W M 55 W F 47 W

     51 differential spots were found: – 7 proteins differed with gender; 17 with age; 15 with race; 12 with

    age and race. – Only one protein, hGH, differed with gender, age, and race.

    – Zhan and Desiderio. Clin. Chem. 49, 1740-1751 (2003).

  • rGH  Genotropin. Commercially available.

    – Expressed in E. coli, strain K12.

     Analyzed with 2DGE; MALDI-MS/MS; LC-MS/MS.  Amino acid substitutions and modifications were found:

    – dimethyl 70K; or 70K  R. – deamidated 149N and 152N. – 127R  K or E. – 14, 125, 170M  partially oxidized. – 14M  I. 14M influences receptor binding.

     Those eight changes (all were partial) occurred at 2% whole protein amount (average = 0.25% = 2.5 ppt).

    Hepner, et al. Proteome Sci. 2005, 3:1 (BioMed Central open access).

  • hGH Tryptic Peptides 9 Lys; 9 Arg

     1-6 7-42 43-45 46-64  65-67 68-90 91-96 97-103  104-120 121-140 141-153 154-160  161-166 167