Identification of Human Pituitary Growth Hormone Variants by Mass

Jan 5, 2009 - and the 17 kDa fragment was shown to be neither growth promoting nor insulin-like but to bind to only one growth hormone (GH) receptor ...
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Identification of Human Pituitary Growth Hormone Variants by Mass Spectrometry Maxie Kohler,† Andreas Thomas,† Klaus Pu ¨ schel,‡ Wilhelm Scha¨nzer,† and Mario Thevis*,† Institute of Biochemistry/Center for Preventive Doping Research, German Sport University Cologne, Germany, and Department of Legal Medicine, University Hospital Hamburg-Eppendorf, Germany Received November 3, 2008

The heterogeneity of human endogenous growth hormone (GH) is used in doping control analysis to distinguish it from the homogeneous recombinant analogue in plasma samples. Pituitary GH variants were characterized by gel electrophoresis and mass spectrometry. Besides 22 and 20 kDa isoforms, fragments of 9 and 12 kDa were identified and a glycosylated 23 kDa GH variant was elucidated to bear a HexHexNac*2 NeuAc modification presumably located at Thr 60. Keywords: 2D-PAGE • orbitrap mass spectrometry • glycosylation • fragment • sport

Introduction Endogenous human growth hormone (hGH) is produced in the pituitary gland and consists of several isoforms and fragments. At the transcription level, different splice variants, especially a 20 kDa variant,1 are generated and different posttranslational modifications further increase the heterogeneity of hGH. Besides phosphorylations that were demonstrated in earlier studies,2-4 glycosylation was assumed using deglycosylation experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).5-7 Furthermore, deamidations,3 acylation, fragments of 17 and 5 kDa8,9 as well as different dimeric and polymeric variants10-12 were identified, and studies on different isoforms and fragments outlined particular functions. The 22 kDa growth hormone, which represents the predominating variant in the pituitary as well as plasma, regulates growth in childhood and has an important impact on various metabolic processes. While skeletal growth as well as other anabolic processes such as protein biosynthesis are mediated via the insulin-like growth factor I (IGF I), which is released from the liver in response to hGH, carbohydrate and lipid metabolism are directly influenced by hGH. In terms of carbohydrate metabolism, hGH antagonizes the effects of insulin. Administration of hGH results in hyperglycemia due to decreased utilization of glucose and increased gluconeogenesis. In contrast, the release of free fatty acids and their oxidation in the liver is increased. Mineral metabolism is promoted by hGH and IGF I resulting in a positive calcium, magnesium and phosphate balance as well as a retention of sodium, potassium and chloride ions.13,14 The 20 kDa splice variant, which covers approximately 5-10% of total growth hormone in the human body, has similar metabolic functions and interacts also with the growth hormone receptor. The splice * To whom correspondence should be addressed. Mario Thevis, Ph.D., Institute of Biochemie/Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark Mu ¨ngersdorf 6, 50933 Cologne, Germany. Tel., 0221-49827070; fax, 0221-49827071; e-mail, [email protected]. † German Sport University Cologne. ‡ University Hospital Hamburg-Eppendorf. 10.1021/pr800945b CCC: $40.75

 2009 American Chemical Society

variant has minor insulin-like and less diabetogenic activity but a longer half-life in the circulation, which is due to a higher affinity to binding proteins.15,16 Different activities were attributed to the proteolytic fragments of 5 and 17 kDa. The N-terminal 5 kDa fragment possesses an insulin-like activity and the 17 kDa fragment was shown to be neither growth promoting nor insulin-like but to bind to only one growth hormone (GH) receptor, which may imply an antagonistic function to the intact GH.15 Growth hormone is pharmaceutically used to medicate dwarfism as well as growth hormone deficiency in adults,17 and possible side effects are mainly known from acromegalic people that suffer from an uncontrolled release of growth hormone from the pituitary.18 Furthermore, recombinant growth hormone (rGH) is supposed to be widespread as a performance enhancing agent in athletes,19 although the effects in healthy humans are controversial.20-22 Clinical and antidoping growth hormone measurements can be optimized by characterization of the protein and its isoforms and fragments. Especially antibody-based assays, which are commonly used in clinics, may react differently to distinct isoforms. This may influence test results if a sample shows unusual ratios of isoforms or unusual modifications. The present study was conducted to identify growth hormone isoforms and fragments from the pituitary by mass spectrometry. The identification was mainly focused on variants which are detected by the 2D-PAGE antidoping method recently described23 to allow a better evaluation and interpretation of assay results, especially if atypical results are obtained.

Material and Methods Materials. Recombinant growth hormone (Genotropin) was from Pharmacia (Karlsruhe, Germany) and 12% Bis-Tris SDS gels, 3-(N-morpholino)propanesulfonic acid (MOPS) running buffer and lithium dodecyl sulfate (LDS) sample buffer were purchased from Invitrogen (Karlsruhe, Germany). The 8-16% gradient Tris-glycine gels were from Proteome Systems (MA), IPG strips were from Bio-Rad (11 cm, pH 4.7-5.9 and 24 cm, Journal of Proteome Research 2009, 8, 1071–1076 1071 Published on Web 01/05/2009

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Figure 1. 2D gel of a pituitary extract. Encircled spots contain growth hormone. Spots numbered 1-10 were further identified by mass spectrometry.

pH 4-7 Munich, Germany) or GE Healthcare (24 cm, pH 4.5-5.5, Munich, Germany) and the Coomassie Blue stain was purchased from Pierce (Rockford, IL). DTT, acrylamide (both analytical grade) and all buffer ingredients (electrophoresis grade) were from Sigma (Deisendorf, Germany). Trypsin was from Promega (Mu ¨nster, Germany) and Glu-C was bought from Roche (Mannheim, Germany). Ultrafree and Amicon centrifugal filters were purchased from Millipore (Schwalbach, Germany). Water, acetonitrile and formic acid for nano-LC were obtained from Biosolve (Valkenswaard, Netherlands). Pituitary Samples. Pituitary glands were provided by the Institute of Legal Medicine, University Hospital HamburgEppendorf, Germany. The fresh tissue was homogenized in 2 mL of multichaotropic sample solution (MCSS, 7.7 M urea, 2.2 M thiourea, 4.4% CHAPS, and 44 mM Tris) and sonicated for 30 min. After centrifugation (5 min, 9000g), the supernatant was transferred to a new tube and frozen at -20 °C until analysis. 2D-PAGE. Proteins were prepared for the isoelectric focusing by addition of 30-50 µL of the pituitary extract to 140-160 µL of MCSS. Disulfide bonds were reduced with 10 µL of 1 M DTT (45 min at room temperature (RT)) and cysteine residues were derivatized with acrylamide (30 µL, 1 M, 45 min at RT). After reaction of the acrylamide excess with further 20 µL of DTT (10 min at RT) and rehydration loading of the samples to the IPG strips, isoelectric focusing was performed overnight in an Ettan IPGphor 3 (GE Healthcare, Munich, Germany) with the following voltage gradient program: 100-300 V in 2 h, 300-10 000 V in 6 h and 10 000 V until 80 000 Vh. The maximum current per strip was set to 50 µA. Strips were equilibrated two times for 10 min in LDS sample buffer and then applied to 11 cm, 8-16% gradient Tris-gylcine gels or to 8 cm, 12% Bis-Tris gels (XCell6 (Invitrogen), 125 V, 90 min) after

Figure 2. Product ion mass spectrum of a peptide uniquely derived from the 20 kDa hGH isoform, which results from alternative splicing and therefore lacks the amino acids 32-46.

cropping the strip to 7.5 cm. SDS-PAGE gels were finally stained withCoomassieBlueaccordingtothemanufacturer’srecommendation. OFFGEL Isoelectric Focusing. For OFFGEL fractionation of pituitary samples, 500 µL of pituitary extract was washed with water in a centrifugal filter (molecular weight cutoff 5 kDa) and diluted in OFFGEL stock solution as recommended by the supplier (Agilent Technologies, Waldbronn, Germany). For fractionation, 24 cm IPG strips with a pH gradient of 4.5-5.5 or 4-7 and the 24 well frame set were used. Samples were run as recommended with a maximum current of 50 µA per strip to a total of at least 50 000 Vh, and a volume of 50-150 µL was recovered from each well. For visualization of the proteins in the fractions, SDS gels were prepared using 20 µL of each fraction, adding 7 µL of LDS sample buffer and 3 µL of DTT (1 M) and heating 10 min at 70 °C. After the SDS-PAGE (125 V, 100 min), gels were stained with Coomassie Blue. Fractions of interest were concentrated in a centrifugal filter (cutoff 5 kDa) and washed with water prior to further separation by capillary LC and identification of intact modified growth hormone isoforms by high resolution/high accuracy mass spectrometry (vide infra). The capillary LC was an Agilent 1100 instrument with a Zorbax 300 SB-C18 precolumn (3.5 µm, 50 × 0.3 mm) and a Zorbax 300 SB-C18 analytical column (5 µm, 5 × 0.3 mm). Solvents were 0.1% acetic acid, 0.01% TFA (solvent A) and 80% acetonitrile, 0.1% acetic acid, 0.01% TFA (solvent B). The following gradient program was applied: 0-2 min 95% A, 2-25 min 60% A, 25-34 min 3% A, 34-44 min 3% A, and reequilibration 16 min 95% A. Ionization voltage was set to 3.5

Table 1. Identified Modifications in the Spots from Figure 1 Growth hormone form

1 2 3 4 5 6 7 8 9 10 1072

22 kDa main form of growth hormone, unmodified 20 kDa splice variant, unmodified 22 kDa, singly phosphorylated or deamidated 23 kDa growth hormone form, glycosylated (HexHexNAc*2NeuAc) 22 kDa, doubly phosphorylated and/or deamidated 20 kDa, singly phosphorylated or deamidated 20 kDa splice variant, modification unknown 12 kDa fragment AS 79-191 12 kDa fragment 9 kDa fragment AS 1-78

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pI (calc)

pI (exp)

5.27 5.39 5.12/5.13

∼5.25 ∼5.35 ∼5.1 ∼5.0

4.99/5.02 5.19 (- 1 (-) charge 5.62) 5.03 (-1 (-) charge 5.27) 5.60

∼5.0 ∼5.15 ∼5.55 ∼5.05 ∼5.25 ∼5.45

Identification of Human Pituitary Growth Hormone Variants by MS

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Figure 3. (a) SDS-gel of OFFGEL fractions 7-15; fractions 9-12 were used for further analysis. (b) MS of the modified (f) and unmodified growth hormone (9) variants. (c) MS/MS spectrum of the 20-fold charged precursor at m/z 1154. Ions marked with asterisk (*) include the modification.

kV in positive mode, the maximum fill time for the orbitrap was 5000 ms, and the resolving power was 100 000 at m/z 400 (fwhm). Prior to mass spectrometry measurements, mass calibration of the instrument was performed with the manufacturer’s calibration mixture (mass accuracy ( 1 ppm, n ) 10). Measurement of Intact Growth Hormone Fragments from Pituitary Extracts. To analyze intact hGH fragments, pituitary extract was filtered using a centrifugal filter (cutoff 30 kDa). The flow through was immediately measured using capillary liquid chromatography-orbitrap mass spectrometry as described for the OFFGEL fractions.

Bottom-Up Sequencing of hGH. After 2D-PAGE or SDSPAGE, spots of interest were excised and the entrapped protein was digested with trypsin. Peptides were extracted with 50% acetonitrile containing 1% of TFA, and analyses were performed on an LTQ Orbitrap (Thermo, Bremen, Germany) after separation by nanoflow liquid chromatography (Waters, Eschborn, Germany). A Waters nano-UPLC instrument was used with a Symmetry C18 precolumn (5 µm, 180 µm × 20 mm, flow 5 µm/ min) and a BEH130C18 peptide column (1.7 µm. 100 µm × 100 mm) at a flow rate of 750 nL/min. A gradient program with 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B) was used as follows: 3 min 97% A, 3-5 Journal of Proteome Research • Vol. 8, No. 2, 2009 1073

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Figure 4. (a) The 9 kDa, N-terminal fragment of 22 kDa growth hormone, mass spectrum with different charge states; (b) MS/MS spectrum of the 9 kDa fragment from precursor [M + 5H]5+ at m/z 1850.

min 80% A, 5-45 min 40% A, 45-48 min 20% A, 48-50 min 3% A, 50.01 min 97% A, 15 min hold-equilibration. The LTQ Orbitrap was used with a nanospray ion source in positive mode with 1.4 kV ionization voltage. Full scan spectra were recorded at a resolution of 60 000 (fwhm) and the collision energy for MS/MS experiments was set to 35% (arbitrary unit, Xcalibur 2.0 SR 2, Thermo) using helium as collision gas. The damping gas used was nitrogen supplied by a CMC nitrogen generator (CMC Instruments, Eschbach, Germany). Tandem Mass Spectrometry of OFFGEL Fractions. For the detection of typical carbohydrate fragments, MS/MS spectra of intact modified growth hormone were recorded on an Applied Biosystems Qtrap 4000 mass spectrometer (Foster City, CA) after separation on an Agilent 1100 liquid chromatograph (Palo Alto, CA). The LC was equipped with a Zorbax StableBond guard column (1 mm × 17 mm, 5 µm) and a Zorbax 300SBC18 analytical column. Solvents used were 1% acetic acid/0.1% TFA (A) and acetonitrile/solvent A (80:20) (B) with the following gradient: 95% A, 0-25 min 15% A, and 10 min re-eqilibration. The collision energy for MS/MS experiments was set to 40 eV. 1074

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Results and Discussion Figure 1 shows the result of a typical two-dimensional gel electrophoresis of a pituitary extract, and all encircled spots contained peptides derived from human growth hormone. Spots that were studied in detail are numbered (1-10), and in Table 1, the different forms and respective modifications are listed. Earlier studies exploring growth hormone isoforms in the pituitary after 2D separation yielded a similar pattern after gel electrophoresis;3 however, different conditions in IEF and SDS-PAGE were applied. The spots 1-4 represent hGH variants that are utilized in a recently published 2D-PAGE growth hormone doping control method.23 Hence, their in-depth characterization was of major interest in the present study. Additionally, new fragments of 9 and 12 kDa were observed (spots 8-10). The 22 kDa Growth Hormone. The 22 kDa isoform (Figure 1, spot 1) without further modification is the main form of human growth hormone consisting of 191 amino acids. It was identified with a total sequence coverage of 90% by bottom-

Identification of Human Pituitary Growth Hormone Variants by MS a

Table 2. Pituitaries Analyzed for the 9 and 12 kDa Fragments

a

Fragments were detected in the pituitaries marked in gray.

up sequencing, and the isoelectric point as well as the molecular weight on the 2D-gel are in accordance with calculated values. Spots 3 and 5 contained phosphorylated variants of the 22 kDa hGH isoform. In spot 3, peptides were found to be phosphorylated at serines 106 and 150, and in spot 5, the phosphorylation at serine 150 was detected.3,24 For both phosphorylations, a typical loss of -98 u was observed in the MS/MS spectra (data not shown), which clearly differentiated the phosphorylation from a sulfation. Further modifications in spots 3 and 5 may be deamidations, which would result in the same pI shift. The 20 kDa Splice Variant. Spot 2 was identified as the 20 kDa splice variant by the detection of the “new” peptide formed as a result of the missing amino acids 32-46.1 The triply charged precursor ion and its MS/MS spectrum are shown in Figure 2. Analysis of the 20 kDa spot shifted to a more acidic pI value (spot 6) resulted in detection of a phosphorylation that was found at serine 150 as for the 22 kDa variants. The 20 kDa peptide detected in spots 2 and 6 was also detected in spot 7. The shift to a more basic pI value could be due to amidation or acetylation of acidic amino acids or the C-terminus, which could not be substantiated after trypsin digestion that yielded the same set of peptides as for the unmodified growth hormone. The “24” (23) kDa Glycosylated Growth Hormone. For spot 4, which has a slightly higher molecular weight than the major variant of hGH and which is the fourth spot detected in the doping control method recently described,23 glycosylation was assumed earlier using deglycosylation experiments and SDSPAGE analysis.6 The determination of the molecular weight of the protein in the spot after OFFGEL fractionation by Orbitrap mass spectrometry resulted in a monoisotopic molecular mass of 23 062.5 Da (average mass 23 077.5 Da). This results in a difference of +947.4 u in comparison to the unmodified human growth hormone (monoisotopic mass 22 115.1 Da), which is smaller than expected for this modified variant as it was described as “24 kDa GH”.6 An SDS gel showing the fractions of the OFFGEL IEF (pH range of 4.7-5.9) is shown in Figure 3a, and in Figure 3b, the full scan mass spectrum of the unmodified and glycosylated hGH is illustrated.

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The OFFGEL fractionation prior to mass spectrometry was necessary to reduce the amount of unmodified growth hormone to allow the measurement of the modified variant. Using OFFGEL fractionation, an appropriate ratio of 1:1 of the 22 and 23 kDa hGH was obtained, and the molecular weight of the modified variant was determined by deconvolution. Figure 3c shows the product ion mass spectrum of m/z 1154 ([M + 20H]20+) after collision-induced dissociation, which yielded diagnostic ions for the carbohydrate moiety such as m/z 292 for neuraminic acid (NeuAc), m/z 274 for NeuAc-H20 and m/z 366 for HexHexNAc. The summation of the observed fragments yielded exactly the additional 947.4 Da (365.1316 + 291.0949 + 291.0949 ) 947.3214; 947.32 + 22115.1 ) 23062.4 (calc), error -4.3 ppm) and the composition of HexHexNAc and two neuraminic acid residues was proposed. This type of carbohydrate structure was described earlier as O-glycosylation of other human proteins.25-27 The prediction of the site of O-glycosylation in the amino acid sequence of human growth hormone by NetOGlc 3.128,29 yielded one possible mucin-like glycosylation at threonine 60. Excision and tryptic digestion of the gel spot supported the prediction, and the peptide containing the amino acids 42-64 (YSFLQNPQTSLCFSESIPTPSNR) was missing in the modified variant but detected in the unmodified spot. Additionally, after treatment with Glu-C for digestion of the modified spot, peptide 57-64 (SIPTPSNREE) was missing in comparison to the unmodified hGH, which further substantiated the theoretical position of modification. The 9 and 12 kDa Fragments. Spots 8-10 were shown to contain fragments from the N- (spot 10) and the C-terminus of 22 kDa growth hormone (spots 8 and 9). Trypsin digestion yielded peptides from either end, and one peptide, amino acids 78-94, was detected in neither of the spots. Measurements of intact analytes after ultrafiltration of the pituitary extract showed spot 10 to be the N-terminal fragment ranging from amino acid 1-78 (M (monoisotopic), 9241.64 Da; error, 1.7 ppm) and spots 8 and 9 contained the C-terminal fragment starting at amino acid 79 which was substantiated by the accurate mass measurements (M (monoisotopic), 12891.43 Da; error, 1.8 ppm). Figure 4a shows different charge states of the 9 kDa N-terminal fragment and Figure 4b the corresponding MS/MS spectrum recorded after collision-induced dissociation of the 5-fold charged precursor ion at m/z 1850. These spots did not appear in all pituitaries but in only 2 out of 7. None of the available information like age, gender, weight, height, autolysis, cause of death, which are listed in Table 2, gave any hint to explain the appearance of these fragments in these specimens.

Conclusion The identification of the growth hormone variants detected here is important for doping control as well as clinical analysis and endocrinology. Antibody-based assays to measure growth hormone levels may react differently to selected isoforms of growth hormone especially if the modification lies within or near the antibody epitope. The better and more comprehensive the characterization of the endogenous protein, the easier it will be to correctly interpret assay results. In terms of doping analysis, the developed assays are based on the measurement of different isoforms, which makes the characterization of the detected variants particularly important. The present study focused on the variants, which are employed in the recently described 2D-PAGE method, and all spots used in that approach were characterized here. Identification of growth Journal of Proteome Research • Vol. 8, No. 2, 2009 1075

research articles hormone variants may also help to improve the understanding of endocrinological functions of the protein and its variants. Additionally, future investigations might reveal the reason for modifications, which are detected only in some subjects as, for example, the 9 and 12 kDa fragments that may also affect normal metabolic actions of growth hormone.

Acknowledgment. The study was carried out with support of the World Anti-Doping Agency, the Federal Ministry of Education and Research of Germany and the Federal Ministry of the Interior of the Federal Republic of Germany. The authors thank Agilent Technologies (Waldbronn, Germany) for the OFFGEL fractionations. Supporting Information Available: Table listing the peptides originating from Trypsin (or GluC) digestion of protein spots shown in Figure 1. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Lewis, U. J.; Bonewald, L. F.; Lewis, L. J. The 20,000-dalton variant of human growth hormone: location of the amino acid deletions. Biochem. Biophys. Res. Commun. 1980, 92 (2), 511–516. (2) Liberti, J. P.; Antoni, B. A.; Chlebowski, J. F. Naturally-occurring pituitary growth hormone is phosphorylated. Biochem. Biophys. Res. Commun. 1985, 128 (2), 713–720. (3) Zhan, X.; Giorgianni, F.; Desiderio, D. M. Proteomics analysis of growth hormone isoforms in the human pituitary. Proteomics 2005, 5 (5), 1228–1241. (4) Giorgianni, F.; Beranova-Giorgianni, S.; Desiderio, D. M. Identification and characterization of phosphorylated proteins in the human pituitary. Proteomics 2004, 4 (3), 587–598. (5) Diaz, M. J.; Dominguez, F.; Haro, L. S.; Ling, N.; et al. A 12-kilodalton N-glycosylated growth hormone-related peptide is present in human pituitary extracts. J. Clin. Endocrinol. Metab. 1993, 77 (1), 134–138. (6) Haro, L. S.; Lewis, U. J.; Garcia, M.; Bustamante, J.; et al. Glycosylated human growth hormone (hGH): a novel 24 kDa hGH-N variant. Biochem. Biophys. Res. Commun. 1996, 228 (2), 549–556. (7) Garcia-Barros, M.; Costoya, J. A.; Rios, R.; Arce, V.; et al. Nglycosylated variants of growth hormone in human pituitary extracts. Horm. Res. 2000, 53 (1), 40–45. (8) Sinha, Y. N.; Jacobsen, B. P. Human growth hormone (hGH)-(44191), a reportedly diabetogenic fragment of hGH, circulates in human blood: measurement by radioimmunoassay. J. Clin. Endocrinol. Metab. 1994, 78 (6), 1411–1418. (9) Such-Sanmartin, G.; Bosch, J.; Segura, J.; Wu, M.; et al. Characterisation of the 5 kDa growth hormone isoform. Growth Factors 2008, 26 (3), 152–162. (10) Brostedt, P.; Luthman, M.; Wide, L.; Werner, S. Characterization of dimeric forms of human pituitary growth hormone by bioassay, radioreceptor assay, and radioimmunoassay. Acta Endrocrinol. 1990, 122 (2), 241–248.

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Kohler et al. (11) Brostedt, P.; Roos, P. Isolation of dimeric forms of human pituitary growth hormone. Prep. Biochem. 1989, 19 (3), 217–229. (12) Lewis, U. J.; Peterson, S. M.; Bonewald, L. F.; Seavey, B. K.; et al. An interchain disulfide dimer of human growth hormone. J. Biol. Chem. 1977, 252 (11), 3697–3702. (13) Jenkins, P. J. Growth hormone and exercise: physiology, use and abuse. Growth Horm. IGF Res. 2001, 11 (Suppl A), S71-77. (14) Godfrey, R. J.; Madgwick, Z.; Whyte, G. P. The exercise-induced growth hormone response in athletes. Sports Med. 2003, 33 (8), 599–613. (15) De Palo, E. F.; Gatti, R.; Antonelli, G.; Spinella, P. Growth hormone isoforms, segments/fragments: does a link exist with multifunctionality. Clin. Chim. Acta 2006, 364 (1-2), 77–81. (16) Baumann, G. Growth hormone heterogeneity: genes, isohormones, variants, and binding proteins. Endocr. Rev. 1991, 12 (4), 424–449. (17) Doga, M.; Bonadonna, S.; Gola, M.; Mazziotti, G.; et al. Growth hormone deficiency in the adult. Pituitary 2006, 9 (4), 305–311. (18) Melmed, S. Medical progress: Acromegaly. N. Engl. J. Med. 2006, 355 (24), 2558–2573. (19) Rickert, V. I.; Pawlak-Morello, C.; Sheppard, V.; Jay, M. S. Human growth hormone: a new substance of abuse among adolescents. Clin. Pediatr. 1992, 31 (12), 723–726. (20) Yarasheski, K. E.; Zachweija, J. J.; Angelopoulos, T. J.; Bier, D. M. Short-term growth hormone treatment does not increase muscle protein synthesis in experienced weight lifters. J. Appl. Physiol. 1993, 74 (6), 3073–3076. (21) Holloway, L.; Butterfield, G.; Hintz, R. L.; Gesundheit, N.; et al. Effects of recombinant human growth hormone on metabolic indices, body composition, and bone turnover in healthy elderly women. J. Clin. Endocrinol. Metab. 1994, 79 (2), 470–479. (22) Berggren, A.; Ehrnborg, C.; Rosen, T.; Ellegard, L.; et al. Shortterm administration of supraphysiological recombinant human growth hormone (GH) does not increase maximum endurance exercise capacity in healthy, active young men and women with normal GH-insulin-like growth factor I axes. J. Clin. Endocrinol. Metab. 2005, 90 (6), 3268–3273. (23) Kohler, M.; Pu ¨ schel; Sakharov, D.; Tonevitskiy, A.; et al. Detection of recombinant growth hormone in human plasma by a 2D-PAGE method. Electrophoresis 2008, 29 (22), 4495–4502. (24) Beranova-Giorgianni, S.; Zhao, Y.; Desiderio, D. M.; Giorgianni, F. Phosphoproteomic analysis of the human pituitary. Pituitary 2006, 9 (2), 109–120. (25) Kobata, A. Structures and functions of the sugar chains of glycoproteins. Eur. J. Biochem. 1992, 209 (2), 483–501. (26) Mann, M.; Jensen, O. N. Proteomic analysis of post-translational modifications. Nat. Biotechnol. 2003, 21 (3), 255–261. (27) Patel, T.; Bruce, J.; Merry, A.; Bigge, C.; et al. Use of hydrazine to release in intact and unreduced form both N- and O-linked oligosaccharides from glycoproteins. Biochemistry 1993, 32 (2), 679–693. (28) Julenius, K.; Molgaard, A.; Gupta, R.; Brunak, S. Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites. Glycobiology 2005, 15 (2), 153–164. (29) Center for Biological Sequence Analysis, NetOGlyc 3.1 Server Home page, http://www.cbs.dtu.dk/services/NetOGlyc/, Technical University of Denmark, Lyngby, Denmark; accessed 02.10.08.

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