probing for characteristics of growth hormone by lc-esi- ms/ms€¦ · 355 mario thevis1, rachel r....

355

Mario Thevis1, Rachel R. Ogorzalek-Loo2, Joseph A. Loo3,4, Michael Bredehöft1 and

Wilhelm Schänzer1

Probing for Characteristics of Growth Hormone by LC-ESI-

MS/MS

Institute of Biochemistry, German Sport University, Cologne, Germany1, and Departments of

Chemistry and Biochemistry3, Biological Chemistry2 and the Mass Spectrometry and

Proteomics Technology Center4, University of California, Los Angeles, CA 90095-1570

Introduction Growth hormone (GH, Figure 1) was identified as the growth-promoting principle of the

human pituitary gland in 1956 [1], and with its isolation and preparation from hypophysis its

structural heterogeneity became evident. Detailed investigations elucidated the actual reasons

for numerous GH variants, which were found to originate from an alternative splice site at the

mRNA level [2, 3], from post-translational modifications such as N-terminal acylation or

deamidation [4], as well as from oligomerization [5-7]. In addition, artificial products such as

scissions within the primary structure supposedly resulting from harsh extraction conditions

or long-term storage have been identified [8, 9]. Several assays enabling the determination of

GH levels as well as the potential misuse of recombinant preparations have been published

primarily utilizing sophisticated immunoassays [6, 10-17]. In order to optimize immunoassays

in terms of cross reactivity as well as specificity, the characterization of antigens and their

natural as well as artificial variants is of paramount importance and has been subject of

considerable research in the past. Objects of investigation have been natural monomers (e.g.

22 kDa and 20 kDa variants) [18, 19], covalent as well as non-covalent oligomers [6, 7, 20-

24], and chemical, proteolytic or metabolic degradation products of growth hormones [9, 25].

In the present study, mass spectrometric investigations regarding quality and composition of

recombinant as well as pituitary growth hormone preparations are described, providing

unambiguous data on degradation after long-term storage and post-translational modification

of GH samples.

In: W Schänzer, H Geyer, A Gotzmann, U Mareck (eds.) Recent Advances In Doping Analysis (13). Sport und Buch Strauß - Köln 2005

356

Phe

Pro

Thr

Ile

ProLeu

SerArg

LeuPhe

AspAsn

Ala Met Leu Arg Ala His Arg

1

10

Leu20

His Gln Leu Ala Phe Asp Thr Tyr Gln GluPhe

Glu

30

GluAla

Tyr

Ile

Pro

Lys

Glu

Gln

Lys

40

Tyr

Ser

Phe

LeuGln

AsnPro

GlnThr50

Ser Phe Leu SerThr Pro Ile Ser Glu60

ProSerAsnArgGluGluThrGlnGln

LysSer

Asn

Leu

Gln

Leu

70

Leu

Arg

Ile

Ser

Leu 80

LeuLeu

IleGln

SerTrp Leu Gln Pro Val

90Val Phe Ala Asn Ser

100

LeuVal

TyrGly

Ala

Ser

Asn

Ser

Asp

Val 110TyrAspLeu

LeuLys

Asp

LeuGlu

Glu

Gly120

Asp Glu Leu Arg Gly Met Leu ThrGln

Ile130

PheIle

GlnGly

ThrArg Pro Ser Gly

Cys

140

SerArgLeuPheGln

Lys

Lys

Ser

Tyr

Thr

Gln

SerAsnThr

AspPhe 150

AspAlaAsp

AsnHis

Leu

LeuLys Asn Tyr Gly

160Leu Leu Tyr Cys

Val Ile ArgLeu

Phe

MetAsp

AspLys

Arg

PheThr

GluVal

Lys

Gln

SerArg

ValGlu Gly

Gly PheCys

Cys

170

180

Ser

190

NH

2

CO

OH

Figure 1: Primary structure of the 22 kDa isoform of human growth hormone

consisting of 191 amino acids and two disulfide bonds between the cysteine

residues 53 and 165 as well as 182 and 189.

Experimental

Chemicals and reagents. Recombinant human GH (Genotropin) was purchased from Pfizer

(Karlsruhe, Germany). Pituitary GH was obtained from the National Institute for Biological

Standards and Control (NIBSC 80/505, South Mimms, UK). NuPage mini gels (12% Bis-Tris,

1.0 mm x 10 well), NuPage LDS sample buffer, NuPage reducing agent and NuPage MOPS

SDS running buffer were bought from Invitrogen (Carlsbad, CA). Tris(carboxyethyl)phospine

(TCEP) and dithiothreitol (DTT) were purchased from Sigma (Steinheim, Germany).

GelCode Blue stain reagent was obtained from Pierce (Rockford, IL).


357

Gel electrophoresis. 1D-gel electrophoresis was conducted on a Hoefer SE 260 gel

electrophoresis using Invitrogen NuPage Bis-Tris (12%) pre-cast gels and MOPS SDS

running buffer. Aliquots of 26 µL of aqueous samples containing 50 pmol of analyte were

mixed with 10 µL of LDS running buffer and, in case of reducing conditions, with 4 µL of

reducing agent and incubated at 70°C for 10 minutes. After cooling to ambient temperature,

20 µL were placed in each mini gel well, and gels were run at 200 V (constant) for 50

minutes. Staining was accomplished by gently shaking the gel in Coomassie GelCode blue

staining reagent for two hours. Subsequently, gels were washed with deionized water for 1

hour to enhance the intensity of visualized bands.

Reduction of intact proteins. Aqueous solutions of GH were treated either with 10 mM TCEP

or DTT for 10 minutes at 60°C.

Liquid chromatography-mass spectrometry. Liquid chromatography-mass spectrometry was

performed on an Agilent 1100 Series capillary LC equipped with a Zorbax SB300 C18

column (0.3mm x 50mm, particle size 3 µm, pore size 300 Å). Solvents used were A: 0.1%

acetic acid containing 0.01% TFA, and B: acetonitrile-water (80% acetonitrile/20% water)

containing 0.1% acetic acid and 0.01% TFA. After loading of 5µL of sample onto a trapping

conlumn (Zorbax SB300 C8, 1.0mm x 17mm) for 3 minutes, analytes were transferred onto

the analytical column at a flow rate of 10 µL/min using a linear gradient from 90% A to 0% A

in 15 minutes. The mass spectrometer was an Applied Biosystems Qtrap 4000 analyzer

operated either in the full scan or product ion scan mode using the linear ion trap at a scan rate

of 1000 u/s. The declustering potential was set to 50V and collision offset voltages varied

according to the stability of selected precursor ions. Positive ionization was used for all

analyses at a spray voltage of 5000V.

Results

In Figure 2a, the ESI mass spectrum of recombinant GH is shown consisting of a charge-

envelope from (M+12H)12+ to (M+23H)23+. In comparison, Figure 2b represents the ESI

spectrum generated from GH extracted from pituitaries. Here, two charge-envelopes are

visible demonstrating the presence of the two most abundant GH variants at 22 kDa and 20

kDa as shown by means of deconvolution in the inset.


358

800 1000 1200 1400 1600 1800 2000m/z

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel

ativ

e ab

unda

nce

1302. 4 1383.7

1230.11581.3

1165.4

1702.9

1107.21844.8

1054.61006.7

1475.9

17+ 16+

18+

19+

20+

21+22+

14+

15+

13+

12+

800 1000 1200 1400 1600 1800m/z

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel

ativ

e ab

unda

nce

1383.81303.1

1475.61581.01231.1 1352.4

1448.91166.41702.81268.5

1689.61108.3

1560.1

1193.6

17+ 16+

18+

19+

20+

14+15+

13+

17+

16+12+

14+

15+ 13+

a)

b)

Figure 2: ESI spectra of a) recombinant GH (Genotropin) and b) GH from pituitary

extracts recorded on an Applied Biosystems Qtrap 4000.

The long-term storage of hGH in solution at +4°C causes a considerable degradation as

described in the literature. In Figure 3, a collage of 1D-gel electrophoreses is depicted

prepared from fresh recombinant GH and pituitary GH after storage for 4 months in a

refrigerator in aqueous solution.

The lanes A-B contain recombinant GH without cleavage of disulfide bonds while lanes C-D

were prepared with disulfide reduction. In both cases only one band at 22 kDa was observed

demonstrating that a freshly prepared solution of GH does not degrade under reducing

conditions. Here, DTT as well as TCEP were employed generating identical results (data not

shown). In contrast to recombinant GH, GH samples from hypophysis gave rise to several

bands upon 1D-gel electrophoresis. Lanes E-F of Figure 3 were prepared without disulfide

20.000 20.500 21.000 21.500 22.000 22.500

22123.7

20266.5

m/z

20 kDa hGH

22 kDa hGH


359

reduction and numerous bands were assigned, the most abundant one of which is the 22 kDa

variant of GH. The 20 kDa isoform was separated and visualized below the 22 kDa band, and

above an intense but artificial “24 kDa” variant was observed. In addition, dimeric structures

are shown at 45 kDa. The naturally occurring 24 kDa variant obtained by glycosylation of GH

is usually low abundant, hence an artificial origin was assumed for the intense band shown in

Figure 3 (E-F). The disulfide reduction of pituitary GH followed by gel electrophoresis gave

rise to the lanes G-H that did not contain the “24 kDa” GH band but a new band at

approximately 16 kDa. The phenomenon of a “24 kDa” GH has been described in the

literature severalfold, and a sequence cleavage between phenylalanine 139 and lysine 140 of

the 22 kDa GH isoform was assumed causing altered migration properties of the protein under

1D-gel electrophoresis conditions due to additional C- and N-termini. The presence of a

disulfide bond between the cysteine residues 53 and 165 prevents a loss of the C-terminal

peptide, but reduction of the protein results in an elimination of the liberated peptide.

Figure 3: 1D-gel electrophoreses of recombinant and pituitary GH without (n-red.) and

with (red.) disulfide reduction.

Mass spectrometric evidence for this proposal was obtained by analyzing the degraded and

disulfide-reduced sample by LC-MS. In Figure 4, two mass spectra are shown representing a

16 kDa (a) as well as 6 kDa (b) fragment of GH, the measured masses of which are shown in

respective insets obtained by deconvolution. Considering an additional water molecule due to

the hydrolyzed peptide bond, the molecular masses are exactly matching a cleavage of the GH

10 kDa15 kDa

20 kDa25 kDa

37 kDa

50 kDa

75 kDa100 kDa

A B C D E F G H

“24-kDa GH“

20-kDa GH

22-kDa GH

recombinant pituitary (NIBSC)

n-red. red. n.-red. red.16-kDa fragment

A B C D E F G H dimeric structures (45 kDa)


360

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel

ativ

eab

unda

nce

m/z800 1000 1200 1400 1600

1004.9 1148.2

1071.7

1236.6

1339.4

945.8

1461.0

1606.8

1.45e4 1.50e4 1.55e4 1.60e4 1.65e4 1.70e4

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

m/z

Rel

ativ

eab

unda

nce

17+

16+ 14+

15+

13+

12+

11+

10+

16061.5

900 1000 1100 1200 1300 1400 1500 1600

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel

ativ

eab

unda

nce

m/z

4000 4500 5000 5500 6000 6500 7000 7500

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

m/z

Rel

ativ

eab

unda

nce

6085.5

1218.2

1015.4

1521.84+

5+

6+

primary structure between Phe139 and Lys140, substantiating the proposed degradation

process. In addition to the determination of protein fragment masses, top-down as well as

bottom-up sequencing provided unambiguous information on the origin of the investigated

signals.

a)

b)

Figure 4: ESI-mass spectra of a 16 kDa fragment (a) and a 6 kDa fragment (b) obtained

from degraded and disulfide-reduced pituitary GH. Masses of analytes were

obtained by deconvolution and represent the first 138 or the last 53 amino acid

residues of GH.


361

Top-down sequencing of the 6 kDa peptide gave rise to the product ion spectrum shown in

Figure 5a allowing the assignment of a sequence tag (NDDALLKN) unambiguously

demonstrating its origin from human GH. In addition, the 16 kDa fragment allowed the

identification of a sequence tag of EAYIPKEQK that represents the N-terminal region of GH

from residue 33 to 41. Moreover, the 16 kDa band was excised from the 1D-gel (Figure 3),

hydrolyzed by means of trypsin, and resulting peptides were measured by MALDI-MS

allowing a peptide mass fingerprint of GH with a sequence coverage of 87%.

In addition to the identification of degradation products of GH generated by long-term

storage, post-translational modifications such as inter-molecular disulfide bonds and

phosphorylation/sulfonation were observed. In Figure 6a a product ion mass spectrum is

shown that was recorded from a doubly charged precursor ion at m/z 784.3 derived from

tryptically digested pituitary GH. The amino acid sequence tag obtained by CID enabled the

assumption of a homodimeric peptide composed by SVEGSCGF, the C-terminus of hGH.

The presence of this homodimer was postulated in the literature earlier but was not proven by

mass spectrometry. In order to substantiate the finding, the peptide SVEGSCGF was

synthesized, dimerized in solution and analyzed under identical conditions as used for the

trypsin-digested hGH. The resulting product ion spectrum is shown in Figure 6b proving

identity with the peptide isolated from pituitary GH.

Finally, a peptide presumably composed by the amino acid residues VETFLR was detected as

shown in Figure 7. However, its molecular mass was incremented by 80 u accounting either

for a phosphorylation or sulfonation. At present, the identification of the modification has not

been finished.


362

KQTYSKFDTN SHNDDALLKN YGLLYCFRKD MDKVETFLRI VQCRSVEGSC GFy40 y35

200 400 600 800 1000 1200 1400 1600 1800

10%

20%

30%

40%

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60%

70%

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90%

100%

m/z

Rel

ativ

e ab

unda

nce

y383+

b7

“NDDALLKN“

1129.4

1217.91214.3

1252.4

1188.6950.91292.6

1435.31076.4

1336.1

239.0 866.3 997.9 1473.1736.0

1511.6

1684.6

y202+

y222+

y333+ y34

3+

y373+

(M+5H)5+

(M+5H – H2O)5+

y172+

y353+

1373.6

b2 – H2O883.0

b8

y393+

1550.0y40

3+

Figure 5: ESI-product ion spectra of fragments derived from degraded pituitary GH after

disulfide reduction. a) 6 kDa fragment; precursor at m/z 1218 (M+5H)5+, and b)

16 kDa fragment; precursor at m/z 1339 (M+12H)12+.

FPTIPLSRLF DNAMLRAHRL HQLAFDTYQE FEEAYIPKEQ K

1000 1100 1200 1300 1400 1500 1600 1700 1800

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1131.0

I

“EAYIPKEQK“

962.4

1085.2

1287.2 1363.81056.81016.0 1309.4

1141.2

1266.8

1173.21446.11238.6

997.9 1353.01205.41397.0

1482.4

1528.4 1563.6 1759.41695.0

1715.0

965.0

EA

Y

PK

EQ

K

b364+

b354+

b344+

b334+

b324+

b384+

Rel

ativ

e ab

unda

nce


363

a)

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500

10%

20%

30%

40%

50%

60%

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100%

m/z

Rel

ativ

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nce

784.3

159.0

1252.4

316.2 691.2 1108.4187.0 1381.4775.4442.1424.1 1030.3 1345.41195.4223.0120.0 943.3 1048.2817.1673.2 1402.4

S V E G S C G FІ

S V E G S C G F

b)

100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500

10%

20%

30%

40%

50%

60%

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m/z

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S V E G S C G FІ

S V E G S C G F

b2 b3 b4 b5

784.2

1252.1691.3159.0 316.1 1108.0

775.4460.0373.1 1381.1187.1 1195.1904.0817.0 1345.0673.3 1087.0943.0598.7 702.1223.0 563.0 1402.1

442.0

a2

b2

b3

y2b4

b5 SG E

Figure 6: Product ion mass spectra of (M+2H)2+ at m/z 784.3 from a) tryptically

hydrolyzed pituitary GH, and b) synthesized homodimeric peptide.


364

V E T F L Ry5 y4 y3 y2 y1

200 300 400 500 600

10%

20%

30%

40%

50%

60%

70%

80%

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100%

120.1

m/z

Rel

ativ

e ab

unda

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536.0

664.9201.0175.1

220.9

646.9

435.1271.2 288.1156.0 229.0492.1418.1

314.0 518.1 629.0359.9 501.1

y2

y1

y3

y4

y5

y5-H2O

Figure 7: Product ion mass spectrum of (M+2H)2+ at m/z 422.5 from tryptically

hydrolyzed pituitary GH.

Conclusion

Mass spectrometric studies enable a valuable insight into target analytes and natural reference

material of doping controls. In particular when using peptides and proteins frequent quality

control analyses are recommended owing to the liability to degradation, and mass

spectrometry supports the identification of degradation products that may possibly interfere

with antibody assays. Moreover, the identification of specific characteristics of target

compounds may support the preparation of complementary or more selective and robust

assays employing antibody-antigen strategies.

Acknowledgments

The authors thank the Manfred Donike Gesellschaft e.V., Cologne, for financial support.


365

References

1. Li, C. H.; Papkoff, H. Preparation and Properties of Growth Hormone from Human

and Monkey Pituitary Glands. Science 1956, 124, 1293-1294. 2. DeNoto, F. M.; Moore, D. D.; Goodman, H. M. Human Growth Hormone DNA

Sequence and Mrna Structure: Possible Alternative Splicing. Nucleic Acids Res 1981, 9, 3719-3730.

3. Masuda, N.; Watahiki, M.; Tanaka, M.; Yamakawa, M.; Shimizu, K.; Nagai, J.; Nakashima, K. Molecular Cloning of Cdna Encoding 20 Kda Variant Human Growth Hormone and the Alternative Splicing Mechanism. Biochim Biophys Acta 1988, 949, 125-131.

4. Lewis, U. J.; Singh, R. N.; Bonewald, L. F.; Lewis, L. J.; Vanderlaan, W. P. Human Growth Hormone: Additional Members of the Complex. Endocrinology 1979, 104, 1256-1265.

5. Stolar, M. W.; Amburn, K.; Baumann, G. Plasma "Big" and "Big-Big" Growth Hormone (Gh) in Man: An Oligomeric Series Composed of Structurally Diverse Gh Monomers. J Clin Endocrinol Metab 1984, 59, 212-218.

6. Brostedt, P.; Luthman, M.; Wide, L.; Werner, S.; Roos, P. Characterization of Dimeric Forms of Human Pituitary Growth Hormone by Bioassay, Radioreceptor Assay, and Radioimmunoassay. Acta Endocrinol (Copenh) 1990, 122, 241-248.

7. Brostedt, P.; Roos, P. Isolation of Dimeric Forms of Human Pituitary Growth Hormone. Prep Biochem 1989, 19, 217-229.

8. Singh, R. N.; Seavey, B. K.; Lewis, U. J. Heterogeneity of Human Growth Hormone. Endocr Res Commun 1974, 1, 449-464.

9. Baumann, G. Growth Hormone Heterogeneity: Genes, Isohormones, Variants, and Binding Proteins. Endocr Rev 1991, 12, 424-449.

10. Baumann, G. Growth Hormone Heterogeneity in Human Pituitary and Plasma. Horm Res 1999, 51, 2-6.

11. Ranke, M. B.; Orskov, H.; Bristow, A. F.; Seth, J.; Baumann, G. Consensus on How to Measure Growth Hormone in Serum. Horm Res 1999, 51 Suppl 1, 27-29.

12. Radetti, G.; Buzi, F.; Tonini, G.; Bellone, J.; Pagani, S.; Bozzola, M. Growth Hormone (Gh) Isoforms Following Acute 22-Kda Gh Injection: Is It Useful to Detect Gh Abuse? Int J Sports Med 2004, 25, 205-208.

13. Bidlingmaier, M.; Wu, Z.; Strasburger, C. J. Doping with Growth Hormone. Journal of Pediatric Endocrinology & Metabolism 2001, 14, 1077-1084.

14. Wallace, J. D.; Cuneo, R. C.; Bidlingmaier, M.; Lundberg, P. A.; Carlsson, L.; Boguszewski, C. L.; Hay, J.; Boroujerdi, M.; Cittadini, A.; Dall, R.; Rosen, T.; Strasburger, C. J. Changes in Non-22-Kilodalton (Kda) Isoforms of Growth Hormone (Gh) after Administration of 22-Kda Recombinant Human Gh in Trained Adult Males. J Clin Endocrinol Metab 2001, 86, 1731-1737.

15. Wu, Z.; Bidlingmaier, M.; Dall, R.; Strasburger, C. J. Detection of Doping with Human Growth Hormone. Lancet 1999, 353, 895.

16. Bidlingmaier, M.; Wu, Z.; Strasburger, C. J. Test Method: Gh. Baillieres Best Pract Res Clin Endocrinol Metab 2000, 14, 99-109.

17. Momomura, S.; Hashimoto, Y.; Shimazaki, Y.; Irie, M. Detection of Exogenous Growth Hormone (Gh) Administration by Monitoring Ratio of 20kda- and 22kda-Gh in Serum and Urine. Endocr J 2000, 47, 97-101.


366

18. Baumann, G.; Stolar, M. W.; Amburn, K. Molecular Forms of Circulating Growth Hormone During Spontaneous Secretory Episodes and in the Basal State. J Clin Endocrinol Metab 1985, 60, 1216-1220.

19. Baumann, G.; Stolar, M. W.; Buchanan, T. A. The Metabolic Clearance, Distribution, and Degradation of Dimeric and Monomeric Growth Hormone (Gh): Implications for the Pattern of Circulating Gh Forms. Endocrinology 1986, 119, 1497-1501.

20. Becker, G. W.; Bowsher, R. R.; Mackellar, W. C.; Poor, M. L.; Tackitt, P. M.; Riggin, R. M. Chemical, Physical, and Biological Characterization of a Dimeric Form of Biosynthetic Human Growth Hormone. Biotechnol Appl Biochem 1987, 9, 478-487.

21. Lewis, U. J.; Peterson, S. M.; Bonewald, L. F.; Seavey, B. K.; VanderLaan, W. P. An Interchain Disulfide Dimer of Human Growth Hormone. J Biol Chem 1977, 252, 3697-3702.

22. Nagatomi, Y.; Ikeda, M.; Uchida, H.; Wada, M.; Kobayashi, H.; Hashimoto, Y.; Mabuchi, K.; Hayakawa, M.; Kusuhara, N.; Honjo, M. Reversible Dimerization of 20 Kilodalton Human Growth Hormone (Hgh). Growth Horm IGF Res 2000, 10, 207-214.

23. Zhan, X.; Giorgianni, F.; Desiderio, D. M. Proteomics Analysis of Growth Hormone Isoforms in the Human Pituitary. Proteomics 2005, 5, 1228-1241.

24. Grigorian, A.; Bustamante, J.; Hernandez, P.; Martinez, A.; Haro, L. Extraordinarily Stable Disulfide-Linked Homodimer of Human Growth Hormone. Protein Sci 2005, 14, 902-913.

25. Singh, R. N. P.; Seavey, B. K.; Lewis, U. J. Human Growth Hormone Peptide 1-43: Isolation from Pituitary Glands. J Protein Chem 1983, 2, 525-536.


probing for characteristics of growth hormone by lc-esi- ms/ms€¦ · 355 mario thevis1, rachel r....

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