Two dimensional gel electrophoresis (2-DE) of 1 mg Genotropin^® showed a multiple spot pattern with masses between 20 000 and 35 000 Da and pIs from 4.5 to 7.0. Several two dimensional (2D) gels with sample amounts of 0.5, 1, 2, 5, 10, 20, 50, 100, 200 and 500 g of Genotropin^® were performed. Decreasing protein load showed reduction of spot size and number and finally, limitation to two spots of 22000 Da with pI of 5.3 and 5.4.

Neither MALDI-TOF-TOF nor LC-MS analysis of picked gel spots indicated any modifications or isoforms in an amount, that would explain differences between the two spots.

Electrospray liquid chromatography – mass spectrometry (LC-MS) measurements of the intact Genotropin^® have shown that the main product was a molecule with an average molecular weight (MW) of 22126.8 Da. The manufacturers had determined the average MW of Genotropin^® to be 22124 Da.

The mass difference of approximately 3 Da may originate from the deconvulation of some broader, lower intensity peaks. Several minor components could be also detected. (Figure 1) The mass differences between the main product (Nr. 1) and components Nr. 2–5 respectively indicate the oxidation of several amino acid residues. Components Nr.6 and Nr.7 show a mass discrepancy of approximately +268 Da and -19 Da respectively. According to the ratios of peak areas (Table 1), the sample consists to 84.4% of the unmodified main component; the oxidation products are present to 13.7% of the whole sample and the ratio of other minor components, which may represent additional modifications or amino acid substitutions, is approximately 2%.

Electrospray tandem liquid chromatography – mass spectrometry (LC-MS/MS) measurements of the samples prepared from one dimensional SDS-PAGE indicated mass differences at several peptides. Doubly or triply charged ions were chosen for all MS/MS experiments due to their better fragmentation pattern. Table 2 shows the sequences of the modified peptides and possible explanations for the mass discrepancies.

A mass difference of +28 Da was detected at the position K₇₀ (Figure 2), could be explained by the di-methylation of this residue, or by the exchange of this lysine to an arginine. These modifications result in a mass difference of 28.03 Da and 28.01 Da respectively. The accuracy of the mass spectrometric detection was not high enough to differentiate between these possibilities. Figure 2 shows the fragment spectrum of the peptide EETQQKSNLELLR. Intensive y ions verify that all residues have unchanged masses except of K₇₀, which makes the localization of the mass discrepancy on that lysine residue unambiguous.

Deamidation of the amino acids N₁₄₉ and N₁₅₂ was also detected. The molecular mass of the peptide RLEDGSPR was decreased with 28 Da. The mass difference could be localized to the N terminus of the peptide and might indicate the substitution of R₁₂₇ with a lysine or glutamine. The mass difference of these residues is only 0.04 Da and the accuracy of the mass spectrometric detection was not high enough to differentiate between these amino acids. Residues M₁₄, M₁₂₅ and M₁₇₀ were observed partly oxidized and in some cases the non oxidized residue showed a mass discrepancy of -18 Da (Table 2). This phenomenon is illustrated by Figure 3, which shows a product ion spectrum of the modified peptide LFDNAMLR. Fragment ions from the y series verify the mass reduction at the M₁₄ residue. This mass difference can be explained by the replacement of these methionines with isoleucines, which can be originate from the substitution of the last base in the genetic codon of methionine (M:ATG; I:ATT/C/A). According to the ratios of the peak areas of the peptides containing the unmodified and possibly substituted methionines, these changes were present at < 2% of the whole protein amount. A mass increase of 57 Da was detected at the peptide LFDNAMLR. It could be localized at the N terminus of the peptide and it is supposed to be an artefact of the alkylation step during sample preparation. All modifications were partial; in each case peptides with both modified and unmodified residues were present. LC-MS/MS spectra for all modified peptides are available as supplementary material.

Approximately 96 spots were excised from a 2D gel with a sample load of 1 mg Gentotropin^® and identified by MALDI-TOF on the basis of peptide mass matching 24 following in gel digestion with trypsin. Those samples which were analysed by peptide mass fingerprinting from MALDI-TOF were additionally analysed using LIFT-TOF/TOF MS/MS from the same target. A maximum of three precursor ions per sample were chosen for MS/MS analysis.

Genotropin^® was unambiguously identified by MS and MS/MS Data (Figure 4 and 5), with a maximum of 24 matching peptides, representing a sequence- coverage of 86% to human growth hormone sequence present in database (Figure 4, Table 3). All picked and analysed spots showed similar peptide mass fingerprints. Only the oxidation status of M varied, represented by a mass difference (ΔM) of 16 Da. Oxidation at M₁₄ was demonstrated in 59,52% of analysed spots, 80,91% of M₁₂₅ and 54,87% of M₁₇₀ showed oxidation too (Table 3). Neither changes in amino acid sequence, nor post translational modifications like phosphorylation or deamidation could be detected by this method.

Genotropin ^© MiniQuick 1,0 mg (Pharmacia & Upjohn; Stockholm, Sweden) was suspended in 0,5 ml of sample buffer consisting of 8 M urea (Merck, Darmstadt, Germany), 4% CHAPS (3- [(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate) (Sigma, St. Louis, MO, USA), 10 mM 1,4-dithioerythritol (Merck, Germany) and 0,5% carrier ampholytes "Resolyte" 3,5–10 (BDH Laboratory Supplies, Electran ^®, England). The suspension was transferred into Ultrafree-4 centrifugal filter units (Millipore, Bedford, MA), for desalting and concentrating proteins. Protein content of the supernatant was quantified by the Bradford protein assay system 38. The standard curve was generated using bovine serum albumin and absorbance was measured at 595 nm.

One dimensional SDS-polyacrylamide gel was performed as described by Laemmli 39. Samples of 0.5, 1, 2, 5, 10, 30, 50 and 100 μg were loaded on the gel. For determination of molecular weight 10 μl of precision plus protein standards, all blue (Bio Rad, California, USA), were applied on the gels.

2 DE was performed essentially as reported 40. Samples of 1 mg protein were applied on immobilized pH 3–10 nonlinear gradient strips in sample cups at their basic and acidic ends. Focusing was started at 200 V and the voltage was gradually increased to 8000 V at 4 V/min and then kept constant for a further 3 h (approximately 150,000 Vh totally). After the first dimension, strips (18 cm) were equilibrated for 15 min in the buffer containing 6 M urea, 20% glycerol, 2% SDS, 2% DTT and then for 15 min in the same buffer containing 2.5% iodoacetamide instead of DDT. After equilibration, strips were loaded on 9–16% gradient sodium dodecylsulfate polyacrylamide gels for second-dimensional separation. The gels (180 × 200 × 1.5 mm) were run at 40 mA per gel. Immediately after the second dimension run, gels were fixed for 12 h in 50% methanol, containing 10% acetic acid, the gels were stained with Colloidal Coomassie Blue (Novex, San Diego, CA) for 12 h on a rocking shaker. Molecular masses were determined by running standard protein markers (Biorad Laboratories, Hercules, CA) covering the range 10–250 kDa. pI values were used as given by the supplier of the immobilized pH gradient strips (Amersham Bioscience, Uppsala, Sweden). Excess of dye was washed out from the gels with distilled water and the gels were scanned with ImageScanner (Amersham Bioscience).

Electronic images of the gels were recorded using Adobe Photoshop and Microsoft Power Point Softwares.

Spots were excised with a spot picker (PROTEINEER sp™, Bruker Daltonics, Germany), placed into 96-well microtiter plates and in-gel digestion and sample preparation for MALDI analysis were performed by an automated procedure (PROTEINEER dp™, Bruker Daltonics) 4142. Briefly, spots were excised and washed with 10 mM ammonium bicarbonate and 50% acetonitrile in 10 mM ammonium bicarbonate. After washing, gel plugs were shrunk by addition of acetonitrile and dried by blowing out the liquid through the pierced well bottom. The dried gel pieces were reswollen with 40 ng/μl trypsin (Promega, U.S.A.) in enzyme buffer (consisting of 5 mM Octyl β-D-glucopyranoside (OGP) and 10 mM ammonium bicarbonate) and incubated for 4 hrs at 30°C. Peptide extraction was performed with 10 μl of 1% TFA in 5 mM OGP. Extracted peptides were directly applied onto a target (AnchorChip™, Bruker Daltonics) that was load with α-cyano-4-hydroxy-cinnamic acid (Bruker Daltonics) matrix thinlayer. The mass spectrometer used in this work was an Ultraflex™ TOF/TOF (Bruker Daltonics) operated in the reflector mode for MALDI-TOF peptide mass fingerprint (PMF) or LIFT mode for MALDI-TOF/TOF fully automated using the FlexControl™ software. An accelerating voltage of 25 kV was used for PMF. Calibration of the instrument was performed externally with [M+H]⁺ ions of angiotensin I, angiotensin II, substance P, bombesin, and adrenocorticotropic hormones (clip 1–17 and clip 18–39). Each spectrum was produced by accumulating data from 200 consecutive laser shots. Those samples which were analysed by PMF from MALDI-TOF were additionally analysed using LIFT-TOF/TOF MS/MS from the same target. A maximum of three precursor ions per sample were chosen for MS/MS analysis. In the TOF1 stage, all ions were accelerated to 8 kV under conditions promoting metastable fragmentation. After selection of jointly migrating parent and fragment ions in a timed ion gate, ions were lifted by 19 kV to high potential energy in the LIFT cell. After further acceleration of the fragment ions in the second ion source, their masses could be simultaneously analysed in the reflector with high sensitivity. PMF and LIFT spectra were interpreted with the Mascot software (Matrix Science Ltd, London, UK). Database searches, through Mascot, using combined PMF and MS/MS datasets were performed via BioTools 2.2 software (Bruker). A mass tolerance of 100 ppm and 2 missing cleavage sites for PMF and MS/MS tolerance of 0.5 Da and 1 missing cleavage sites for MS/MS search were allowed and oxidation of methionine residues was considered. The probability score calculated by the software was used as criterion for correct identification.

The algorithm used for determining the probability of a false positive match with a given mass spectrum is described elsewhere 43.

Genotropin^® MiniQuick 0.6 mg (Pharmacia & Upjohn; Stockholm, Sweden) was suspended in the solution provided in the two-chamber cartridge and diluted with 1% formic acid (Merck; Darmstadt, Germany) in water (Maxima, Elga; High Wycombe, UK) to 1 pM/μl. 1 μl of this solution was used for the nano-electrospray LC-MS investigation. The HPLC used was an UltiMate™ system (Dionex Corporation; Sunnyvale, CA, USA) equipped with a PepMap C18 purification column (300 μm × 5 mm) and a 75 μm × 150 mm analytical column of the same material. 0.1% TFA (Pierce Biotechnology Inc.; Rockford, IL, USA) was used on the Switchos module for the binding of the peptides and a linear gradient of acetonitrile (Chromasolv^®, Sigma-Aldrich; Seelze, Germany) and 0.1% formic acid in water was used for the elution. The gradient was (A = 5% acetonitrile / 0.1% formic acid in water; B = 80% acetonitrile / 0.1% formic acid in water) 0% B for 12 min, 80% B in 30 min, 100 % B in 3 min, 100% B for 10 min, 0% B in 2 min, 0% B for 23 min. The flow rate was 240 nl/min. The LC-system was coupled on-line to a QSTAR Pulsar hybrid mass spectrometer (Applied Biosystems; Foster City, CA, USA). The nanospray source of Proxeon (Odense, Denmark) was used with the distal coated silica nanospray capillaries of New Objective (Woburn, MA, USA). The electrospray voltage was set to 1800 V. Spectra were acquired over the mass range of m/z 600–1600. The accumulation time was 1 sec. Protein spectra were deconvoluted by Analyst^® (Applied Biosystems; Foster City, CA, USA). LC-MS/MS analyses were carried out also with the UltiMate™ system interfaced to the QSTAR Pulsar or to an LTQ (Thermo; San Jose, CA, USA) linear ion trap mass spectrometer. The gradient was (A = 5% acetonitrile / 0.1% formic acid in water B = 80% acetonitrile / 0.1% formic acid in water) 0% B for 12 min, 60% B in 88 min, 100 % B in 5 min, 100% B for 10 min, 0% B in 5 min, 0% B for 20 min. Peptide spectra were recorded over the mass range of m/z 450–1300, MS/MS spectra were recorded in information dependent data acquisition over the mass range of m/z 50–1600. One peptide spectrum was recorded followed by two MS/MS spectra on the QSTAR Pulsar instrument; the accumulation time was 1 sec for peptide spectra and 2 sec for MS/MS spectra. The collision energy was set automatically according to the mass and charge state of the peptides chosen for fragmentation. One full spectrum was recorded followed by 3 MS/MS spectra on the LTQ instrument, automatic gain control was applied and the collision energy was set to the arbitrary value of 35. Doubly or triply charged ions were selected for product ion spectra. MS/MS spectra were interpreted by Mascot (Matrix Science Ltd, London, UK).