Protein sample preparation

A 36 µM rhGH stock was prepared by solubilisation of the lyophilised RM in water and vortexing for 1 hour at room temperature. Following this the stock was aliquoted and stored at -20 °C to be removed for single use.

2.2.1.1 Sample preparation for HDX-MS and SEC measurements

A 0.9 mM zinc acetate stock solution was also freshly prepared in water and further diluted in 10mM KPBS (pH 7.4) to obtain a series of solutions; solution C (180 µM zinc acetate), solution D (90 µM zinc acetate) and solution E (18 µM zinc acetate). Prior to analysis, 36 µM stock aliquots of rhGH were defrosted and further diluted to 7.2 µM (0.16 mg/mL) with 10 mM KPBS or solution D or E to obtain 1:10 and 1:2 respectively. Samples were left to equilibrate at room temperature for 1h before analysis.

2.2.1.2 Sample preparation for IMS-MS measurements

For native IMS-MS analysis, zinc acetate solutions were prepared from dilutions of a 0.9 mM zinc acetate stock solution with a 20 mM ammonium acetate solution. A second series of solutions were prepared; solution F (90 µM zinc acetate) and solution G (18 µM zinc acetate).

Prior to buffer exchange and desalting steps, aliquots of 36 µM rhGH stock were removed from the freezer and allowed to defrost and equilibrate for 45min. Desalting and buffer exchange of the stock sample was performed using Bio-spin micro P6 pre- packed size exclusion columns (Bio-rad, California, U.S.A) as per the manufacturer’s

instructions. In short, excess packing buffer was removed using an 5810R centrifuge (Eppendorf, Stevenage, U.K) at 1000 g for 2min. Buffer exchange was then performed by loading 500 µL 20 mM ammonium acetate buffer and centrifuging for 2 min at 1000 g, repeating this step 4 times to ensure 99.9% buffer exchange. 75 µL of rhGH stock was then loaded directly onto the centre of the column and centrifuged for 4min, collecting the eluent. To maximise desalting the buffer exchange process was repeated as described and the sample loaded for a second time.

The desalted and buffer exchanged rhGH was then diluted to 7.2 µM using 20 mM ammonium acetate or zinc acetate solutions F and G to obtain rhGH:Zn 1:10 and 1:2 respectively. A final sample was prepared by dilution of the desalted rhGH stock using a solution of 180 µM of magnesium acetate to give rhGH in the presence of magnesium (rhGH:Mg, 1:10). Samples were left to equilibrate at room temperature for 1h before analysis.

Calibrant solutions of 10 µM Beta-lactoglobulin (B-LAC) in 200 mM ammonium acetate were also prepared as described by Bush et al [52].

2.2.1.3 Preparation of deuterated and undeuterated peptide mixture

A peptide mixture containing MRFA [74 µM], bombesin [24 µM], angiotensin II [angio II, 191 µM], and glu-1-fibrinopeptide B [glufib, 64 µM], insulin chain B [ICB, 77 µM] was prepared from stock solutions. This mixture was then diluted 1200-fold in either deuterated 10mM phosphate buffer, (pH 7.0) or phosphate buffer, (pH 7.4) to act as deuterated and undeuterated peptide mixtures respectively. Both samples were shaken at 25 °C overnight to ensure complete exchange occurred for the deuterated sample and to take into account potential stability issues.

2.2.1.4 Preparation of deuterated and undeuterated α1-Antitrypsin

A 18.5 µM deuterated α1-Antitrypsin stock was prepared by solubilisation of lyophilised α1-Antitrypsin in a deuterated buffer containing pre-deuterated 2M urea, 10 mM potassium phosphate (pH 7.0) solution. To facilitate complete deuteration the protein stock was then heated at 30 °C overnight. Prior to analysis protein stock was then to 3.7 µM using the pre-deuterated buffer. An undeuterated α1-Antitrypsin control stock was also prepared in parallel using undeurated buffers.

2.2.3. HDX-MS

2.2.3.1 Instrumentation

Sample handling and mixing steps were performed using a first generation LEAP PAL system set up for HDX analysis [LEAP Technologies, Carrboro, USA]. Samples were injected onto a refrigerated nanoACQUITY UPLC System with HDX technology [Waters, Milford, USA] for on-line pepsin digestion and chromatographic separation. MS experiments were performed on a Synapt G2Si QToF instrument [Waters, Milford, USA].

2.2.3.2 HDX-MS experiments

For the generation of peptide maps and time zero exchange experiments, 15 µL of rhGH (0.16 mg/mL) was diluted 10-fold in 10 mM KPBS (pH 7.4). For exchange experiments, deuterium labelling was performed by diluting 15 µL of rhGH 10-fold in 10 mM KPBS (pH 7.0) prepared in D2O. Exchange times were 0.5, 5, 60, 240 and

480 min, with the exchange occurring at room temperature (21±2 °C). Triplicate analyses were carried out for each incubation. Following the appropriate incubation period, exchange was quenched by a 2-fold dilution with 50 µL of 300 mM TCEP, 2 M Guanidine Hydrochloride (Gnd.HCl) in 100 mM KPBS (pH 2.5) at 4°C. Following a 0.5 min quench delay, a 50 µL sample loop was overfilled with 95 µL of quenched sample.

On-line digestion was performed using an Enzymate BEH pepsin column (5 μm, 2.1 x 30 mm) [Waters, Milford, USA] at 25 °C and a flow rate was 80 µL/min (mobile phase 0.05% v/v formic acid). Proteolytic peptides were trapped on an ACE C18 guard cartridge (5 µm, 2.1 mm i.d.) and sequentially chromatographically separated on an ACE Excel Super C18, (2 µm, 2.1 x 100 mm i.d, both Hichrom Reading, U.K), both trap and analytical column were held at 0 °C. Digestion pressure was held at ~ 4000 psi using a PEEK restrictor placed post-trap column. Chromatographic separation was carried out at 100 µL/min by application of a 7 min linear gradient from 92% A / 8% B to 65% A / 35% B. Mobile phases consisted of aqueous, 0.1% v/v formic acid (A) and CH3CN, 0.1% v/v formic (B).

MS experiments were performed in positive ESI mode using an 80 °C source temperature, 2.8 kV capillary voltage, 80 V cone voltage and a desolvation temperature of 250 °C. For peptide map generation, data were acquired in MSE

continuum mode over a range of 50 – 2000 amu, with a scan time of 0.2 s, inter scan time of 0.1s and a transfer ramp of 15-40 V. Exchange experiments acquired in MS mode under the same conditions but with no collision energy applied. Mass calibration was performed using a 100 fmol solution of Glu-fibrogen, (50% MeOH, 0.1% formic) with a lockmass of 785.8426 m/z, mass correction was applied post acquisition.

2.2.4 Data analysis

2.2.4.1 Peptide identification

ProteinLynx Global Server software v 3.02 [PLGS, Waters, Milford, USA] was used to generate peak lists by inputting MSE data and searching against a database consisting of both FASTA sequences of porcine Pepsin A (P00791, Uniprot) and Somatotropin (P01241, Uniprot), see Table A-24, in the appendix for parameters. The PLGS outputs were imported in DynamX v3.0 [Waters, Milford, USA] to generate peptide maps, file thresholds (peptides observed in x files out of y, n=x/y) of n=3/3 and n=4/5 were used for digestion optimisation experiments and HDX-MS peptide maps respectively. Additional processing parameters included 0.01, minimum products per amino acid and 20 ppm, maximum MHP+ error.

2.2.4.2 Deuterium uptake measurements

Peptide deuterium levels were calculated as described by Burkitt et al. [138], based on the difference between the calculated centroid mass measurement of the isotopic distribution of the undeuterated control sample and the deuterated sample at t=x. No correction was made for back exchange during deuterium uptake calculations. Measurements are expressed as deuterium uptake (in Da) or as percentage relative deuterium uptake (rDU), calculated by the division of deuterium uptake (in Da) by the theoretical maximum number of exchangeable backbone amide protons in the peptide (using the formula, N-2-P, where N is the number of amino acid residues in the sequence and P is the number of proline residues not including the first two N- terminal residues [120] and expressed as a percentage). Differential calculations were performed using deuterium uptake (in Da) measurements.

2.2.4.3 Qualitative assessment of peptide map

To make a qualitative assessment of the efficiency of digestion under different conditions, a parameter called digestion factor (DF) [221], shown in Equation 26 was used, this factor incorporates a weighted average of the sequence coverage achieved

(y1), the percentage of potential cleavage sites accessed (y2) and the redundancy (y3),

a measure of overlapping peptide sequences..

Equation 26

DF = 8y1 + 5y2 + 0.6y3

The sequence coverage (y1) is the proportion of the primary sequence which is present

in the peptides identified (S) relative to the number of amino acids present in the sequence (N).

Equation 27

𝑦1 =

𝑆 𝑁

The cleavage rate (y2) shows the proportion of peptide bonds cleaved (C) relative to

N.

Equation 28

𝑦₂ = 𝐶 𝑁

The redundancy rate (y3) is the sum of the number of times that an individual amino

acid is seen across multiple peptides (R) relative to N.

Equation 29

𝑦₃ = ∑ 𝑅 𝑁

The digestion factor calculation allows for the three components contributing to efficient digestion to be evaluated simultaneously, whist still allowing some definition in the contribution of these factors, for instance the factor of highest importance, sequence coverage, y1, is weighted more than redundancy, y3.