Mass Spectrometry Analysis

2.7.1 Introduction

To verify the presence of BHMT and to confirm that the observed activity was a due to BHMT protein, a mass spectrum of the final purified BHMT solution was obtained. Mass spectrometry gives a total mass of the subunits present in the reaction mixture, and would also identify if there were any post-translational modifications of BHMT, including glycosylation (which would increase the mass of the BHMT subunit) and cleavage of the protein (which would give a decreased mass).

2.7.2 Method

A sample of the BHMT (0.8 mg/mL, 2 mL), purified as described in Section 2.6 was de-salted against distilled water (3 x 500 mL) using dialysis at 4 °C for 8 h each exchange. The resultant solution was submitted for electrospray mass spectral analysis with Professor Stephen Brennan in the Christchurch School of Medicine.

Urea was added to the sample to 8 M and the sample was heated to 90 °C for 30 min to denature the protein. Iodoacetamide (150 μL, 55 mM) was added to block the cysteine residues.

The sample was then analysed directly by electrospray ionisation (ESI) mass spectrometry (MS) on a Platform II quadrupole analyser (Micromass). Injections of 10-20 μl were introduced to the ion source at 5 μl/min. The probe was charged at +3500 volts and the source maintained at 60 °C. The mass range 400–1200 m/z was scanned every 3 s with a cone voltage ramp of 30–60 volts and up to 80 scans were averaged in acquiring the raw data. Calibration was made over this same m/z range using the charge series generated by

human α globin and data was acquired and processed using MassLynx software and

transformed onto a true molecular mass scale using maximum entropy (MaxEnt) software supplied with the instrument.

2.7.2.1 Tryptic Digest of BHMT Subunit

Tryptic digests were prepared from approximately 0.25 mg of the white precipitate BHMT as described in the mass spectral work above. The individual chains were dried under N₂ and redissolved in 50 μl of 50 mM NH₄HCO₃. Trypsin (1.5 μg) was added and the reaction

incubated for 16 h at 37 °C. After drying under vacuum with P₄O₁₀, digests were redissolved in 100 μl of 0.1% HCOOH, 50% acetonitrile and 10 μl was analyzed by ESI MS as above. The m/z range 300–1900 was scanned every 4 s.

2.7.3 Results

Initial attempts to obtain a mass of the entire BHMT subunit were unsuccessful. Trace amounts of albumin were identified in the mass spectra, but the expected BHMT subunit mass was not identified. It was noted, however, that small amounts of precipitate were forming at the bottom of the Eppendorf tubes following dialysis. Tryptic digest of the solution and these precipitates gave sufficient fragments to map 75% of the BHMT subunit, including two pairs of fragments which were also identified as conjoint sequences [Figure 2.4].

Figure 2.5 Predicted rat BHMT amino acid sequence showing (marked) the observed tryptic fragments of BHMT, as identified by electrospray mass spectrometry. Cyan indicates the possible N-linked glycosylation site. These fragments are summarised in Table 2.2 below.

Table 2.2 Observed fragments from tryptic digest of BHMT subunit (complete listing of tryptic fragments is in Appendix 1.

Fragment Fragment Sequence Mass (Daltons) Observed M/Z (3 s/f)

T5 GILER 586.34 587 T6 LNAGEVVIGDGGFVFALEK 1934.01 968 T9 AGPWTPEAAVEHPEAVR 1815.89 606, 909 T10/11 QLHREFLR 1115.62 530 T11 EFLR 563.31 564 T12/13 AGSNVMQTFTFYASEDKLENR 2425.12 803 T16 VNEAAJDIAR 1117.52 560, 1117 T16/17 VNEAAJDIARQVADEGDALVAGGVSQTPS YLSJK 3568.68 593 T17/18 QVADEGDALVAGGVSQTPSYLSJKSETEVK 3412.47 625, 782, 1042 T20 IFHQQLEVFMK 1418.74 710 T22 NVDFLIAEYFEHVEEAVWAVEALK 2820.4 941 T23 TSGKPIAATMJIGPEGDLHGVSPGEJAVR 2966.4 476 T25 AGAAIVGVNJHFDPSTSLQTIK 2285.15 446, 558, 743 T27 EGLEAAR 744.38 745 T29/30 AYLMSHALAYHTPDJGKQGFIDLPEFPFGLE PR 3794.82 756, 945 T34/35 EAYNLGVR YIGGJJGFEPYHIR 2648.04 658, 877 T35 YIGGJJGFEPYHIR 1727.75 577, 864 T36 AIAEELAPER 1097.57 550, 1098 T36/37 AIAEELAPERGFLPPASEK 2042.07 676 T41/42 KEYWQNLR 1153.59 569, 1140 T43 IASGRRPYNPSMSKPDAWGVTK 2261.13 607 T45 EATTEQQLR 1074.53 538 T48/49 FKSAQ 597.31 580

2.8 N-terminal Sequencing

2.8.1 Introduction

To confirm that the detected BHMT activity was due to the rat BHMT protein, N-

terminal sequencing was conducted on the 45 kDa band seen in the SDS-PAGE gel, suspected to be the BHMT subunit, comparing it to the rat cDNA data available.

2.8.2 Method

Rat BHMT extract, obtained after size exclusion chromatography and concentrated to 1 mg/mL was analysed using gradient SDS-PAGE electrophoresis (5-20%, Sigma) and stained for 1 h with Coomassie Blue stain. The stain was removed using 10% acetic acid in methanol, washed twice for 2 h each time. A second SDS-PAGE was conducted using the same extract and markers, but was not stained. The second gel was electro-blotted at 100 mA constant current (voltage ~45 V) for 15 h to PVDF membrane and stained for 1 min with amido black stain, followed by washing twice with 10% acetic acid in water.

The 45 kDa band was identified on the PVDF membrane and excised. The sample was submitted to the Protein Microchemistry Facility in the Department of Biochemistry at the University of Otago, Dunedin, New Zealand. For the rat sequence only 10 amino acids were sequenced to identify BHMT. The same procedures were then performed on the hagfish liver extract, post size-exclusion chromatography, and concentration to 1 mg/mL. Sigma wide-range markers (M 4038, Sigma) were used to confirm and identify the ~45 kDa band in the SDS- PAGE and PVDF blot [Chapter 6].

2.8.3 Results

The Protein Microchemistry facility obtained the predicted Rat cDNA sequence of ‘‘A P I A G K K A K R G I L E R’’ [Figure 2.4] together with low yields of sequences with staggered

starting N-termini into the chain. Despite a clear dark 45 kDa band being excised in rat BHMT it gave lower yields than expected, approximately 30 pmol. There was evidence of amino peptidase activity cleaving N-terminal amino acids, giving a staggered starting point and making interpretation of the sequence data difficult.