Ultraflextreme maldi tof ms

The exconjugants of S. lividans TK24 and S. albus J1074 containing mpa, sca, or the empty pBE45 vector were picked and restreaked onto fresh MS (mannitol 20 g/L, soybean flour 20 g/L, agar 20 g/L) and ISP4 (soluble starch 10 g/L, K₂HPO₄ 1 g/L, MgSO₄·7H₂O 1 g/L, NaCl 1 g/L, (NH₄)₂SO₄ 2 g/L, CaCO₃ 2 g/L, FeSO₄ 1 mg/L, MnCl₂ 1 mg/L, ZnSO₄ 1 mg/L, agar 20 g/L) agar medium supplied with apramycin at a final concentration of 50 μg/mL and incubated under 30 °C for 5 d. A portion of cell mass (pinhead-sized) was picked from the plate, placed in 20 μL methanol and incubated at room temperature for 1 h. Methanol extract was then mixed equally with 1 μL of 15 mg/mL 70% aq. MeCN solution of α-cyano-4-hydroxycinnamic acid (CHCA) with 0.1% trifluoroacetic acid (TFA) (v/v) on a ground steel MALDI target, and the droplet was dried under ambient conditions. Samples were analyzed using a Bruker UltrafleXtreme MALDI-TOF MS using manufacturer methods for reflector positive mode. The MALDI LIFT-TOF/TOF mass spectra were acquired in the positive ion mode. Metastable fragmentation was induced by a nitrogen laser (337 nm) without the further use of collision gas. Precursor ions were accelerated to 8 kV and selected in a timed ion gate. In the LIFT-cell the fragments were further accelerated to 19 kV. The reflector potential was 29 kV.

A refactored plasmid containing genes mpaA1, mpaB, and mpaC was heterologously expressed as described above. After desalting by SPE column, 1 mL (sample in aq. 70% acetonitrile/0.1% TFA) was added each to two scintillation vials (reaction vs. control). The pH of the eluent was adjusted to pH 4 using 0.1 M NaOH and checked by pH paper. To one vial, O-benzylhydroxylamine was added to 10 mM and scintillation vials were left overnight (~16 h) at room temperature to ensure maximal oxime formation. Reaction products were mixed 1:1 with 50 mg/mL Super-DHB (Sigma-Aldrich) in aq. 60% acetonitrile/0.1% formic acid and dried under ambient conditions on a polished steel MALDI target. Samples were analyzed using a Bruker UltrafleXtreme MALDI-TOF MS using manufacturer’s methods for reflector positive mode.

Freshly prepared 1 M NaBH₄ (Sigma-Aldrich 213462) solution in water was added to 5-mer RNA oligo to a final concentration of 10–200 mM and incubated for an hour at rt. After filtration, the reaction mixture was injected onto a C18 reverse column (Higgins Analytical) using Waters e2695 equipment and the desired peaks were collected. One microliter of the collected fraction was mixed with 2 μL matrix (9:1 [vol/vol] ratio of 2′,4′,6′-trihydroxy acetophenone [THAP; 10 mg/mL in 50% acetonitrile and water]:diammonium citrate [50 mg/mL in water]), loaded onto a MALDI plate and MS spectra were collected using Bruker UltrafleXtreme MALDI-TOF-MS in a positive, reflector mode.