Lcq fleet electrospray ion trap mass spectrometer

A further 10 mg of lyophilised skin secretion were dissolved in 1.5 mL of 0.05/99.95 (v/v) trifluoroacetic acid (TFA) /water and then clarified by centrifugation. The supernatant (1 mL) was then subjected to reversed phase HPLC using a Waters gradient reversed phase HPLC system, fitted with an analytical column (Jupiter, C₅, 300 Å, 5 μm, 4.6 mm × 250 mm, Phenomenex, Macclesfield, Cheshire, UK). Column elution was achieved with a gradient formed from 0.05/99.95 (v/v) TFA/water to 0.05/29.95/70.00 (v/v/v) TFA/water/acetonitrile in 240 min at a flow rate of 1 mL/min, and the effluent was monitored by UV absorbance at 214 nm 280 nm. The eluted fractions were collected at 1 min intervals. The molecular masses of peptides in each fraction were further analysed by use of a MALDI-TOF mass spectrometer (Voyager DE, PerSeptive Biosystems, Foster City, CA, USA) in positive detection mode using α-Cyano-4-hydroxycinnamic Acid (CHCA) as the matrix. Fractions with peptide molecular masses coincident with those of the mature peptides predicted from the cloned cDNA, were then infused into an LCQ Fleet ion-trap electrospray mass spectrometer (Thermo Fisher Scientific, San Francisco, CA, USA) followed by trapping of appropriate ions for MS/MS fragmentation.

Lyophilized skin secretion was dissolved and then subjected to reverse-phase HPLC using a Waters gradient reverse phase high-performance liquid chromatography (HPLC) system as detailed in the previously published article [11 (link)]. The molecular masses of peptides in each fraction were further analysed by use of a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (Voyager DE, PerSeptive Biosystems, Foster City, CA, USA), and selected fractions were then infused into an LCQ Fleet ion trap electrospray mass spectrometer (Thermo Fisher Scientific, San Francisco, CA, USA) followed by trapping of suitable ions for MS/MS fragmentation [11 (link)].

Five mg of lyophilised skin secretion from Pithecopus hypochondrialis were dissolved in 1 mL of trifluoracetic acid (TFA)/water (0.05/99.95, v/v) and centrifuged at 5000× g for 20 min to clarify. The supernatant was injected into an RP-HPLC system (Waters, Miford, MA, USA) fitted with a column (Jupiter C-5, 5 μM, 4.6 mm × 250 mm, Phenomenex, Macclesfield, Cheshire, UK) and eluted with a linear gradient mobile phase from TFA/waster (0.05/99.95, v/v) to TFA/water/acetonitrile (0.05/19.95/80.00, v/v/v) at a flow rate of 1 mL/min in 240 min, detected by the ultraviolet absorbance at 214 nm. Each fraction was collected automatically at 1 min intervals, and then analysed by a MALDI-TOF-MS (Voyager DE, Perspective Biosystems, Foster City, CA, USA) in positive detection mode, using α-cyano-4-hydroxycinnamic acid (CHCA) as the matrix. The system was calibrated by standards with precision reaching ± 0.1%. Fractions containing identical molecular masses correspond with the deduced mature peptide from the cloned cDNA were injected into to an LCQ-Fleet electrospray ion-trap mass spectrometer (Thermo Fisher Scientific, San Francisco, CA, USA) to analysis sequence structure by MS/MS fragmentation sequencing technique.