果蝇差异神经肽组的快速 MALDI 质谱工作流程

A rapid MALDI-TOF mass spectrometry workflow for Drosophila melanogaster differential neuropeptidomics

Molecular Brain；Salisbury et al. Molecular Brain 2013, 6:60 http://www.molecularbrain.com/content/6/1/60

实验部分：

MALDI-TOF MS analysis of single dissected fly brains Mass spectra were acquired on a microflex MALDI-TOF mass spectrometer (Bruker Daltonics Inc., Billerica, MA) equipped with a 337 nm N2 laser. Positive ion mass spectra were acquired from 500 m/z – 4000 m/z in reflectron mode. The acceleration voltage was set at 20 kV and the pulsed-ion extraction was set at 200 ns.

One thousand laser shots were acquired for each spectrum. External mass calibration was achieved using a standard peptide mixture of Angiotensin I and II, Substance P, Renin Substrate, and ACTH (Bruker Daltonics Inc.). The externally calibrated mass accuracy of the instrument was approximately 100 parts-per-million (ppm) at m/z 1500.

MALDI-TOF/TOF MS/MS analysis for identification of detected peptides from single brain on-target extraction sample preparation Fragmentation spectra were acquired in LIFT mode on an autoflex III and an ultraflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics Inc., Billerica, MA).

MS spectra were acquired in positive ion and reflectron modes. For MS/MS analysis, the source acceleration voltage was set to 8.0 kV and the reflectron voltage was set to 29.5 kV. Mass spectra were acquired with approximately 3000 laser shots summed in 200 to 400 shot increments. 

External mass calibration was achieved using a standard peptide mixture of Angiotensin I and II, Substance P, Renin ubstrate, and ACTH (Bruker Daltonics Inc.). The external calibration mass accuracy of the instrument was approximately 20 ppm in MS mode and <400 ppm in LIFT (MS/MS) mode. MS/MS spectra were not internally calibrated. All spectra were processed with FlexAnalysis software (Bruker Daltonics Inc.).