Respective control and LPS samples (75 μL per sample, n=3) were concentrated and desalted using C18 tips (NT1C18; Glygen) as per the manufacturer’s protocol, except that the binding and washing steps were repeated five times before elution.13 (link) The samples were eluted and spotted on a MALDI 384 target plate, dried, and overlaid with an equal volume of sinapinic acid (10 mg/mL in 0.1% FA in 30% of ACN). The spots were analyzed using an Ultraflex II MALDI-TOF/TOF instrument (Bruker Daltonics) in a positive ion linear mode. The instrument was calibrated using a 5–17.5 kDa protein standard (Bruker Daltonics), and the MS data for peptides in the range of 1–10 kDa were collected in an automated mode using the Bruker Flex control software with a constant laser power and 800 laser shots per spot.
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MALDI-TOF/TOF Analysis of Peptides
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Corresponding Organization :
Other organizations : Agricultural Research Service, University of Arkansas at Fayetteville, U.S. National Poultry Research Center
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Variable analysis
independent variables
- Respective control and LPS samples
- Peptides in the range of 1–10 kDa
- Sample volume (75 μL per sample)
- Number of samples (
n =3) - C18 tips (NT1C18; Glygen) binding and washing steps repeated five times before elution
- Elution and spotting on a MALDI 384 target plate
- Overlaying with an equal volume of sinapinic acid (10 mg/mL in 0.1% FA in 30% of ACN)
- MALDI-TOF/TOF instrument (Ultraflex II; Bruker Daltonics) operated in positive ion linear mode
- Instrument calibration using a 5–17.5 kDa protein standard (Bruker Daltonics)
- MS data collection in an automated mode using the Bruker Flex control software with a constant laser power and 800 laser shots per spot
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