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.
5 17.5 kda protein standard
Manufactured by Bruker
The 5–17.5 kDa protein standard is a laboratory tool used to measure the molecular weight of proteins within the specified range. It provides a reference for calibrating and verifying the performance of protein separation techniques such as gel electrophoresis.
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3 protocols using 5 17.5 kda protein standard
1
MALDI-TOF/TOF Analysis of Peptides
2
MALDI-TOF/TOF Protein Analysis Protocol
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One hundred microliters of HLTH and FHN samples (n = 9 each) from previous step were dried, reconstituted in the same volume of 0.1% FA, and desalted using reverse-phase (RP) C18 tips (NT1C18; Glygen) as per the manufacturer’s protocol with some minor modifications. The binding and washing steps were repeated five times before final elution. One microliter of each sample was spotted on a MALDI 384 target plate and dried, and the spots were overlaid with an equal volume of sinapinic acid (10 mg/mL in 0.1% FA in 50% of ACN) and analyzed using Ultraflex II MALDI-TOF/TOF instrument (Bruker Daltonics) in positive ion linear mode. The instrument was calibrated using a 5–17.5 kDa protein standard (Bruker Daltonics), and the MALDI spectra were collected in a range between 1 and 10 kDa in a fully automated mode using Bruker Flex control software with a constant laser power and 800 laser shots per spot.
3
MALDI-TOF/TOF Protein Analysis
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The eluted samples from both procedures were spotted (1 μL per spot) on a MALDI 384 target dried and overlaid with an equal volume of sinapinic acid (10 mg/mL 0.1% FA in 50% ACN). The spots were analyzed using an Ultraflex II MALDI-TOF/TOF instrument (Bruker Daltonics) in positive ion linear mode. The instrument was calibrated using a 5–17.5 kDa protein standard (Bruker Daltonics), and the MS data for peptides between the 1–10 kDa range 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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