Mucins are a family of high molecular weight, heavily O-glycosylated proteins. The high molecular weight (0.25-50 MDa) and high sugar content (30-80% of weight) have been exploited for the separation of mucins from other cellular components. Typical purification steps of mucins from complex secretions and tissues often require: 1. Two-step density gradient centrifugation using CsCl to partition mucins from other proteins and nucleic acids; 2. Gel filtration to separate mucins from smaller proteins; 3. Anion-exchange chromatography to enrich mucins. The aforementioned purification steps require a very large starting material in a low throughput way. Faster protocols with micro-scale purification are clearly needed with increasing demands to study mucins in severely limited amounts. This exploratory study is aimed to exploit 3 well-validated high-throughput and micro-scale devices for mucin purifications. The Adaptive Focused Acoustics (AFA) technology has been routinely used to fragment ng to µg genomic DNA in less than a minute. DNase-treatment of samples followed by the application of centrifugal filtration with appropriate molecular weight cut-off (MWCO) membrane could separate biomolecules easier and faster than gel filtration. Strong anion exchange (SAX) spin columns could be used to improve speed and recovery over the conventional resin-based column chromatography. By combining the three high-throughput devices (sonicator, MWCO and SAX spin columns), we may shorten the time required for the above steps 1-3 typically from a day down to less than one hour with better recoveries. Instead of the commonly used trichloroacetic acids, we also explored the use of perchloric acid (PCA) to precipitate proteins, leaving more mucins in the supernatant. An additional benefit of PCA is that it exerts negligible oxidizing power at ≤ 10% when cold. Upon heating (by sonication in our study), PCA becomes a powerful oxidizer to release glycans from mucins. This innovative way is expected to offer an easier control than the bleach method proposed by other researchers.

High throughput and micro-scale purification of mucins

256th National Meeting (2018)

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