AlbuVoid™ On Bead Digestion Results

Serum proteomics can be challenging for two reasons: 1) Albumin accounts for about 50% of the total protein mass, and 2) much of the remaining protein is glycosylated, a particularly proteolytic resistant class of proteins. To overcome the first challenge, we have developed AlbuVoid™, a surface chemistry and method that can deplete the albumin by a voidance strategy, enriching the remaining low abundance proteins on the bead. To improve the workflow, we have previously reported a comparable proteolytic efficiency when comparing bead-bound proteolysis to conventional in-solution proteolysis. However, these tests used a pH 8 system, optimal for in-solution. Subsequently, we have found that for bead-bound digestions, pH 7 is optimal. We suspect that at this pH much of the protein remains bound to the bead during early stage proteolysis, and such destabilized higher order protein structure improves protease access to the interior regions of the polypeptide chains. Trypsin remains in solution and within its optimal pH condition.

On Bead AlbuVoid™ Digestion Results

In the Venn diagrams below, we report on the total protein identifications from both pooled human and rat sera. We processed each serum the same way using AlbuVoid™ for on-bead enrichment, depleting the albumin. Trypsin digestion was performed at two time intervals, 4 hours and overnight (O/N). Each was then analyzed by a single LC-MS/MS 3 hour gradient run.

The results demonstrate high efficiency digestions, even at short digestion times. Also, as has been reported by others, Trypsin can over-digest some proteins, so protein identifications from both pools overlap, while others remain in one or the other population. This suggests that for on-bead digestion, three populations of proteins are obtained:

Those that are digested efficiently at short digestion times, but may be prone to non-specific digestion.

Those that are digested efficiently even at short digestion times, and are “bullet-proof” to non-specific digestion.

Those that require overnight digestion for identification.

So, as can be seen with this data, for quantitative proteomics, exploratory studies can establish optimal conditions for digestion assessing the level of specific limit peptides of interest.Brownridge and Beynon have reported a similar strategy, in that a rapid reaction minimizes the influence of nonspecific degradation events, while later stage proteolysis may minimize the number of mis-cleaved peptides.

P. Brownridge, R.J. Beynon, The importance of the digest: Proteolysis and absolute quantification in proteomics Methods 54 (2011) 351-360.

As with all digestion procedures, there is not one ideal method, and care must be taken to choose the right digestion time and condition for the application, whether it be for discovery or quantitation.

About Biotech Support Group LLC

Biotech Support Group LLC is a leading provider of genomics and proteomics sample preparation products and enrichment reagent kits as well as integrated biotechnology services for life sciences research, biomarker and drug discovery. Based in New Jersey, it’s principal products include: AlbuSorb™ & AlbuVoid™ for albumin depletion,Cleanascite™ for lipid adsorption and clarification, HemogloBind™ & HemoVoid™ for hemoglobin removal, NuGel™ for functional & chemical proteomics, andProCipitate™ & ProPrep™ for nucleic acid isolation. For more information, go to http://www.biotechsupportgroup.com

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Dr. Swapan Roy & Matthew Kuruc
Biotech Support Group LLC
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