New on-bead digestion for LC-MS applications for proteomic studies
MONMOUTH JUNCTION, NJ.– Biotech Support Group LLC introduces a new on-bead digestion for liquid chromatography–mass spectrometry (LC-MS) applications for proteomic studies. We have demonstrated that the NuGel™ silica-based supports perform optimally when using tryptic digestion of the bound proteins, prior to LC-MS analysis; most commonly called "on-bead digestion" in the literature. Key benefits of on-bead digestion are much simpler workflows, eliminates elution, no precipitations, shorter preparation time.
Equivalent or better peptide recovery, compared to elution protocol:
# of peptides, Elution 8398 vs. # of peptides, On-Bead 10132
Quality control for the peptides: On left is the standard AlbuVoid™ elution protocol followed by tryptic digestion and LC-MS/MS peptide analysis. On right, using equivalent operating parameters, the on-bead digestion protocol generates comparable peptide output.
AlbuVoid™ On-Bead Digestion Protocol
- After the final wash steps from AlbuVoid™ protocol step 7, add 100 µls of 5 mM DTT solution to the beads for
complete immersion, mix and incubate at 60°C for ½ hour.
- After cooling, add 100 µls of 25 mM iodoacetamide to the DTT/bead suspension, mix and incubate in the dark for 1 hour.
- Centrifuge at 5000xg (medium setting, not max) for 3 mins, and discard filtrate. Transfer the filter slurry of beads, DTT and iodoacetamide to a clean Eppendorf tube.
- On-bead digestion is done by adding 100 µls of a 0.025 ug/uL solution of MS-grade. Trypsin to the beads. Digest overnight at 37°C.
- Centrifuge at 5000xg (medium setting, not max) for 3 mins, and retain peptide filtrate.
- To further extract remaining peptides, add 100 µls of 10% solution of formic acid to the beads.
- Incubate for 15 minutes at 37°C, centrifuge at 5000xg (medium setting, not max) for 3 mins, and add this volume to the first volume.
- Reduce to a final volume of 100 µls using a SpeedVac and store at -80 °C until LC-MS/MS.
On-bead digestion protocols can be used with these products:
AlbuVoid™ Albumin Removal and Low Abundance Enrichment
HemoVoid™, Hemoglobin Depletion and Low Abundance Enrichment
UPCK™ Urine Protein Enrichment/Concentration Kit
KinaSorb™ Kinase (& ATP binding proteins) Enrichment Reagent
NuGel™ Glycoprotein Enrichment PBA Kit - With Phenyl Boronic Acid NuGel™ Matrix
NuGel™ immobilized ligands, coupling chemistries include
About Biotech Support Group LLC
Biotech Support Group LLC is a leading provider of proteomics and genomics 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™ for albumin depletion, AlbuVoid™ for albumin depletion plus low abundance serum protein enrichment, Cleanascite™ for lipid adsorption and clarification, NuGel™ for passivated silica-based affinity chromatography, ProCipitate™ & ProPrep™ for nucleic acid isolation, and for depletion and purifying hemoglobin; HemoVoid™ for depletion from red blood cells and HemogloBind™ primarily for hemolyzed serum. Biotech Support Group LLC and ProFACT Proteomics Inc., are collaborating on the development of a functional proteomics separations platform used in biomarker and functional proteomic prospecting. For more information, go to: http://www.biotechsupportgroup.com/