Though unglamorous, sample prep is widely regarded as the most critical step in performing robust mass spectrometry. Any number of problems - contamination, incomplete digestion, unwanted side effects, technician error, etc. - may arise during sample prep to undermine a mass spec run. Recently, advances in reagents, protocols and automation are helping to mitigate sample-prep problems, speeding throughput and improving reproducibility.
Detergent removal is a case-in-point. Used to isolate proteins from various sample types, detergents (e.g., SDS, CHAPS, Triton X-100, NP-40) are notoriously incompatible with mass spec (MS) analysis. Successful removal of detergents with good protein or peptide recovery, especially for low-abundance proteins, is critical for accurate MS analysis. Earlier this year, at the ASMS conference in Vancouver, Thermo Fisher Scientific presented a new approach for detergent removal.
“Our goal was to identify a resin or workflow that would allow removal of detergents that would be detergent independent, so it wouldn’t just work with anionic detergent, for instance, but would also be compatible with any sample type,” said John Rogers, Ph.D., manager R&D, mass spec reagents.
Thermo identified a resin with a strongly hydrophobic pocket; detergents bind within the pocket but the pocket is small enough that it excludes proteins or peptides.
“What’s unique about this product is that it works with either protein or peptide samples. The advantage is that you can perform a digestion with a complex sample in the presence of detergents and then easily remove the detergent,” added Babu Antharavally, Ph.D., senior scientist.
Thermo reports the method successfully removed >95% of nonionic, ionic and zwitterionic detergents from 0.5−1% solutions with high recovery of proteins or peptides. Tandem mass spectrometric analysis of 2.5−10 micrograms of BSA enzymatic digested at 25−100 microgram/mL — prepared in the presence of detergents and processed to remove detergent — revealed sequence coverage and MASCOT scores as good as or better than control BSA samples processed without detergent.