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 reactions, technician error, etc.—may arise during sample prep to undermine a mass spec run. Recently, advances in reagents, protocols, and automation are helping 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 it works with either protein or peptide samples. The advantage is 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 digests 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.
How Good Is Your Trypsin?
Trypsin digestion is a common step in protein sample prep, but there are few standardized methods for determining trypsin efficiency. JPT Peptide Technologies has developed a kit to monitor trypsin efficiency and showed supporting work at ASMS.
“When you do a trypsin cleavage there’s always an efficiency question. How much do you add? At what temperature should the reaction run and for how long? What are the buffers,” said Ulf Reimer, Ph.D., head, R&D. “Some people do it overnight in the cold room. Others do it at 37º. But there’s no easy way to measure trypsin cleavage efficiency.”
JPT is one of few peptide providers using a paper-based spot synthesis instead of beads, noted Dr. Reimer. Part of JPT’s manufacturing process involves putting a trypsin cleavage tag on peptides enabling JPT to accurately quantify production. The same principle is used in the kit.
“To quantify the completion of the trypsin, you would put the peptides in this kit —several different target peptides (different lengths and sequences) with different tags on the N-terminus—in with your sample. On the basis of recovery of the cleaved controlled peptides you can determine the efficiency of the trypsin in your prep process,” said Dr. Reimer.
He cites the kit’s advantages as accurate in situ determination of trypsin cleavage efficiency and reproducibility; compatibility with routine sample digestion workflows; analysis using standard LC-MS protocols; and absolute quantification of cleavage products in the samples using heavily labeled tags. Kit availability is planned before the end of the year.
Miniature HPLC Columns
Automating sample prep in modest scale settings has proved difficult because of cost (equipment) and skill (automation expertise) requirements. Agilent Technologies presented an approach it calls “Rapid, Automated Sample Preparation for Peptide Mapping,” based on the company’s AssayMAP Bravo platform. The result, said Agilent, is improved reproducibility, robustness, and increased throughput.
“We have developed a disposable cartridge that has a 5-microliter packed bed in it and along with technology for packing any resin in there with high chromatograph efficiency. These are like miniature disposable HPLC columns,” said Scott Fulton, MS bioengineering, and head, AssayMAP operations and workflow development.
Built into the robotic head is a probe syringe and when a cartridge is connected, it provides positive displacement flow control so there’s no air gap. “We are able to control the flow at an appropriate rate for running through this tiny packed bed,” said Fulton.
Typical flow rates are on the order of 2-to-10 microliters per minute, “so it’s really slow and we’re able to do that using 96 little syringe pumps operating in parallel.”
A technician is still required for some steps. The platform is “not hugely expensive but it has some limited deck space, and in between the digestion and purification steps you might need to go in and spend two minutes putting different plates on the deck and moving a couple of things around,” according to Fulton.
Binding is quantitative, he continued, producing a high product yield from a small volume. He said the approach is aimed at applications such as biomarker validation efforts “where you need to run something on the order a thousand patient samples to do a study and you really want to do that campaign in a week or two.”