Agilent reduces the noise in proteomics experiments

The new Human-14, Multiple Affinity Removal System (MARS) removes between 95 and 99 per cent of the 14 most abundant of these proteins, halving the number of proteins that can mask potential biomarkers during LC-MS (liquid chromatography - mass spectrometry) and electrophoresis gel analysis.

Proteins associated with diseases can be hard to isolate and identify because they usually occur in small amounts that are often swamped by the presence of the high-abundant proteins that account for up to 94 per cent of the protein mass.

"Many people will spend months studying their data [from LC-MS proteomics studies] and trying to identify all of the proteins present, they are going to save time because they have less noise and miscellaneous material to filter through," said Maureen Joseph, LC product manager at Agilent.

The new MARS products make use of specific rabbit antibodies and genetically engineered 'Affibody' ligands to remove the high-abundant proteins.

The system is available in two formats, either as an HPLC (high performance liquid chromatography) pre-column or as a spin cartridge that can be used in a conventional centrifuge.

"Customers who like to use the spin-cartridges may be customers who don't own their own mass spectrometer - they are preparing a sample and preparing it for someone else to do the analysis," said Joseph.

The HPLC pre-columns will be used by customers with their own in-house LC-MS systems and because the pre-columns are used in-line can be part of an automated analysis system.

By a clever selection of buffers, the MARS devices first mop up the high abundance proteins and allow the low-abundant proteins to be eluted before the systems are regenerated by washing off those proteins that have stuck to the column.

This allows the columns to be reused again and again, with Agilent guaranteeing the system can be regenerated up to 200 times.

"There is a lot of discussion in the field as to whether you want to get rid of these proteins or not, but when you consider that the majority of biomarkers that have been discovered to date are in the low abundant proteins, being able to study what's there easily is a big issue for many companies," said Maureen Joseph, LC product manager at Agilent.

The potential problem with this is that proteins of interest may be removed from the sample when separating the 'wheat' from the 'chaff' and detecting these in an enriched high-abundant protein sample could be beyond the detection limits of current technologies.

However, many groups have successfully used protein- and immuno-depletion methods.

Researchers led by Dr Gary Siuzdak, director of the Scripps Center for Mass Spectrometry, have shown that such techniques can be very powerful and used the MARS system before profiling the blood plasma of patients suffering from sepsis.

Sepsis is an often fatal systemic inflammatory response to overwhelming infection or trauma that has no single biomarker that can predict the onset reliably.

The researchers published results in the Journal of Proteome Research (2006, 5, p3154) that showed that a combination of immunodepletion, 3D LC separation and MS/MS analysis allowed 10 potential biomarkers to be identified from the 3000 identified.

While some may be concerned about the potential loss of proteins of interest during such depletion experiments, Joseph said that they had thoroughly studied all the proteins collected and had been unable to find traces of low-abundant proteins.