Protein therapeutics is a fast-growing segment within the global pharmaceutical industry. Six thousand biologic drugs were in clinical trials in 2009 compared to 1,200 in 2005. By 2014, six of the top 10 drugs on the market will be biologics, according to a 2011 Thompson Reuters–Newport study of U.S. drug trends.
For more than a decade, LC-MS has been used for small molecule absolute quantitation. As the role of protein-based biologics grows, this technology is becoming attractive for absolute quantitation of proteins. The challenge is the selective isolation of the protein of interest from the large amount of irrelevant protein species naturally present in biological matrices. This issue was one of the main topics discussed at the recent “Pittcon” conference in Orlando.
“Complicated samples contain a lot of proteins, especially high-abundance proteins. The mixtures are so complex that you need to clean them up before you use the LC-MS,” explains Richard King, Ph.D., laboratory director, PharmaCadence Analytical Services.
Absolute quantitation of proteins is difficult, and even more so, because there are no real standards as with small molecules. The assumption is every molecule of protein generates one molecule of peptide but, in reality, the peptides can come from inactive versions of the protein, fragments, precursors, or degradants. PharmaCadence Analytical Services uses electrophoresis as a protein sample-preparation technique to distinguish between those species.
The advantage of electrophoresis is that almost all proteins will run through a gel with consistent, high recovery rates. In addition, phospholipids, a main cause of matrix effects in the MS, run right through the gel. The limitation is loading capacity so electrophoresis might not be the preferred technique for a low-abundance protein in plasma.
“Our goal is separation at the whole protein level. The Expedeon/Protein Discovery 8100 Fractionation Gelfree® System (gel-eluted liquid fraction entrapment electrophoresis system from Protein Discovery), eight-channel, parallel-tube SDS-PAGE allows us to track our protein of interest with molecular markers, elute our fractions of interest off the gel, keep them in solution, then do the digestion. As long as we catch the protein we are interested in, we can live with impurities at that point; we will select against those with the LC-MS,” continues Dr. King.
The Gelfree system is an antibody-free separation technique and can fill in when there are no antibodies or antibodies are under development. Antibody development can be a daunting and time-consuming task, taking from two months to two years.
PharmaCadence Analytical Services combines electrophoresis with reverse-phase LC and MS. Each of the techniques has a different separation mechanism and, when combined, produces a very powerful system when working with complex mixtures.
More than One Approach
“Sample preparation is challenging because you are looking for a specific protein in a global protein soup. There is more than one approach. It depends on the variables, such as the concentration level, the location, solubility, and the tools available in the laboratory,” discusses Nalini Sadagopan, Ph.D., south-west regional LC-MS product specialist, Agilent Technologies.
“Many protein sample-preparation techniques have been around for a while. Manufacturers continually evaluate the market needs and develop new formats, such as 96-well formats for high-throughput screening and parallel processing. Plus there have been considerable improvements in abundant protein removal protocols and immunoaffinity purification in the last several years.”
“The next hurdle is to marry the sample-preparation techniques to the LC-MS workflow because of the desire to quantitate very low-level proteins, unknown proteins, or proteins for which antibodies do not exist.”
The two broad approaches to protein sample preparation are to remove the extraneous high-abundance proteins and to let the protein of interest pass through or to enrich the protein subgroups by capturing, then eluting, the protein of interest.
For human serum, specific removal of six high-abundance proteins depletes approximately 85–90% of the total protein mass. The Multiple Affinity Removal System, spin cartridges, and LC columns can be used to remove the top 7 or 14 high-abundance proteins from a sample.
The AssayMAP® platform for high-throughput micro-chromatography allows integrated protein sample-preparation workflows from crude samples and incorporates purification, digestion, and peptide clean-up using automated liquid-handling systems. The disposable cartridges contain a 5 μL bed, which can be packed with 15–100 μm particle-size chromatography resins or immobilized enzymes, such as protein A, streptavidin or trypsin, for use in subgroup enrichment.
Advances in MS technology have also simplified protein sample preparation. The high sensitivity of the Agilent 6490 triple-quadrupole LC-MS with iFunnel technology provides attogram (10–18) limits of detection and zeptomole (10–21) sensitivity with six logs of linearity. This allows, in some circumstances, the detection of proteins at low levels without extensive sample preparation by just diluting the sample.