Hardware and software advances have greatly improved the reliability of mass spectrometry. In addition, its versatility and movement into the clinic has expanded the number of users and applications. Also affecting the MS market are increased efforts to screen patients through identification of biomarkers and to develop more effective drugs based on structural properties of targets.
Researchers at the Fred Hutchinson Cancer Research Center (www.fhcrc.org) are working to discover cancer biomarkers using patient plasma samples. “The protein-concentration range in plasma is very large, 10 to 12 orders of magnitude. There’s no single analytical technique to handle so wide a dynamic range,” explains Hong Wang, Ph.D., director of the center’s mass spec facility.
The proteins that really reflect disease development are low-abundance proteins, which are below the nanogram/mL level. To detect these rare proteins, Dr. Wang’s group uses chromatogram-based protein fractionation first, followed by MS analysis.
There are four electron-spray ionization (ESI)-MS systems in Dr. Wang’s lab. They were chosen instead of LC-MALDI because of their high-sample throughput. “After 2-D HPLC intact-protein fractionation and trypsin digestion, I pool the digested fractions for LC-ESI-MS analysis.” Up to 720 samples can be analyzed in a single experiment. Dr. Wang adds that it is key to reduce carryover from run to run with the ESI system in order to increase the HPLC separation efficiency and to analyze protein isoforms.
Dr. Wang also uses a linear, ion-trap MS analyzer because it has a fast scanning reader. “If scanning of data acquisition is slow, you will miss peptide analysis.” Sensitivity is really not an issue for MS, it’s more important to have a dynamic range of three orders of magnitude, he adds. Since reproducibility depends on the HPLC system, Dr. Wang’s group uses a nanofluor HPLC and they pack their own columns to ensure stability.
Cerebral Spinal Fluid
Perhaps even more difficult to analyze than plasma, cerebral spinal fluid (CSF) poses different challenges. “We haven’t done the fundamental studies, like others have done with plasma, to know what the interfering compounds are,” explains Mark Hayward, Ph.D., associate director of analytical chemistry at Lundbeck Research USA (www.lundbeckresearch.lundbeck.com).
Triple quadrupole MS-MS is used to analyze CSF samples because it is the “tried-and-true best way to do quantitative analysis in complex matrices. These analyzers provide predictable performance, so you get a lot of dynamic range that’s not available with other types of MS systems.”
Dr. Hayward’s group is focusing on small peptides and neurotransmitters like dopamine and acetylcholine to discover what the best biomarkers are and to measure quantitative levels. “We’re able to get high-precision levels to three percent RSD (relative standard deviation), which is very precise for picogram/mL concentrations.” This is important because “it allows the information to be useful, so you can actually screen patients,” he adds.
Another hurdle has been to make nano-sized 2-D liquid chromatography reproducible in order to get the really low concentrations. “The chromatography is advanced enough for nanoscale, but getting it all to work is quite a technical challenge.” In addition, Dr. Hayward says, “as you move downstream and have greater impact, the reproducibility gains more importance.”
Detecting Protein Changes
Merck & Co’s(www.merck.com) proteomics research group is using high-resolution MS to identify and quantify changes in peptide and protein levels related to disease or therapeutic agents.
“Our goal is to facilitate Merck’s basic research and to impact clinical medicine by providing a scientific approach to address these questions without antibody reagents,” states Ronald Hendrickson, Ph.D., director of proteomics, molecular profiling, Merck Research Labs (www.merck.com/mrl). A main advantage to this approach is the ability to perform unbiased analyses in complex biological systems without having to prespecify the analyte being measured, adds Dr. Hendrickson.
Since MS can rapidly cross species boundaries, it enables quick movement from cell-based experiments to preclinical models and then to humans. Dr. Hendrickson’s lab utilizes a triple quadrupole MS-based assay that uses stable isotope-labeled internal standards to improve precision and provide absolute quantification in lieu of developing an ELISA assay, which is time limiting.
The group is currently focusing on peptide and protein markers of proximal target engagement. This class of markers helps to address the question “did treatment engage the desired biochemical target?” Knowing this information in both preclinical models and human trials is critical and can help inform early go or no-go decisions. Another interesting application involving this type of MS is looking at protein turnover rates, where an understanding of protein dynamics is especially important to some diseases.
Key characteristics to look for in a MS system, says Dr. Hendrickson, include resolution, mass accuracy, sensitivity, dynamic range, and compatibility with the chromatography timescale. “I look for instruments that can maintain mass accuracy of better than five parts per million over a three-week period without requiring recalibration and without an internal standard.”