Enhancing Biomarker Discovery
“SRM delivers a unique fragment ion that can be monitored and quantified in the midst of a very complicated matrix,” said Melissa Radabaugh, senior R&D scientist, Sigma-Aldrich. “However, to achieve the desired result, the column, ionization source, and the flow rate need to be optimized.”
Previously, while at Pfizer, Radabaugh and her team greatly improved the sensitivity of detection of nitrotyrosine, a marker of many pathological conditions resulting from oxidative/nitrative cell damage, by developing an on-line immunoaffinity SRM LC-MS/MS method. Optimization of the process parameters enabled detection of nitrotyrosine in the low picomolar levels. Coupling with an antibody column further improved the specificity of detection.
The assay was tested on a variety of biological fluids including urine, plasma, and cerebrospinal fluid, ensuring that the method is robust enough to support evaluation of nitric oxide-driven pathologies and their response to treatment.
Radabaugh’s most recent study optimized a system for quantification of peptides. In order to translate these studies into clinic-based diagnostics, the serum samples were spiked with a nonhuman synthetic peptide. These defined standard mixtures were used for optimization of liquid chromatography (LC) and MS parameters.
The team tested various separation parameters such as flow rates, peak shape, retention time, and delay-time volumes using several separation columns. The goal was to achieve the optimal separation using capillary flow rates as opposed to a more commonly used nanoflow. In the context of analyzing proteins by SRM, capillary flow has a number of advantages. This flow rate prevents issues of clogging when complex protein mixtures are used.
The column with the best outcomes was coupled with different ionization sources. The ion source is the first part of the mass spectrometer and is used to ionize the sample. Under the testing conditions, the Michrom Advance source demonstrated the strongest signal. As a whole, the system was able to uptake a larger amount of sample, making this a useful system for biomarker discovery, quantitation, and validation.
“The advantages of microflow includes the ability to load a larger sample and to achieve better separation,” said Tina Settineri, Ph.D., director, HPLC products, Eksigent division of AB Sciex.
“However, nanoflow provides the absolute best sensitivity for mass spectrometry, because the sample becomes highly concentrated. For added flexibility between nanoflow and microflow LC, nanoLC customers can effortlessly convert between nano- and microflow by interchanging the flow channels in our 2D nanoLC systems, such as the NanoLC-Ultra® 2Dplus system. Furthermore, Eksigent’s ekspert™ microLC 200 system enables two different flow-rate ranges.”
The most recent enhancement coupled the nanoflow liquid chromatography with high-performance mass spectrometry to achieve the best possible combination of high sensitivity and high acquisition speeds. This combination of hardware is essential to deal with samples of high complexity, such as serum or cellular digests.
“The key to increasing protein separation on the nanoLC was to increase the column length,” continued Dr. Settineri. “This in turn increased the separation time, and hence the time available for MS/MS acquisition, resulting in the most proteins identified utilizing just under 50 MS/MS scans per second. By going from a 15 cm column to a 30 cm column, we were able to increase the number of identified proteins by 20%, while keeping the identification errors to less than 1%.”
The company is exploring ways to further increase column length to 50 cm in order to extend coverage to a more complete proteome. The analysis was performed on the TripleTOF™5600 system, a top of the line hybrid quadrupole time of flight (TOF) mass spectrometer.
This instrument is a central piece of the proteomic technique that quantifies nearly all proteins in a sample in a single analysis. SWATH™ Acquisition does not simply detect a single precursor ion. Instead, it uses a certain predefined mass range to perform MS/MS acquisition. SWATH technique generates highly specific fragment ion data for all peptides within this mass range.
Resulting fragment ion chromatograms uniquely identify each peptide of interest, just like in SRM. The technique provides a complete quantitative profile of all proteins in the sample.