Craig R. Aurand1, David S. Bell1, Teresa Lamb2 and Deborah Bell-Pedersen2
1Sigma Aldrich/Supelco, 2Texas A&M University
Complex metabolome profiling by LC-MS can be facilitated using advanced instrumentation and software. The choice of the HPLC column is also important. This article shows the benefit of choosing highly-efficient Ascentis Express phases with orthogonal selectivities to provide the most information from the LC-MS experiments.
The general aim of metabolomic profiling is to document the set of metabolites from a defined sample for determination of physiological changes. The specific sample can be characterized by a variety of descriptors or parameters; such as cell type, organelle, age, tissue, treatment, etc. In this study, Neurospora crassa cultures grown over a specified time period in the dark were compared and contrasted for a set of identified components. Much is known regarding the genome of Neurospora crassa, specifically in the determination of circadian rhythms. However, little is known regarding how the metabolome changes over the course of the day under control of the circadian clock. Endogenous circadian biological clocks program 24 h rhythms in biochemical, physiological and behavioral processes of living entities; including animals, plants, and fungi. These cyclic processes typically occur with an approximate 24-hour period, but this period can be impacted by light-to-dark and temperature cycles. When the organism is maintained in constant environmental conditions, such as constant dark, they will free run with an endogenous period. For Neurospora, the free running period is 22.5 hrs. The goal of this study was to profile the change in the metabolome of Neurospora crassa as a function of the circadian clock. The small molecule metabolites from time series experiments are evaluated to determine possible influences or artifacts of the circadian rhythms in the neurospora cultures.
Time-series experiments were conducted at Texas A&M University. Each time series was performed in triplicate from the same starting culture, labeled series A, B, or C. The experiments consisted of an initial culture that was subdivided into 27 cores; all individual cores were then inoculated at the same time. Culture sets were shifted from a light environment to a dark environment at various times (three per time period) as described in Table 1 to obtain cultures representing different times of the day. For example, 12 hours of darkness represents dawn and 28 hours of darkness represents midnight. Total incubation period for the experiment was 48 hours. After the incubation period, tissue samples were processed, packaged in dry ice, and shipped to the laboratory. Tissue samples were kept frozen until they were extracted. The tissue samples were extracted by placing 20 mg of tissue into to a 2 mL centrifuge tube. Next, 100 μL of 50:50 methanol:water was added making the final concentration for all samples 200 mg extracted tissue per mL. Samples were then vortexed to thoroughly mix sample and placed in refrigerator for 1 hour. The samples were then vortexed and centrifuged for 3 minutes at 15,000 rpm. The resulting supernatant was collected and analyzed directly.
Table 1: Neurospora crassa Incubation Period
Profiling of the Neurospora samples was conducted using high performance liquid chromatography (HPLC) in both reversed phase (RP) and hydrophilic interaction (HILIC) modes utilizing accurate mass time-of-flight (TOF) mass spectrometry. The concept behind utilizing both RP and HILIC HPLC is to facilitate a more accurate determination of an actual sample component versus a chromatographic artifact, without relying specifically on accurate mass resolution. By leveraging the selectivity differences between two (or more) different or orthogonal chromatographic modes, sample components that co-elute, do not retain, or do not elute on one mode may be resolved using the other mode. In this study, RP and HILIC separations were carried out using Fused-Core™ Ascentis Express RP-Amide and Ascentis Express HILIC columns, respectively. The polar embedded group of the amide was chosen over traditional C18 phases to increase the retention of the polar components. The Ascentis Express HILIC was chosen for alternative selectivity for polar analytes. Because of the large amount of unknown components in the samples, using orthogonal chromatographic separation in combination with accurate mass enabled better dissemination of components of interest from sample matrix and chromatographic anomalies.
Samples were analyzed by LC-MS RP (Figure 1) and HILIC (Figure 2) modes. The data were deconvoluted and pushed into the Mass Profiler™ software programs. Mass Profiler enables sets of experiments to be compared to each other. This can be performed using individual data files or batch files. By performing batch processing, samples can be compared for common components within all samples from the batch. Batch processing can also identify components that are common to only one set of samples, or attributed to a subset within the batch. There are several permutations of the comparisons that can be made. In this particular example, all samples from series A, B, and C were compared to each other for components common to all samples. The study also incorporated a blank chromatographic run to cancel out anomalies from the chromatographic system.
Figure 1: RP-HPLC Separation of Neurospora crassa Extract on Ascentis Express RP-Amide (product referenced - 53914-U)
Figure 2:HILIC Separation of Neurospora crassa Extract on Ascentis Express HILIC (product referenced - 53946-U)
The comparison was based upon the accurate mass of the components along with chromatographic retention time of either the RP or HILIC separations.
Approximately 310 components, or features, were observed in the RP separation (Figure 1), while approximately 670 components were observed in the HILIC separation (Figure 2). When data from both RP and HILIC methods were compared, twelve major components were found in all sample extracts. The goal of the study was to determine if there was a correlation between the intensity of the major components and the time of day. The experiment was designed to track intensity changes of the major common components throughout the time series. To simplify the experiment, this study did not target components that decreased completely, nor did it track the formation of new components. The signal intensity vs. incubation period was plotted for the twelve common components to determine if intensity levels exhibited circadian rhythm behavior. An example of this data for one component is shown in Figure 3. Here, the signal intensity of component m/z 326.1945 was plotted as a function of incubation period for series A. The trend in the time series is a cyclic change in the intensity of m/z 326.1945. At this stage of the study, the exact identity of m/z 326.1945 has yet to be determined nor has the influence from the circadian rhythm been identified. This work is still ongoing.
Figure 3: Time Series A for 326.1945 Component
Profiling of metabolic changes in biological samples can be a complex and tedious task, even with the most advanced instrumentation and software. Every advantage should be utilized to help simplify the deconvolution process, including sample preparation and enhanced chromatographic resolution. The approach of using orthogonal chromatographic separation modes greatly increases the opportunity for distinguishing true sample components from chromatographic anomalies. By doing so, this simplifies the data interpretation while increasing the confidence level of tracking components of interest. The use of the high-resolution Ascentis Express HPLC columns greatly aids in the resolution of components in even the most complex sample matrix. An added benefit of Ascentis Express columns is their durability, which makes them less susceptible to fouling and therefore highly suited for the long-term analysis of complex biological matrixes, such as those encountered in this study.
For additional information on sample profiling using orthogonal chromatographic modes, please see the Reporter 27.2 article on “Profiling of Stevia rebaudiana Extract by Accurate Mass Using HILIC and Reversed-Phase Chromatography.”