A Benchtop Approach To Measuring S-AdenosylMethionine Metabolite Levels Using HILIC UPLC-MS

Abstract

S-adenosylmethionine (SAMe) is the most common methyl donor found in living organisms. It is the second most common cellular enzyme substrate and is an essential molecule in the carbon metabolic cycle. SAMe is widely used in both a clinical setting and as a nutritional supplement to treat a variety of disorders ranging from liver disease to osteoarthritis in both humans and animals. Demand for SAMe is rapidly expanding and the yeast Saccharomyces cerevisiae is often used as an industrial production host. Increasing clinical research into SAMe, as well as increasing commercial demand, has led to the need for rapid, accurate measurement of SAMe levels from a variety of sources. We report here a novel hydrophilic interaction liquid chromatography (HILIC) UltraPerformance liquid chromatography – mass spectrometry (UPLC-MS) benchtop method to measure intracellular levels of SAMe from S. cerevisiae. Research is ongoing to understand the cellular impact of variations in SAMe levels as well as mutation screens to identify yeast strains with increased SAMe production. This UPLC method allows researchers to measure SAMe with increased resolution, speed, and sensitivity while decreasing both cost and environmental impact, and has the potential to be adapted for the measure of many other metabolites.

INTRODUCTION

S-adenosylmethionine (SAMe) is the primary cellular methyl donor for all organisms and its use in the cell as an enzyme substrate is second only to ATP (Cantoni 1975; Chiang et al. 1996). In addition, this versatile molecule functions in three primary metabolic pathways: aminopropylation, transmethylation, transsulfuration (Hidese et al. 2017; Tehlivets et al. 2013). SAMe is widely used as a nutritional supplement and prescription drug for a variety of clinical disorders including liver disease (Guo et al. 2015; Lu and Mato 2012), osteoarthritis (Rutjes et al. 2009), depression (Sharma et al. 2017), and dementia (Montgomery et al. 2014) in both humans and animals. SAMe was approved by the FDA as an over-the-counter supplement in 1999 (Liou et al. 2014), leading to an even larger demand for commercially produced SAMe. A variety of industrial production methods have been investigated, the most common being the use of microorganisms such as Saccharomyces cerevisiae (Hayakawa et al. 2016; Kanai et al. 2017) and Escherichia coli (Wei et al. 2014) as SAMe “factories”. As advances are made in the study of SAMe, both its role in disease as well as its production, advances in the ability to rapidly and accurately measure SAMe are also necessary.

A wide variety of methods have been used to measure SAMe levels including capillary electrophoresis (Mizunuma et al. 2004), high performance liquid chromatography (HPLC) with a variety of detectors (Hayakawa et al. 2015; Huang et al. 2012; Liou et al. 2014; Sturgess 2014), liquid chromatography-tandem mass spectrometry (LC-MS/MS)(Iglesias González et al. 2015; Kamarthapu et al. 2013), nuclear magnetic resonance (NMR) (Liou et al. 2014; Vinci and Clarke 2010), and immunoassay (Hao et al. 2017). We have developed a robust and reproducible method for quantitating intracellular levels of SAMe in the yeast S. cerevisiae via hydrophilic interaction liquid chromatography (HILIC) UltraPerformance Liquid Chromatography Mass Spectrometry (UPLC-MS) using a benchtop system consisting of an Acquity HClass and QDa Mass Detector (Waters Corporation, Milford, MA). UltraPerformance liquid chromatography (UPLC) improves upon HPLC in resolution, speed, sensitivity, and separation efficiency. It also reduces the volume of solvent used, improving both cost and environmental impact (Swartz 2005).

MATERIALS AND METHODS

Strains and growth conditions

Our parental strain (hereafter referred to as wildtype) genotype is: MAT a/alpha, leu2–3/leu2–3 his3-Δ200/his3-Δ200 trp1-Δ1/trp1-Δ1 lys2–801/LYS2 ura3–52/ura3–52 can1–100/CAN1 ade2–101/ade2–101 2x [CF:(ura3:: TRP1, SUP11, CEN4, D8B)]. Deletions of SAM alleles were created utilizing the homologous recombination switch-out method to create mutant strains M1–M7 (Wach et al. 1994). All strains were grown to saturation overnight at 30°C in Synthetic Complete media with appropriate auxotrophic selection. Saturated cultures were diluted 1:1000 into 200 mL selective media and grown shaking at 30°C until they reached log phase (OD₆₀₀ of 0.5–0.8, Thermoscientific Genesys^™20). Cells were pelleted by centrifugation (2 min, 2500 rpm), washed with deionized water, and stored at −80°C.

Sample preparation

Cell pellets were thawed on ice and spun at 20,000 rpm for 1 min. Excess liquid was removed and the pellet was weighed. Cells were added or removed until the cell pellets weighed between 100–160 mg. 350 μL of freshly prepared cold 25:75 deionized H₂O:acetonitrile (Fisher Scientific HPLC grade) and ~200 μL glass microbeads (Sigma Aldrich 425–600 μm) were added and cells were homogenized (BioSpec Mini-BeadBeater-8) for 30 sec followed by a 5 min rest on ice. This cycle of homogenization and ice incubation was performed a total of three times. The supernatant was transferred to a new microcentrifuge tube and cell debris was pelleted by centrifugation (5 min, 20,000 rpm). The supernatant was syringe filtered (Restek^™ 0.22 μm PVDF) into LC/GC certified glass vials (Waters 186000384c) and stored at 4°C. Samples were analyzed within 24 hours.

HILIC UPLC-MS Analysis

Analysis of extract was performed by UltraPerformance Liquid Chromatography Mass Spectrometry (UPLC-MS); the system consisted of an Acquity HClass and QDa Mass Detector (Waters Corporation, Milford, MA). The sample compartment was maintained at 10°C. The mass detector (QDa) was operated in electrospray positive mode with a capillary voltage of 0.8V and probe temperature of 500°C. Analysis of the SAMe was done by monitoring the protonated molecular ion (M+H⁺) at mass 399 in selected ion recording (SIR) mode. Separation of the compound was done using an Amide column (BEH Amide column, 2.1×50 mm (Waters Corporation, Milford, MA)) at 45°C. A HILIC separation method employing a gradient of ammonium acetate, formic acid, and acetonitrile at a flow rate of 0.5 mL/min was used. SAMe eluted at 2.5 min in the 5 min analysis. A calibration curve was generated by injecting 1 μL of eight SAMe standards (32 mM New England Biolabs) in 25:75 deionized H₂O:acetonitrile from a concentration of 2 μg–100 μg/mL; resulting in a correlation coefficient (r²) of 0.994 on an 8 point curve. (For samples that were above the calibrated range, further dilutions in 75:25 deionized H₂O:acetonitrile were utilized.)

Data analysis

The SAMe μg/mL measurements for each sample were converted to μmol/g wet cell pellet weight. The average, standard deviation, and relative standard deviation for intraday technical replicates (n = 2) were calculated using Microsoft Excel.

RESULTS

The Limit of Detection (LOD) for SAMe is <0.1ug/mL, calculated as a signal to noise value of 12:1 using Empower 3 software, extrapolated from the lowest standard injected (Empower 3 Chromatography Software, Waters Corporation, peak-to-peak signal to noise calculation).

We were able to accurately measure SAMe within a range of 2–100 μg/mL with a correlation coefficient (r²) of 0.994. The results show that a well-defined peak of SAMe appears at 2.5 min in the 5 min analysis from both a 10 μg/mL standard (Fig 1) as well as from a representative sample of intracellular yeast extract (Fig 2).

Figure 1.

SAMe standard peak. A representative ion recording (SIR) at mass 399.1 Da of a 1 μL injection of a 10 μg/mL SAMe standard.

Figure 2.

SAMe S. cerevisiae sample peak. A representative ion recording (SIR) at mass 399.1 Da of a 1 μL injection of a S. cerevisiae sample found to contain 20.3 μg/mL SAMe.

In order to analyze our ability to detect differences in intracellular SAMe levels we prepared samples for measurement from S. cerevisiae strains with deletions in a variety of S-adenosylmethionine synthetase alleles. The products of these genes (SAM1 and SAM2) catalyze the formation of SAMe from methionine and ATP. Deletion of one or more alleles in various combinations results in variations in intracellular SAMe levels. We could accurately measure intraday technical replicates (n = 2) of SAMe in a range of 175–911 μmol/g wet cell pellet weight with a standard deviation range of 2–59 μmol/g wet cell pellet weight and a relative standard deviation range of 0.8–12.8% (Table 1).

Table 1.

SAMe technical replicates (Intraday).

	SAMe concentration (μmol/g wet cell pellet)
Strain	cell pellet 1	cell pellet 2	Avg ± StDev	RSD (%)
wildtype	405	394	400 ± 8	1.90
Ml	939	884	911 ± 39	4.32
M2	728	746	737 ± 13	1.72
M3	499	496	498 ± 2	0.39
M4	256	254	255 ± 2	0.80
M5	643	651	647 ± 6	0.86
M6	181	168	175 ± 9	5.31
M7	505	421	463 ± 59	12.8

Data presented as the average of two intraday replicates ± the standard deviation. RSD: relative standard deviation.

DISCUSSION

The method described allows precise and sensitive determination of intracellular SAMe levels in S. cerevisiae. Polar compounds such as amino acids demonstrate good retention and separation by HILIC (Buszewski and Noga 2012). This method allows the use of a mobile phase that does not contain ion pairing buffers, which can suppress ionization by mass spectrometry (MS), decrease sensitivity, and dirty the mass spectrometer, resulting in more time spent cleaning the equipment between uses. HILIC, combined with the smaller particle sizes, smaller columns, and greater pressure ranges used with UPLC, allows increases in both speed and peak capacity (# of peaks resolved/unit time). The higher chromatographic efficiency of UPLC results in increased selectivity with MS detection (Tolley et al. 2001) and the use of the amide HILIC column adds an additional level of peak detection and confirmation over traditional UV based methods.

SUMMARY

Our method improves upon other methodologies in several ways. First, the sample volume needed is only 1 μL, and second, the total run time is 7 min. The smaller sample volume and faster run time could increase the efficiency of several high-throughput methods, including clinical screening of patient samples and industrial screening of gene mutations in microorganisms for increased SAMe production. The equipment used here, a single quad MS (QDa), is affordable and includes a benchtop detector that is self-aligning and calibrating with fewer user setting and using minimal bench space. This equipment is designed for routine, high-throughput analysis and the method presented should be easily adapted to measuring additional metabolites.

Availability of data and material

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.