Quantitative Amino Acid Profiling Using the Kairos Amino Acid Kit and Metaboquan-R

[ TECHNOLOGY BRIEF ]

Quantitative Amino Acid Profiling Using the Kairos Amino Acid Kit and MetaboQuan-R

Myriam Kabu and Billy Joe Molloy Waters Corporation, Wilmslow, UK

Amino acids are extremely important biomolecules and are relevant in many branches of biomedical research. Here, we demonstrate the use of the Kairos Amino Acid Kit with the high-throughput, targeted, multi-omics LC-MS/MS platform, MetaboQuan-R. By doing so, we are able to produce a rapid workflow that delivers GOAL reproducible, quantitative, amino acid analysis. The aim of this experiment was to Compound name: Glutamine demonstrate the use of the Kairos™ Amino Correlation coefficient: r = 0.997652, r^2 = 0.995310 Calibration curve: 0.0231114 * x + 0.00420411 Acid Kit with the MetaboQuan-R™ Response type: Internal Std (Ref 30), Area * (IS Conc./IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None LC-MS/MS platform for the quantitative, high-throughput profiling of amino acids. 90.0 80.0

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60.0 BACKGROUND on se 50.0

Amino acids are the constituent building Re sp 40.0 blocks of proteins and are, therefore, 30.0 central to all aspects of biological function. 20.0 The accurate, precise, and reproducible 10.0 measurement of amino acids is critical in -0.0 Conc many branches of biomedical research. -0 500 1000 1500 2000 2500 3000 3500 4000 The analysis of these compounds is Figure 1. Calibration curve produced for Glutamine using the Kairos Amino Acid Kit and typically performed using derivatization MetaboQuan-R. followed by flow injection analysis (FIA) tandem mass spectrometry (MS/MS) standards, combined with a reproducible, high-throughput UPLC-MS/MS or FIA-MS/MS. A major limitation of platform, provides a solution to these two issues. FIA-MS/MS methods for amino acid analysis is the inability to distinguish isobaric species, leading to degraded THE SOLUTION accuracy and precision. Another limitation of many available amino acid analyses Sample preparation is the lack of standardized calibration Human serum samples were prepared as per the instructions provided in standards and quality control samples, the Kairos Amino Acid Kit. In summary, samples were subjected to protein key components for reproducible studies. precipitation using sulfosalicylic acid that contained SIL internal standards. The use of quality-controlled consumables Samples were then derivatized using the AccQTag™ reagent before dilution kit and stable-isotope labelled (SIL) and injection into the MetaboQuan-R LC-MS/MS workflow.

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LC conditions Cone Collision RT Amino acid MRM transition voltage energy UPLC separation was performed with an (mins) (V) (eV) ACQUITY™ UPLC™ I-Class System (Fixed 4-hydroxyproline 302.20 > 171.10 0.17 Loop), equipped with a CORTECS™ T3 2.7 µm Alanine 260.20 > 171.10 0.57 (2.1 x 30 mm) analytical column. A sample of α-Aminobutyric Acid 274.20 > 171.10 0.96 2 µL was injected at a flow rate of 1.3 mL/min. β-Aminobutyric Acid 274.20 > 171.10 0.79 Mobile phase A was 0.01% formic acid (aq) γ-Aminobutyric Acid 274.20 > 171.10 0.65 20 containing 0.2 µM ammonium formate and Aminoadipic acid 332.20 > 171.10 0.70 mobile phase B was 50% isopropanol in Asparagine 303.20 > 171.10 0.20 acetonitrile containing 0.01% formic acid Beta-Alanine 260.20 > 171.10 0.47 and 0.2 µM ammonium formate. The derivatized Citrulline 346.20 > 171.10 0.41 amino acids were eluted from the column and Ethanolamine 232.20 > 171.10 0.34 14 separated with a gradient of 1–8% mobile Glutamine 317.10 > 171.10 0.30 phase B over 2.4 minutes, followed by a 0.9 20 Glycine 246.20 > 171.10 0.30 minute column wash at 98% mobile phase B. Histidine 326.20 > 171.10 1.31 25 The column was then re-equilibrated to initial Homocitrulline 360.20 > 171.10 0.67 conditions. The analytical column temperature Isoleucine 302.20 > 171.10 2.27 30 20 was maintained at 60°C. Kynurenine 379.20 > 171.10 2.07 MS conditions Lysine 244.10 > 171.10 1.59 25 Methionine 320.20 > 171.10 1.32 20 Multiple Reaction Monitoring (MRM) analyses Ornithine 237.10 > 171.10 1.32 were performed using a Xevo™ TQ-S micro 25 Phenylalanine 336.20 > 171.10 2.30 Mass Spectrometer. All experiments were Proline 286.20 > 171.10 0.74 20 performed in positive electrospray ionization Sarcosine 260.20 > 171.10 0.37 25 (ESI+) mode. The ion source temperature and Serine 276.20 > 171.10 0.25 20 capillary voltage were kept constant at 150°C Taurine 296.20 > 171.10 0.25 15 and 2.0 kV, respectively. The cone gas flow Threonine 290.20 > 171.10 0.24 20 rate was 50 L/hr and desolvation temperature Tryptophan 375.20 > 171.10 2.43 was 650°C. 25 Tyrosine 352.20 > 171.10 1.30 Valine 288.20 > 171.10 1.49 Calibration lines 20 Leucine 302.20 > 171.10 2.33 Calibration lines were created for the 29 amino acids detailed in Table 1 using the peak area Table 1. List of MS/MS conditions and retention times for derivatized amino acids analyzed ratio to a SIL internal standard. The SIL internal using the Kairos Amino Acid Kit and the MetaboQuan-R workflow. standard used for each analyte was either the heavy labelled analog of the analyte itself or Compound name: Lysine Correlation coefficient: r = 0.999931, r^2 = 0.999862 Calibration curve: 0.0528634 * x + -0.02915 the closest internal standard to the analyte Response type: Internal Std (Ref 32), Area * (IS Conc./IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None in terms of chromatographic retention time. 45.0 Figures 1–3 show examples of the calibration 40.0 lines produced. These are for Glutamine, 35.0 Homocitrulline, Lysine, and Tyrosine.

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Figure 2. Calibration curve produced for Lysine using the Kairos Amino Acid Kit and MetaboQuan-R. 2 [ TECHNOLOGY BRIEF ]

Compound name: Tyrosine SUMMARY Correlation coefficient: r = 0.999766, r^2 = 0.999532 Calibration curve: 0.0438988 * x + 0.0484731 Response type: Internal Std (Ref 40), Area * (IS Conc./IS Area) The Kairos Amino Acid Kit was successfully Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

used on the MetaboQuan-R LC-MS/MS 160 workflow for the quantitative analysis of amino 140 acids. Excellent analytical specificity, linearity, 120 and dynamic range were demonstrated. e The combination of a standardized kit and 100 80

LC-MS/MS workflow provides an optimized Respons

platform for the accurate, precise, and 60

reproducible analysis of amino acids in biological 40 samples and is ideally suited to a wide variety of 20 biomedical research studies. -0 Conc -0 400 800 1200 1600 2000 2400 2800 3200 3600

Figure 3. Calibration curve produced for Tyrosine using the Kairos Amino Acid Kit and MetaboQuan-R.

For Research Use Only. Not for use in diagnostic procedures.

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. Waters, The Science of What’s Possible, Kairos, MetaboQuan-R, ACQUITY, UPLC, CORTECS, and AccQTag are trademarks of Waters Corporation. All other trademarks are the property of their respective owners. T: 1 508 478 2000 F: 1 508 872 1990 ©2019 Waters Corporation. Produced in the U.S.A. April 2019 720006532EN TC-PDF www.waters.com