<p> Supporting Information</p><p>Identification and quantitation of malonic acid biomarkers of in-born error metabolism</p><p> by targeted metabolomics</p><p>Chandra Shekar R Ambati1, Furong Yuan2, Lutfi A Abu-Elheiga,2 Yiqing zhang1 and</p><p>Vivekananda Shetty*1</p><p>Metabolomics Core Facility1, Molecular and Cellular Biology1, Alkek Center for Molecular</p><p>Discovery1, Department of Biochemistry2, Baylor College of Medicine, Houston, TX 77030 Figure S-1: Product-ion mass spectra of 3NPH derivatized malonic acids</p><p>Figure S-2. Proposed fragmentation mechanism for 3NPH derivatized malonic acids Figure S-3. LC-MRM-MS of malonic acid (A) metabolites extracted from plasma and (B) standards after 3NPH derivatization and ( C ) free malonic acid standards (10 μM each) showing the efficiency of 3NPH chemical derivatization Figure S-4: Standard curves of 3NPH derivatized malonic aicds Figure S-5 : Representative extracted MRM chromatograms of 3NPH derivatized standards and QC plasma malonic acid metabolites Figure S-6: Work-flow for the metabolomics analysis of malonic acids using </p><p>HILIC and mass spectrometry</p><p>HILIC Method a) Extraction of MA, MMA and EMA from plasma </p><p>Same set of three groups of mice plasma samples were used in this study [WT (n=3), MCD</p><p>(n=3) and QC (n=3)]. A master mixture of internal standard (ISTD) solutions was freshly made</p><p>13 each time using the three isotopic labeled ISTD compounds (MA- C2 -5 µg/µl ,MMA-D3 -</p><p>1µg/µl and EMA-D3 -5µg/µl) and equal volume of this master mixture was spiked into each plasma test sample. A 80 µl of each plasma sample was transferred into a 4ml glass vial, spiked with 50 µl of ISTD master mixture solution (14 µl of ISTDs +36 µl H 2O). Then all the plasma samples were filtered by using 3kDa amicon filter devices. To obtain maximum recovery, an additional 500µl of 15% formic acid in methanol solution was used in the second round filtration. Flow-throµgh samples were combined and dried in a speed vacuum instrument. The dried sample was suspended in 100µl of mobile phase (20 mM NH4Ac in 90% acetonitrile and 10% water (PH 5.7), vortexed (5 min) sonicated (5 min) and centrifuged for 5min at 40C.</p><p>Supernatant solution was recovered and 20 µl of this extracted solution was injected for LC-</p><p>MRM-MS analysis. b) Preparation of calibration curve </p><p>In order to generate a standard calibration curve for each compound, a stock solution of isotopic</p><p>13 labeled compounds [MA- C2 (240ng/µl); MMA-D3 (160ng/µl); EMA-D3 (80ng/µl)] was first made in water. This stock solution was used to prepare respective unlabeled standard compound solutions as well as for serial dilutions to keep isotopic compounds concentrations constant (for the subsequent data normalization). Final concentrations of unlabeled standard solution were MA</p><p>-240 ng/µl, MMA -160ng/µl and, EMA - 80 ng/µl and starting from these concentrations eight serial dilutions were made using isotope labeled compounds solution. A 5 µl of each dilution was used for the identification and quantitation of these three target molecules by LC-MRM-MS analysis. c) LC-MRM-MS and data analysis </p><p>Chromatographic separation of all the target molecules was achieved on ZIC-HILIC metal free column (peek 100 mm x 2.1 mm) using isocratic buffer composed of 20mM NH4Ac in 90% acetonitrile and 10% water (PH 5.7). The initial flow rate was 0.3 mL/min from 0 to 7 min and then it was increased to 0.8 min and maintained until 12 min. The total run time was 20 min.</p><p>Similar LC-MRM-MS platform and data analysis tools were used as described in case of 3NPH method. The HPLC column temperature was set at 300C and tandem mass spectrometry experiments were carried out under negative ion ESI mode. The optimized collision energies and</p><p>MRM transitions of all the compounds investigated in this study are given in Table S-1. Figure S-7: HILIC based LC-MRM-MS analysis malonic acids</p><p>Figure S-8: Linearity of Malonic acids analysis in HILIC method Figure S-9: Separation of MMA and SA after 3NPH derivatization Table S-1: Optimized parameters and MRM transitions of malonic acids, malonic acid derivatives and corresponding ISTDs</p><p>Precursor MS1 Product MS2 Collision Cell Accelerator Compound Name ISTD Ion Res Ion Res Dwell Fragmentor Energy Voltage Polarity MALONIC ACID × 103 Unit 45 Unit 30 380 3 3 Negative MALONIC ACID × 103 Unit 59 Unit 30 380 3 3 Negative</p><p>13 MALONIC ACID- C2  105 Unit 60 Unit 30 380 3 3 Negative 13 MALONIC ACID- C2  105 Unit 46 Unit 30 380 3 3 Negative METHYL MALONIC ACID × 117 Unit 73 Unit 30 380 5 3 Negative METHYL MALONIC ACID × 120 Unit 76 Unit 30 380 5 3 Negative</p><p>METHYL MALONIC ACID-D3 × 120 Unit 76 Unit 30 380 5 3 Negative ETHYL MALONIC ACID × 131 Unit 87 Unit 30 380 6 5 Negative</p><p>ETHYL MALONIC ACID-D3  134 Unit 90 Unit 30 380 6 5 Negative MALONIC ACID (DER)-1 × 373.2 Unit 178 Unit 30 380 20 4 Negative MALONIC ACID (DER)-2 × 373.2 Unit 150 Unit 30 380 20 4 Negative</p><p>13 MALONIC ACID- C2 (DER)-1  375.2 Unit 179 Unit 30 380 20 4 Negative 13 MALONIC ACID- C2 (DER)-2  375.2 Unit 151 Unit 30 380 20 4 Negative METHYL MALONIC ACID (DER)-1 × 387.2 Unit 178 Unit 30 380 20 4 Negative METHYL MALONIC ACID (DER)-2 × 387.2 Unit 150 Unit 30 380 20 4 Negative</p><p>METHYL MALONIC ACID-D3 (DER)-1  390.2 Unit 178 Unit 30 380 20 4 Negative</p><p>METHYL MALONIC ACID-D3 (DER)-2  390.2 Unit 150 Unit 30 380 20 4 Negative ETHYL MALONIC ACID (DER)-1 × 401.2 Unit 178 Unit 30 380 20 4 Negative ETHYL MALONIC ACID (DER)-2 × 401.2 Unit 150 Unit 30 380 20 4 Negative</p><p>ETHYL MALONIC ACID-D3 (DER)-1  404.2 Unit 178 Unit 30 380 20 4 Negative</p><p>ETHYL MALONIC ACID-D3 (DER)-2  404.2 Unit 150 Unit 30 380 20 4 Negative</p><p>Table S-2: Optimized parameters and MRM transitions of malonic acid, its derivatives and corresponding ISTDs </p>