Supplemenatry Figure S1-A. Determination of the chemical structure of up-regulated metabolites in urine sample from diabetic subjects by tandem mass spectrometry. Top panel shows the MS/MS fragmentation of the ion with m/z 206.0804 from urine samples in the negative electrospray ion mode. The bottom panel shows the fragmentation for standard N-acetyl-D-phenylalanine under the same conditions.

Urine extract TOF MSMS 206.08ES-

N-acetyl-D-phenylalanine TOF MSMS 206.08ES-

Urine extract TOF MSMS 101.03ES-

2-Ketobutyric acid TOF MSMS 101.03ES-

Supplementary Figure S1-B. Determination of the chemical structure of down regulated metabolite in urine samples of diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 101.03 from urine extract in the negative ion mode. The bottom panel shows the fragmentation for standard 2-Ketobutyric acid under the same conditions.

Urine extract TOF MSMS 145.02ES-

2-Ketoglutaric acid TOF MSMS 145.02ES-

Supplementary Figure S1-C. Determination of the chemical structure of down regulated metabolite in urine samples of Diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 145.02 from urine extract in the negative ion mode. The bottom panel shows the fragmentation for standard 2-Ketoglutaric acid under the same conditions.

Urine extract TOF MSMS 168.08 ES-

1-Methylhistidine TOF MSMS 168.08 ES-

Supplementary Figure S1-D. Determination of the chemical structure of down regulated metabolite in urine samples of Diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 168.08 from urine extract in the negative ion mode. The bottom panel shows the fragmentation for standard 1-Methylhistidine under the same conditions.

Urine extract TOF MSMS 188.04 ES-

Kynurenic acid TOF MSMS 188.04 ES-

Supplementary Figure S1-E. Determination of the chemical structure of down regulated metabolite in plasma samples of Diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 188.04 from urine extract in the negative ion mode. The bottom panel shows the fragmentation for standard Kynurenic acid under the same conditions.

Urine extract TOF MSMS 206.08ES-

Xanthurenic acid TOF MSMS 206.08ES-

Supplementary Figure S1-F. Determination of the chemical structure of down regulated metabolite in plasma samples of Diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 206.08 from urine extract in the positive ion mode. The bottom panel shows the fragmentation for standard Xanthurenic acid under the same conditions.

Plasma extract TOF MSMS 124.02ES-

Taurine TOF MSMS 124.02ES-

Supplementary Figure S1-G : Determination of the chemical structure of down regulated metabolite in plasma samples of Diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 124.02 from plasma extract in the negative ion mode. The bottom panel shows the fragmentation for standard Taurine under the same conditions.

Plasma extract TOF MSMS 167.03ES-

Uric acid TOF MSMS 167.03ES-

Supplementary Figure S1-H. Determination of the chemical structure of down regulated metabolite in plasma samples of Diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 167.03 from urine extract in the negative ion mode. The bottom panel shows the fragmentation for standard Uric acid under the same conditions.

Plasma extract TOF MSMS 267.07ES-

Inosine TOF MSMS 267.07ES-

Supplementary Figure S1-I. Determination of the chemical structure of downregulated metabolite in plasma samples of Diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 267.07 from plasma extract in the negative ion mode. The bottom panel shows the fragmentation for standard Inosine under the same conditions.

Supplementary Figure S1-J. Determination of the chemical structure of upegulated metabolite in urine samples of Diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 177.10 from plasma extract in the positive ion mode. The bottom panel shows the fragmentation for standard Serotonin under the same conditions.

Urine extract TOF MSMS 177.10 ES+

Serotonin TOF MSMS 177.10 ES+

Urine extract TOF MSMS 87.00 ES-

Pyruvic acid TOF MSMS 87.00 ES-

Supplementary Figure S1-K. Determination of the chemical structure of upregulated metabolite in plasma samples of Diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 87.00 from urine extract in the negative ion mode. The bottom panel shows the fragmentation for standard Pyruvic acid under the same conditions.

Urine extract TOF MSMS 130.08 ES-

Leucine TOF MSMS 130.08 ES-

Supplementary Figure S1-K. Determination of the chemical structure of upregulated metabolite in plasma samples of Diabetic subject by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 130.08 from urine extract in the negative ion mode. The bottom panel shows the fragmentation for standard Leucine under the same conditions.

Supplementary Figure S1-L. Determination of the chemical structure of up-regulated metabolite in plasma samples of by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 380.2552 from plasma extract in the positive ion mode. The bottom panel shows the fragmentation for standard Sphingosine-1-phosphate under the same conditions.

Plasma extract TOF MSMS 380.2552 ES+

Sphingosine-1-Phosphate TOF MSMS 380.2552 ES+

Plasma extract TOF MSMS 117.0189 ES-

Succinate TOF MSMS 117.0189 ES-

Supplementary Figure S1-M. Determination of the chemical structure of downregulated metabolite in plasma samples by tandem mass spectrometry. Top Panel shows the MS/MS fragmentation of the ion with m/z 117.0189 from plasma extract in the positive ion mode. The bottom panel shows the fragmentation for standard Succinate under the same conditions.

Supplementary Figure S1-N. Determination of the chemical structure of upegulated metabolite in plasma samples of Diabetic subjects by tandem mass spectrometry and matching the fragments with standard fragmentation patern . The top panel shows the fragmentation for standard PC(18:0/0:0) taken from Lipid maps. The bottom panel shows the MS/MS fragmentation of the ion with m/z 524.37 from plasma extract in the positive ion mode.

H

H

18:0

LPC

+ H

18:0

LPC

–H

2O +

H

Plasma Extract TOF MSMS 524.37 ES+

MS/MS spectra of PC(18:0/0:0) Lipid maps m/z 524.37 ES+

Supplementary Figure S1-O. Determination of the chemical structure of down regulated metabolite in plasma samples via tandem mass spectrometry. The top panel shows the fragmentation for standard PE(P-16:0/22:6). The bottom panel shows the MS/MS fragmentation of the ion with m/z 746.51 from plasma extract in the negative ion mode.

Supplementary Figure S1-P. Determination of the chemical structure of up-regulated metabolite in plasma samples by tandem mass spectrometry. The top panel shows the fragmentation for standard PG(18:0/18:1) taken from Lipid maps. The bottom panel shows the MS/MS fragmentation of the ion with m/z 775.54 from plasma extract in the negative ion mode.

(281

) 18:

1 FA

-H(2

83) 1

8:0

FA-H

36:1

GPG

ro -H

MS/MS spectra of PG(18:0/18:1) Lipid maps m/z 775.54 ES-

Plasma Extract TOF MSMS 775.54 ES-

Supplementary Table ST1. UPLC-MRM-MS Parameters used for quantitative measurement of metabolites in plasma and urine samples. Metabolites were quantified using Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer (Xevo TQ, Waters). For each metabolite, a MRM method was optimized using the “IntelliStart” function on the Xevo TQ for determining the optimal cone voltages, collision energy and dwell time for maximal sensitivity and ion transmission for the selection of Q1/Q3 transitions.

Urine MetabolitesIonization Mode Compound Name Parent (m/z) Transition (m/z)Dwell (s) Cone (V) Collision (V)Molecular FormulaES+ Xanthurenic Acid 206.0694 131.9828 0.043 36 28 C10H7NO4

Xanthurenic Acid 206.0694 159.9844 0.043 36 18 C10H7NO4ES- Kyuneric acid 188.0745 116.0088 0.043 18 26 C10H7NO3

Kyuneric acid 188.0745 144.0125 0.043 18 16 C10H7NO3ESCi- 2- Ketoglutaric Acid 145.0534 100.9649 0.217 16 8 C5H6O5ES+ Serotonin 177.1025 132.1017 0.025 40 16 C10H12N20

Serotonin 177.1025 115.7523 0.025 40 20 C10H12N20ES- Succinate 117.0189 99.922 0.217 16 8 C4H6O4

Succinate 117.0189 73.0332 0.217 16 8 C4H6O4ES- Pyruvic Acid 87.0071 43.001 0.217 16 8 C3H4O3

Plasma MetabolitesIonization Mode Compound Name Parent (m/z) Transition (m/z)Dwell (s) Cone (V) Collision (V)Molecular FormulaES+ Sphingosine- 1- phosphate 380.1125 264.199 0.03 60 23 C18H38NO5PES+ PC(18:0/0:0) 524.3717 506.3611 0.037 20 38 C26H55NO7P

PC(18:0/0:0) 524.3717 184.0739 0.037 20 22 C26H55NO7PES- PG(18:0/18:1) 775.5504 419.6802 0.031 25 24 C42H81O10P

PG(18:0/18:1) 775.5504 281.2859 0.031 25 24 C42H81O10P

Supplementary Table ST2. UPLC-MRM-MS Parameters of stable-isotope-labeled standards used for SID-MRM-MS in plasma and urine samples. For each metabolite, a MRM method was optimized using the “IntelliStart” function on the Xevo TQ for determining the optimal cone voltages, collision energy and dwell time for maximal sensitivity and ion transmission.

Ionization Mode

Compond Name Parent (m/z) Transition (m/z) Dwell (s) Cone (V) Collision (V)

121.0319 76.9714 0.011 22 10121.0319 101.9437 0.011 22 10

193.0957 120.997 0.011 18 26

193.0957 149.0005 0.011 18 16

ES- 13C3- Pyruvic Acid 89.9442 45.0122 0.011 22 8

ESCi- D6 - α-Ketoglutaric Acid 149.0681 104.969 0.011 16 8

387.34 81.95 0.05 18 30387.34 271.31 0.05 18 18

D6 - SuccinateES-

D5- Kynurenic AcidES-

ES+ D7-Sphingosine - 1- Phosphate

Supplementary Figure S2. Multiple reaction monitoring yields ion traces of stable isotope labeled and unlabeled metabolites that show retention time reproducibility. The urine and plasma samples were processed by spiking a known concentration of the stable isotope labeled metabolites and injected for UPLC-SID-MRM-MS analysis as described in the Methods section. MRM runs were performed by monitoring Q1/Q3 transitions. The peak area of the endogenous metabolite was normalized to that of the respective isotope labeled standard and the relative ratios in T2DM and controls were calculated.

13C3-Pyruvic acidm/z 89.94 45.01

D5-Kynurenic acidm/z 193.09 149.00

D6-Succinatem/z 121.03 76.97

D6-Ketoglutaric acidm/z 149.06 104.96

D7-Spingosine-1-phosphatem/z 387.34 271.31

Spingosine-1-phosphatem/z 380.11 264.19

2-Ketoglutaric acidm/z 145.05 100.96

Succinatem/z 117.01 73.03

Kynurenic acidm/z 188.07 144.01

Pyruvic acidm/z 87.00 43.00

Supplementary figure S3. Random forests analysis of T2DM and Control subjects. Panel A: Heat map visualization of the top 50 features rankings comparing relative levels in control and T2DM in plasma samples. Panel B. Heatmap of the top 50 features in urine samples. Each row represents a unique feature with a characteristic mass to charge and retention time (minutes).

T2DM Control T2DM Control

AB

S.No. m/z (mode) RT(min) Relative Fold Change(T2DM/Control) P-value

1 399.1639(-) 3.4176 52.2 0.002

2 429.0847(-) 3.0324 27.7 0.01

3 404.0421(-) 2.8961 14.5 0.01

4 455.0450(-) 2.9946 9.3 0.02

5 391.0627(-) 3.5508 6.6 0.03

6 273.0720(-) 0.4020 6.04 0.01

7 557.0609(-) 3.9133 5.6 0.006

8 171.0646(-) 2.541 0.4 0.02

9 1.6.0286 2.2618 0.4 0.0110 176.934(-) 0.3436 0.3 0.02

11 236.0644(-) 0.2644 0.1 0.01

12 429.0820(-) 3.5082 0.04 0.03

13 470.2229(+) 3.0051 44.7 0.005

14 308.0307(+) 3.2817 22.6 0.04

15 121.0290(+) 1.6804 21.9 0.02

16 113.0819(+) 0.3239 17.4 0.001

17 148.041(+) 3.1763 0.4 0.002

18 144.0438(+) 1.8838 0.2 0.02

19 399.0439(+) 2.831 0.2 0.0120 431.0959(+) 3.4517 0.04 0.02

Supplementary Table ST3: Unidentified putative markers of diabetes

Supplementary Figure S4. KEGG pathway for tryptophan metabolism. Functional pathway analysis for changes in metabolite levels in diabetes revealed tryptophan metabolism to be one of the major pathways involved with a significant representation of metabolites participating in this pathway. (Green: down-regulated in diabetes; Red: up-regulated in diabetes)

Supplementary Figure S5: OPLSDA plot generated for the nine target metabolites.

(T2DM)(Control)