Large-Scale Metabolite Analysis by Laser Desorption Ionization Mass Spectrometry from Silicon Nanopost Arrays

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1 Supporting Information Large-Scale Metabolite Analysis by Laser Desorption Ionization Mass Spectrometry from Silicon Nanopost Arrays Andrew R. Korte, Sylwia A. Stopka, Nicholas Morris, Trust Razunguzwa, and Akos Vertes * Department of Chemistry, George Washington University, Washington, DC 20052, USA Protea Biosciences, Inc., Morgantown, WV 26505, USA *Corresponding Author vertes@gwu.edu (A. Vertes). Phone: Fax: Address: Department of Chemistry, The George Washington University, nd Street, N.W., Washington, DC 20052, USA. S-1

2 Table of Contents Supplement for EXPERIMENTAL SECTION S-3 Supplementary Figures S-4 to S-8 Supplementary Tables S-9 to S-18 REFERENCES... S-19 IN SEPARATE FILES: Spreadsheet S1. All metabolite ions detected in NAPA-LDI-MS analysis of standard mixtures. Spreadsheet S2. Detected metabolites within the metabolic pathways presented in Figure 3. S-2

3 Supplement for EXPERIMENTAL SECTION Serum extraction and analysis. Human Serum Type AB, Male (Cat. No. H4522 and Lot No. SLBN9196V) was purchased from Sigma-Aldrich (St. Louis, MO). Proteins were precipitated from 100 µl aliquots of the serum by the addition of 400 µl of -20 C methanol. After methanol addition, the samples were vortexed briefly to mix, sonicated for 5 min, and incubated at -20 C for 1 h. They were then centrifuged for 10 min at 14,000 g and 4 C. The supernatant was transferred to a fresh sample tube, and 400 µl of -20 C chloroform and 100 µl of 4 C water were added to each sample, inducing separation into organic and aqueous phases. The samples were then briefly vortexed and centrifuged for 10 min at 14,000 g and 4 C. The organic and aqueous phases from each sample were removed to separate sample tubes and dried in a vacuum centrifuge at 4 C, then stored at -80 C until use. Aqueous extracts were reconstituted in 100 µl of water and 0.5 µl was spotted onto each nanopost array. Organic extracts were reconstituted in 10 µl of 1:1 acetone:water and 0.2 µl was spotted onto each nanopost array. All extract samples were analyzed using a fluence of 100 mj/cm 2, three laser shots per scan, and one scan per raster point. The full area of each array was sampled at a raster pitch of 100 µm. Spectra were acquired in the orbitrap analyzer using a resolving power setting of 30,000, and m/z ranges of for aqueous extracts and for organic extracts. For standard addition experiments in which serum samples were spiked with known concentrations of metabolites, standards of glucose or an amino acid mixture (arginine, phenylalanine, and proline) were prepared in water and diluted to a range of concentrations. Concentrations of the spike solutions were selected such that final serum concentrations of the metabolites were ~1 (unspiked), ~1.5, ~2, ~3, ~5, and ~10 the reported natural concentrations 1. Prior to protein precipitation, 10 µl of the standard solution was added to the raw serum and samples were processed as above. S-3

4 Figure S1. Mass spectra obtained from NAPA-LDI-MS of (a-h) 3,5 -cyclic adenosine monophosphate (camp) and (i-p) acetylcholine at a range of laser fluences. Intact metabolite ions ([M] + for acetylcholine and [M H] for camp) are denoted by ( ), whereas ( ) denotes fragment ions. camp and acetylcholine spectra are normalized to absolute intensities of au and au, respectively. S-4

5 Figure S2. (a) MS spectrum of camp standard at 100 mj/cm 2 fluence showing structure specific fragmentation (SF) to adenine ion. (b) MS/MS spectrum of adenine generated by structure specific fragmentation of camp. (c) MS 3 spectrum of adenine generated by CID fragmentation of camp. S-5

6 Figure S3. Positive ion mode NAPA-LDI-MS spectra of L-amino acid standards. S-6

7 Figure S4. Negative ion mode NAPA-LDI-MS spectra of L-amino acids standards. S-7

8 Figure S5. Summed absolute ion intensities for (a) glucose, (b) proline, (c) phenylalanine, and (d) arginine ions as a function of serum spike concentration. Glucose signal includes [M+Na] + and [M+K] + species and amino acid signals include [M+2Na H] +, [M+Na+K-H] +, and [M+2Na-H] + species. Glucose-spiked standards were spiked with glucose in water, while amino acid-spiked standards were spiked with a mixture of proline, phenylalanine, and arginine in water. R 2 coefficients are 0.97 for glucose in glucose-spiked serum and >0.99 for the three amino acids in amino-acid spiked serum. S-8

9 Table S1. Identified ions from positive ion mode analysis of individual L-amino acid standards Amino acid m/z ID Composition Error (mda) Glycine [M+2Na H] + C 2 H 4 O 2 NNa Alanine [M+2Na H] + C 3 H 6 O 2 NNa Serine [M+Na] + C 3 H 7 O 3 NNa [M+2Na H] + C 3 H 6 O 3 NNa Proline [M+H] + C 5 H 10 O 2 N [M+Na] + C 5 H 9 O 2 NNa [M+2Na H] + C 5 H 8 O 2 NNa Valine [M+Na] + C 5 H 11 O 2 NNa [M+2Na H] + C 5 H 10 O 2 NNa Threonine [M+H] + C 4 H 10 O 3 N [M+Na] + C 4 H 9 O 3 NNa [M+2Na H] + C 4 H 8 O 3 NNa Cysteine [M+H] + C 3 H 8 O 2 NS 0.2 Isoleucine [M+H] + C 6 H 14 O 2 N [M+Na] + C 6 H 13 O 2 NNa [M+2Na H] + C 6 H 12 O 2 NNa Leucine [M+H] + C 6 H 14 O 2 N [M+Na] + C 6 H 13 O 2 NNa [M+2Na H] + C 6 H 12 O 2 NNa Asparagine [M+H] + C 4 H 9 O 3 N [M+Na] + C 4 H 8 O 3 N 2 Na [M+2Na H] + C 4 H 7 O 3 N 2 Na Aspartic acid [M+H] + C 4 H 8 O 4 N [M+Na] + C 4 H 7 O 4 NNa [M+2Na H] + C 4 H 6 O 4 NNa Glutamine [M+H NH 3 ] + C 5 H 8 O 3 N [M+H] + C 5 H 11 O 3 N [M+Na H 2 O] + C 5 H 8 O 2 N 2 Na [M+Na] + C 5 H 10 O 3 N 2 Na [M+2Na H] + C 5 H 9 O 3 N 2 Na Lysine [M+H NH 3 ] + C 6 H 12 O 2 N [M+H] + C 6 H 15 O 2 N [M+Na] + C 6 H 14 O 2 N 2 Na 0.6 Glutamic acid [M+H H 2 O] + C 5 H 8 O 3 N [M+H] + C 5 H 10 O 4 N [M+Na] + C 5 H 9 O 4 NNa [M+2Na H] + C 5 H 8 O 4 NNa S-9

10 Methionine [M+H NH 3 ] + C 5 H 9 O 2 S [M+H] + C 5 H 12 O 2 NS [M+Na] + C 5 H 11 O 2 NNaS [M+2Na H] + C 5 H 10 O 2 NNa 2 S 0.5 Histidine [M+H H 2 CO 2 ] + C 5 H 8 N [M+H] + C 6 H 10 O 2 N Phenylalanine [M+H H 2 CO 2 ] + C 8 H 10 N [M+H] + C 9 H 12 O 2 N [M+Na] + C 9 H 11 O 2 NNa [M+2Na H] + C 9 H 10 O 2 NNa Arginine [M+H CH 3 NO] + C 5 H 12 ON [M+H NH 3 ] + C 6 H 12 O 2 N [M+H] + C 6 H 15 O 2 N Tyrosine [M+H] + C 9 H 12 O 3 N [M+Na] + C 9 H 11 O 3 NNa [M+2Na H] + C 9 H 10 O 3 NNa Tryptophan [M+H C 2 H 5 NO 2 ] + C 9 H 8 N [M+H CH 2 O 2 ] + C 10 H 11 N [M+H NH 3 ] + C 11 H 10 O 2 N [M+H] + C 11 H 13 O 2 N [M+Na] + C 11 H 12 O 2 N 2 Na [M+2Na H] + C 11 H 11 O 2 N 2 Na S-10

11 Table S2. Identified ions from negative ion mode analysis of individual L-amino acid standards Amino acid m/z ID Composition Error (mda) Glycine [2M+Na 2H] C 4 H 8 O 4 N 2 Na -0.1 Alanine [2M+Na 2H] C 6 H 12 O 4 N 2 Na 0.5 Serine [2M+Na 2H] C 6 H 12 O 6 N 2 Na 1.4 Proline [M H] C 5 H 8 O 2 N [2M+Na 2H] C 10 H 16 O 4 N 2 Na 2.0 Valine [M H] C 5 H 10 O 2 N -0.3 Threonine [M H] C 4 H 8 O 3 N -0.2 Cysteine Isoleucine [M H] C 6 H 12 O 2 N -0.2 Leucine [M H] C 6 H 12 O 2 N -0.2 Asparagine [M H] C 4 H 7 O 3 N Aspartic acid [M H] C 4 H 6 O 4 N 0.1 Glutamine [M H] C 5 H 7 O 2 N [M H] C 5 H 9 O 3 N Lysine [M H] C 6 H 13 O 2 N Glutamic acid [M H] C 5 H 6 O 3 N [M H] C 5 H 8 O 4 N 0.1 Methionine [M H] C 5 H 10 O 2 NS -0.1 Histidine [M H] C 6 H 8 O 2 N Phenylalanine [M H NH 3 ] C 9 H 7 O [M H] C 9 H 10 O 2 N -0.3 Arginine [M H CH 2 N 2 ] C 5 H 11 O 2 N [M H NH 3 ] C 6 H 10 O 2 N [M H] C 6 H 13 O 2 N Tyrosine [M H] C 9 H 10 O 3 N [M 2H+Na] C 9 H 9 O 3 NNa -0.3 Tryptophan [M H C 3 H 5 NO 2 ] C 8 H 6 N [M H C 2 H 3 N] C 9 H 8 O 2 N [M H] C 11 H 11 O 2 N S-11

12 Table S3. Tentatively assigned ionic species detected in positive ion mode NAPA-LDI-MS analysis of aqueous phase serum extracts. m/z Assignment Ion Error (mda) Betaine [M+H] Phosphoric acid [M+Na] Formic acid [M+2K H] Methionine [M+H H 2 O] Pyruvic acid [M+2Na H] Alanine [M+2Na H] Lactic acid [M+2Na H] Creatinine [M+Na] Phosphoric acid [M+K] Phosphoric acid [M+2Na H] ,4-dihydroxybutyric acid [M+Na] Acetoacetic acid [M+2Na H] Pyruvic acid [M+Na+K H] hydroxybutanoic acid [M+2Na H] Creatinine [M+K] Creatinine [M+2Na H] Proline [M+2Na H] N-Acetylglycine [M+2Na H] Valine [M+2Na H] Pyruvic acid [M+2K H] Alanine [M+2K H] Lactic acid [M+2K H] Pyroglutamic acid [M+2Na H] Phosphoric acid [M+2K H] Citraconic acid [M+2Na H] Citric acid [M+H H 2 O] Trans-4-hydroxyproline [M+2Na H] Creatine [M+2Na H] Leucine/isoleucine [M+2Na H] Ornithine [M+2Na H] Salicylic acid [M+2Na H] Glutamine [M+2Na H] Lysine [M+2Na H] Proline [M+2K H] Valine [M+2K H] Histidine [M+2Na H] Hexose [M+Na] Pyroglutamic acid [M+2K H] Trans-4-hydroxyproline [M+2K H] S-12

13 Leucine/isoleucine [M+2K H] Phenylalanine [M+2Na H] Hexose [M+K] Arginine [M+2Na H] Glutamine [M+2K H] Tyrosine [M+2Na H] N-alpha-acetyl-L-lysine [M+2Na H] Phenylalanine [M+2K H] Tryptophan [M+2Na H] Arginine [M+2K H] N-alpha-acetyl-L-lysine [M+2K H] Myristic acid [M+2Na H] Palmitic acid [M+Na] Palmitelaidic acid [M+2Na H] Palmitic acid [M+2Na H] Linoleic acid [M+Na] Oleic acid [M+Na] Linoleic acid [M+2Na H] Oleic acid [M+2Na H] Stearic acid [M+2Na H] Maltose [M+2Na H] S-13

14 Table S4. Tentatively assigned ionic species detected in negative ion mode NAPA-LDI-MS analysis of aqueous phase serum extracts. m/z Assignment Ion Error (mda) Benzoic acid [M H] Glutamine [M H H 2 O] Lysine [M H H 2 O] Pyroglutamic acid [M H] Creatine [M H] Leucine/isoleucine [M H] Salicylic acid [M H] Glutamine [M H] Phenylalanine [M H H 2 O] Acetaminophen [M H] Histidine [M H] Hexose [M H H 2 O] Tyrosine [M H H 2 O] Phenylalanine [M H] Leucine/isoleucine [M 2H+K] N-alpha-acetyl-L-lysine [M H H 2 O] Tyrosine [M H] Tryptophan [M H H 2 O] Uric acid [M 2H+Na] Tyrosine [M 2H+Na] Tryptophan [M H] Capric acid [M 2H+K] Myristic acid [M H] Dodecanoic acid [M 2H+K] Palmitelaidic acid [M H] Palmitic acid [M H] Heptadecanoic acid [M H] Linoleic acid [M H] Oleic acid [M H] Stearic acid [M H] -0.3 S-14

15 Table S5. Tentatively assigned ionic species detected in positive ion mode NAPA-LDI-MS analysis of organic phase serum extracts. CPA: cyclophosphatidic acid; LPA: lysophosphatidic acid; PA: phosphatidic acid; LPC: lysophosphatidylcholine; PC: phosphatidylcholine; LPE: lysophosphatidylethanolamine; PE: phosphatidylethanolamine; LPG: lysophosphatidylglycerol; DG: diacylglycerol; SM: sphingomyelin. m/z Assignment Ion Error (mda) Glycerophosphocholine [M+H] Palmitic acid [M+2Na H] Dihydroxyeicosanoic acid [M+Na] Cholesterol [M+H H 2 O] CPA (16:0) [M+Na] CPA (18:1) [M+H] CPA (16:0) [M+2Na H] CPA (18:2) [M+Na] CPA (18:1) [M+Na] CPA (18:0) [M+Na] LPA (18:0e) [M+Na] LPA (16:0) [M+2Na H] LPA (18:2) [M+Na] LPA (18:1) [M+Na] CPA (18:2) [M+2Na H] CPA (18:1) [M+2Na H] LPA (18:0e) [M+2Na H] LPA (18:0) [M+2Na H] LPC (P-16:0) [M+Na] LPC (P-18:1) [M+H] LPE (20:0) [M+H] LPC (16:0) [M+Na] LPC (18:0) [M+H] LPC (18:2) [M+Na] LPC (18:1) [M+Na] LPC (18:0) [M+Na] LPG (18:0) [M+2Na H] DG (34:3) [M+Na] DG (36:5) [M+Na] DG (36:4) [M+Na] DG (38:5) [M+Na] PA (36:2) [M+Na] SM (d34:1) [M+Na] PA (36:2) [M+2Na H] PE (38:2) [M+H] PE (38:1) [M+H] S-15

16 PC (34:1) [M+Na] PC (34:1) [M+Na] PE (40:2) [M+H] PC (34:1) [M+2Na H] PC (36:3) [M+Na] PC (36:2) [M+Na] PC (38:4) [M+Na] S-16

17 Table S6. Tentatively assigned ionic species detected in negative ion mode NAPA-LDI-MS analysis of organic phase serum extracts. LPA: lysophosphatidic acid; PA: phosphatidic acid; LPE: lysophosphatidylethanolamine; PE: phosphatidylethanolamine; LPI: lysophosphatidylinositol; PI: phosphatidylinositol; CerP: Ceramide phosphate; ST: sulfatide; PG: phosphatidylglycerol. m/z Assignment Ion Error (mda) Azelaic acid [M+Na 2H] Dodecanedioic acid [M H H 2 O] Myristic acid [M H] Inositol phosphate [M H H 2 O] Palmitelaidic acid [M H] Palmitic acid [M H] Inositol phosphate [M H] Heptadecanoic acid [M H] Linoleic acid [M H] Oleic acid [M H] Stearic acid [M H] Hydroxyoctadecadienoic acid [M H] Arachidonic acid [M H] Eicosatrienoic acid [M H] Eicosatrienoic acid [M+Na 2H] Docosapentaenoic acid [M H] Dehydroepiandrosterone sulfate [M H] Androsterone sulfate [M H] Tetracosahexaenoic acid [M+Na 2H] Dihydroxyandrostenone sulfate [M H] LPA (16:0) [M H H 2 O] LPA (16:0) [M H] LPA (18:1) [M H H 2 O] LPA (18:0) [M H H 2 O] LPA (18:1) [M H] LPA (18:0) [M -H] LPA (18:0) [M+Na 2H] Cholesterol sulfate [M H] LPE (18:0) [M H] LPE (20:0) [M H] LPI (18:0) [M H H 2 O] CerP (34:1) [M H H 2 O] LPI (18:0) [M H] CerP (34:1) [M H] CerP (36:1) [M H] PA (34:2) [M H] PA (34:1) [M H] 2.3 S-17

18 PA (36:4) [M H] PA (36:2) [M H] PA (36:2) [M+Na 2H] CerP (42:1) [M H] PE (36:2) [M H] PE (36:1) [M H] PE (38:4) [M H] PE (38:3) [M H] PE (38:2) [M H] ST (34:1) [M H] PE (42:9) [M H H 2 O] PG (36:2) [M+Na 2H] PI (38:4) [M H] 0.5 S-18

19 REFERENCES (1) Psychogios, N.; Hau, D. D.; Peng, J.; Guo, A. C.; Mandal, R.; Bouatra, S.; Sinelnikov, I.; Krishnamurthy, R.; Eisner, R.; Gautam, B.; Young, N.; Xia, J. G.; Knox, C.; Dong, E.; Huang, P.; Hollander, Z.; Pedersen, T. L.; Smith, S. R.; Bamforth, F.; Greiner, R.; McManus, B.; Newman, J. W.; Goodfriend, T.; Wishart, D. S. Plos One 2011, 6, e S-19

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