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Analysis of Biomolecules through Nanomaterials Based Mass Spectrometry

Analysis of Biomolecules through Nanomaterials Based Mass Spectrometry. Huan-Tsung Chang. Department of Chemistry National Taiwan University changht@ntu.edu.tw. 07, 15, 2015. Optical. Solar cells. Optical. Catalytic Activity. Fuel cells. Sensors (Abs, Fluor., and SERS ).

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Analysis of Biomolecules through Nanomaterials Based Mass Spectrometry

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  1. Analysis of Biomolecules through Nanomaterials Based Mass Spectrometry Huan-Tsung Chang Department of Chemistry National Taiwan University changht@ntu.edu.tw 07, 15, 2015

  2. Optical Solar cells Optical Catalytic Activity Fuel cells Sensors (Abs, Fluor., and SERS) Energy andEnvironment Analytical Chemistry Decontaminants Biocompatible, Stable, and Low Cost CellImaging Antibacterial Agents Health Drugs

  3. Nanomaterials are used as the matrix in MS. High absorption coefficient Concentration effect More than thousand (at least 10X) molecules per NPs Easy sample preparation Minimum problems associated with sweet spots Large analytes peptides, proteins, ONDs, polysaccharides Small analytes ATP, thiols, melamine, carbohydrates Cell imaging

  4. Mixtures Au Au Au m/z hν hν m/z Au Au Au : Arg : GSH, Cys, and HCys; Gold nanoparticles as a matrix for the quantitation of thiol compounds (A) (B)

  5. 80 (A) 60 R2 = 0.997 40 20 0 0 10 20 30 40 50 60 140 (B) 120 R2 = 0.996 80 40 0 0 10 20 30 40 50 60 70 80 90 160 (C) 120 R2 = 0.995 80 40 0 0 20 40 60 80 100 120 Calibration Curves • [GSH + Na]+: 330.07 (B) [Cys + H]+: 143.98 (C) [HCys + H]+: 173.98 Linear Range Signal Intensity (a. u.) • GSH : 2.5 －50 mM • Hcys : 2.5 －100 mM • Cys : 5.0 －75 mM Limit of Detection • GSH :1.0 mM • Hcys : 2.0 mM • Cys : 1.3 mM Concentration (mM)

  6. 330.07 330.07 346.03 341.26 341.26 Concentration effect 1 (A) 20 μL 1.0 mM GSH 0 1 (B) 0.1 mM GSH Signal Intensity (a. u.) 0 1 (C) 1 mL 0.1 mM GSH LOD: nM level 0 335 340 345 350 325 330 A-B: (NR)Au NPs (3) as matrices without concentration C: 0.1 (NR)Au NPs -- concentration factor of 50 m/z

  7. m/z Detection of ATP using Apt-AuNPs Au Au Au Au ATP AuNP Au Au (NH4)3citrate Au Au Au Au hν : ATP-aptamer : ATP

  8. Determination of ATP and GSH in Red Blood Cells 1.9 (± 0.3) mM (n = 3) Apt-AuNP + AuNP ATP DHB 0.94 (± 0.06) mM (n = 3) Apt-AuNP + AuNP GSH DHB

  9. Improved quantitation of CAP using 4-MBA as an internal standard A drugfor treatment of hypertension CAP: CAP 4-Mercaptobenzoic acid (4-MBA) 4-MBA

  10. [MBA －H]－ Linearity: 2.5–25 μM LOD: 1 uM [CAP －H]－ SALDI Mass spectrum of CAP, MBA and 14-nm Au NPs (7.5 nM) prepared in 0.1 mM ammonium citrate (pH 6.00).

  11. Detection of Proteins through MS using HgTe UV-Vis Energy-dispersive X-ray (EDX) Existence of HgTe Absorbed light over UV-Vis range 5 mM BSA SA as matrix Dominated peak: [M + H]+ Laser shots : 300; laser pulse energy :62.5 μJ (power density :2 × 109 W/cm2). LOD: 14 nM

  12. Mass limit : IgG (m/z ~ 150 kDa) Quasimolecular ions of two identical light chains of IgG HgTe has a low melting temperature and low thermal conductivity. Generation of higher temperature can be achieved for efficient desorption/ionization of the analytes. 150 kDa ions detected 12 IgG (5 mM)

  13. Detection of protein-protein complexes Enhance 6-fold w/o Brij 76 w/ 1% Brij 76 IgG 5 uM a1-antitrypsin w/o Brij 76 protein G 1.7 uM trypsin w/ 0.1% Brij 76 IgG + protein G 5 uM a1-antitrypsin + 1.7 uM trypsin w/ 0.1% Brij 76 a1-antitrypsin: 3 uM (1.5 pmol) Trypsin: 0.5 uM (0.25 pmol) IgG: 5 uM (2.5 pmol) protein G: 2 uM (1 pmol) LOD:

  14. Detection of carbohydrates in vrious honey samples Longan honey Maltose Fructose LODs (from standard solutions) Fructose: 15μM Maltose:10 μM

  15. longan honey 1, (b) litchi honey, (c) osmanthus honey, • (d) longan honey 2, (e) pomelo honey

  16. pomelo honey m/z 650–2700 Da Oligosaccharides?

  17. 3000 365.3 [Sucrose + Na]+ 0 340 400 SALDI mass spectra of sucrose, -CD, -CD, and -CD 8000 995.8 [-CD + Na]+ Signal intensity (a. u.) 0 1030 970 4000 1320.3 [-CD + Na]+ 1158.0 [-CD + Na]+ 996.1 [-CD + Na]+ 0 900 1400 m/z 17 0.5 mM ammonium citrate, pH 7.0 + 0.1 mM NaCl, 1X HgTe

  18. 1000 100 40 50 mM ammonium citrate, pH 9.0 8321 7838 8803 7356 Signal intensity (a. u.) 0.5 mM ammonium citrate, pH 7.0 Signal intensity (a. u.) 9285 5893.9 5406.6 6871 6377.4 0 4919.7 9767 0 8000 4000 6387 6000 12000 m/z 4432.3 m/z 10248 6861.1 10727 7347.5 11211 0 0 7000 8000 6000 9000 10000 11000 12000 6000 7000 8000 5000 4000 Pullulan is a polysaccharide polymer consisting of maltotriose units. 1500 Matrix : 1x HgTe 100 nM PL-6k 10 M PL-6k Laser : 75 J Signal intensity (a. u.) 100 nM PL-10k Laser : 120 J 10 M PL-10k m/z

  19. Polydispersity indices (PDIs) PDI = Mw/Mn Mn: Number-average molecular weights Mn = (NiMi) / Ni Mw: Weight-average molecular weights Mw = (NiMi2) / (NiMi) PL-6k : 1.03 (1.09 was determined from GPC) PL-10k : 1.05 (1.10 was determined from GPC)

  20. Linear range & LODs LR R2 LODs (nM) PL-6k 0.01–5 0.9919 8.5 Intradaya Interdayb PL-10k (Mw 7353) 0.05–10 0.9931 28 Mean (RSD) Mean (RSD) PL-10k (Mw 9765) 0.1–10 0.9924 100 Mw 7353 764 (5.2%) 798 (7.6%) Mw 9765 420 (10.7%) 421 (11.9%) Reproducibility a: n=6 ; b: n=3

  21. Dextran (19500) Signal intensity (a. u.) m/z = 162 m/z ~ 12,000 m/z 50 mM ammonium citrate, pH 9.0, 3x HgTe

  22. Analyses of PEG–Au NPs Through HgTe NMs/SALDI-MS and Monitor Biointeractions  CM-PEG-Au NPs NH2-PEG-Au NPs Biotin-PEG-Au NPs Biotin-PEG-Au NPs avidin (5.0 nM) This method also facilitated the analyses of various functional-PEG–modified Au NPs without the need for any sample pretreatment and monitoring biointeractions .

  23. Functional Microgels Assisted Tryptic Digestion and Quantification of Cytochrome c Through Internal Standard Mass Spectrometry

  24. Tryptic digest of Cyt c (500 nM) microwave irradiation for 15 s.

  25. LOD: nM

  26. Conclusion HgTe are useful for small and large analytes, while AuNPs are only good for small analytes. Internal standard MS methods allow quantitation of analytes. MS using HgTe allows detection of proteins and their complexes with other proteins. MS using HgTe allows determination of MW of polysaccharides.

  27. Thank you for your attention

  28. Detect Proteins and Protein-Protein Complexes • IgG (~150 kDa, pI~5.8) & • protien G (~26 kDa, pI~4.2) • 9.5~12.5 mg/ml • α1-antirypsin (~50 kDa, pI~5) • trypsin (~23 kDa, pI~10.5) - 2 - 4 mg/mL in blood Brij 76 (polyoxyethylene(10) stearyl ether) Anal. Chem.2012, 84, 1924-1930.

  29. Scheme : Microwave-accelerated enzymatic digestion of Cyt c using TR/Au NPs/MGs Preparation and Characterization of Au NPs/MGs

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