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Yong-Li Pan, Dec. 28, 2011 ( Biomaterial Chemistry C ourse)

Invasive candidiasis ( 念珠菌病 ). Invasive aspergillosis ( 麴菌病 ). Diagnosis of infectious disease and pathogens based on protein biomarkers of MALDI-TOF mass spectrometry. Discovery/Development of Biomarker panels by Systems Biology.

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Yong-Li Pan, Dec. 28, 2011 ( Biomaterial Chemistry C ourse)

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  1. Invasive candidiasis (念珠菌病) Invasive aspergillosis (麴菌病) Diagnosis of infectious disease and pathogens based on protein biomarkers of MALDI-TOF mass spectrometry Discovery/Development of Biomarker panels by Systems Biology Yong-Li Pan, Dec. 28, 2011(Biomaterial Chemistry Course) Genomics; Functional genomics Proteomics Metabolomics Oligonucleotide array ESI mass spectrometry SERS Raman spectroscopy cDNA microarray MALDI mass spectrometry Normal Raman spectroscopy (Riboflavin, Pigment, etc.) 1

  2. Corneal ulceration Infectious corneal ulcer Noninfectious corneal ulcer A typical presentation of noninfectious corneal ulcer is more peripheral distribution, a clear zone between lesion and adjacent limbus. Infectious corneal ulcer is more centrally located, more discharge and greater vision-threatening than noninfectious. However, sometimes they are difficult to differentiate in several cases without typical presentation.

  3. Species identification of lethal Aspergillus by detecting single conidiospores base on Raman spectroscopy Single spores acquired before the significant sporulation on the whole agar plate Stage II of A. fumigatus Stage I of A. fumigatus 2nd day 1st day On SDA plate, 30~35 ℃ Sporulation

  4. Definition of Biomarkers • Medical or clinical biomarker • FDA: A characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. • Environmental or pollution biomarker • Microorganisms (指標微生物) • For monitoring pollution • Drug target marker • For target therapy (neutralization/blocking by humanized antibody) • With drug releasing or photothermalmateial/device

  5. Molecular types of biomarkers • Proteins: proteomics, mass spectrometry • mRNA expression profiling: cDNAmicroarray, qPCR • Ribosomal DNA or SNP (single nucleotide polymorphisms) • Small molecules: metabonomics

  6. Clinical applicationof biomarker • Screening(CEA,AFP,CA125,PSA) • Diagnosis (Tumor, infectious disease, cardiovascular disease,etc.) • Monitoring treatment • Detection of recurrence • Prognosis • Personalized medicine 個人化醫療(pharmacogenomics and proteomics/molecular diagnostics)

  7. Types of tumor markers • Circulating tumor markers • Enzymes, serum proteins and hormones • Associations with metastases • Prognostic factors • Monoclonal-defined tumor markers • Mucin and blood group substances • Nonspecific tumor markers • Urinary and CSF tumor markers • Cellular tumor marker

  8. a b c d e f g h 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 Raman shift (cm-1) Discovery/development and validation of biomarkers by biosensing technology Cell physiology/dielectrophresis Proteomics/mass spectrometry Biochemical component/Raman spectroscopy

  9. Workflow of developing reliable methods for rapidly screening lethal fungal species based on DNA microarray, Raman spectral and MALDI-TOF mass biosensing technologies Collection of target specimens according to purpose Optimization of rapid and cheap microbial preparation methods Identification by conventional methodsbased on culture for checking (short-term culture) Optimization of rapid microbial detection methods Characterization of different species n=5~8 (3 SD) Establishment of reliable diagnostic criteria Validation of biomarkers Statistical calculation and interpretation of experimental data Discussions Diagnostic performance evaluation Poor diagnostic ability n=46 (3 SD) Excellent diagnostic ability and reliability Application in clinical microbial laboratories

  10. Detecting valid biomarkers by probes immobilized on biosensors

  11. Detection of Tear Proteins Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry The early stage of keratitis Cocrystalization with matrix Isolation/Cultivation Bottom layer method Tear fluid Contact lens solution Soft ionization

  12. Different ion sources for mass spectrometry 表面張力 電場 小分子 • Electro Spray Ionization (ESI) • Easily coupling with other chromatographic and separation technologies (LC-MS) on line • Without sample preparation • Small m/z ratio signal (<1000 m/z) • Matrix-Assisted Laser Desorption Ionization (MALDI-TOF) • Sample preparation with matrix(off line, dried droplet) • Large m/z ratio signal and range (1000~>10000 m/z) Spray 毛細管液體 Ionization ＋ 氧化 (陽極) 多電荷 － 還原 (陰極)

  13. MALDI: Matrix Assisted Laser Desorption Ionization Laser Sample plate 大分子 hn • 1. Sample (A) is mixed with excess matrix (M) and dried • on a MALDI plate. • 2. Laser flash de-ionizes matrix molecules (acid). • 3. Sample molecules are ionized by proton transfer from matrix: • MH+ + A  M + AH+. 固體 電場 AH+ Desorption Cocrystalization Ionization Laser Variable Ground Grid Grid +20 kV Sandler Mass Spectrometry User’s Group, UCSF, US

  14. Time-of-flight mass analyzer 電場加速 無電場 (等速飛行) Ion Source Drift region (flight tube) ESI MALDI + + detector + + V • Ions are formed in pulses. • Small ions reach the detector before large ones. • Measures the time for ions to reach the detector. Sandler Mass Spectrometry User’s Group, UCSF, US

  15. Sample Linear Extraction plate Reflector detector grids Timed ion detector Attenuator selector Reflector Prism Laser Collision cell Camera Pumping Pumping Voyager-DE STR MALDI-TOF MS Sandler Mass Spectrometry User’s Group, UCSF, US

  16. BrukerDaltonics MALDI-TOF mass spectrometry Steel target plate Autoflex, Bruker Autoflex, Bruker Co-crystalization of matrix and protein Desorption and ionization by UV Laser (355 nm ND-YAG Laser)

  17. MALDI TOF MS applied in pathogenic fungi First MALDI TOF MS of filament fungal spores (2000) First soft ionization technique for high mass molecules(1987) Nobel prize in chemistry (Koichi Tanaka, 田中耕一, and J.B. Fenn, 芬恩) History of MALDI TOF MSapplied in medical microbiology First MALDI TOF MS of whole fungal cell (2001) John B. Fenn：將ESI (Electro Spray Ionization) 電灑游離法應用在蛋白質.生化大分子的分析上Koichi Tanaka：以MALDI (Matrix Assisted Laser Desorption Ionization) 基質輔助雷射脫附法來分析生化大分子 Piseth Seng, et. al., Clinical Infectious Diseases 2009; 49:543–51

  18. Application of MALDI TOF MS in rapid detection of bacterial isolation Piseth Seng, et. al., Clinical Infectious Diseases 2009; 49:543–51 Isolation from blood culture Traditional methods MALDI TOF MS

  19. Delays, costs, and Level of training for isolate identification methods Piseth Seng, et. al., Clinical Infectious Diseases 2009; 49:543–51

  20. Basic workflow of mass spectrometry Separated, degraded peptide or lysed microorganisms Ion detection ESI or MALDI TOF-MS or FT-MS Mass spectral pattern Amino acid sequencing http://www.peptide2.com/peptide/Wikipedia_Mass_Spectrometry_files/Ms_block_schematic.gif 20 http://pubs.niaaa.nih.gov/publications/arh26-3/images/israel1.gif

  21. Sample Preparation and Measurement Condition - I 1. Microorganism inactivation Bio-safety Lab 3. Discriminatory ability analysis (cluster analysis and peak comparison) Isolated colonies Wash, centrifuge, freeze drying 100℃ for 5 mins Wet heating inactivation UV Laser (337 nm) Living yeast Dead yeast Safe transportation and experiment Sinapic acid cocrystalization Dried droplet 2. MALDI TOF MS experiment Common Standard Lab Beavis, R. C.; Chait, B. T. Rapid Commun. Mass Spectrom.1989, 3, 432-435. Small amount of pellet Extracted protein Homogenization in 5% formic acid and 35% ACN Disrupt, lyse cells and extract proteins 21

  22. MALDI TOF mass spectral patterns of TFA extracted proteins of Aspergillus species at growth stage I (A) and growth stage II (B) of sporulation (A) (B)

  23. MALDI-TOF MS of living and wet-heating inactivated C. albicans Homogenization with 5% formic acid and 35% ACN ＊: Specific reproducible m/z in living and inactivated yeast cells (more than 2 strains) BCRC 20518 Inactivated yeast cells 2588 * 3398 * 2990 * * 8542 2851 * 4821 6620 6875 13745 8770 4175 13240 6346 1008 11399 7449 9510 Heat-extracted proteins may be removed by washing BCRC 20513 2588 * * 1751 * * * 6624 12848 * * * * * 6474 * 13242 1010 10391 4175 8772 11997 7573 5998 14052 9512 11419 * * BCRC 20519 1779 3399 6626 * * 4524 2632 * * 4176 * 2935 7576 9517 2369 1010 6475 6880 5057 5958 * * Living intact yeast cells 6616 BCRC 20512 * 6470 * * * 6872 * * 8559 8764 * 13262 10380 11988 11412 * * BCRC 21538 * 6879 6476 * * * * 8770 * 8567 * 10391 11997 6624 13275 * 11391 * BCRC 22063 * * 6878 * 6476 * * 8770 * 8566 10390 * 11994 6623 11413 * 13269 * (m/z) 23 23

  24. Strain-to strain reproducibility of Candida albicans (Reference strains from BCRC) ＊: Specific reproducible m/z in more than 2 strains of the same Canddia species (small variation range) * * * BCRC 20518 * * 2588 3398 * 2990 * * * * 8542 2851 4821 6620 * 6875 13745 * 8770 4175 13240 6346 1008 11399 7449 9510 * BCRC 20513 * 2588 * * * 1751 * * * * 6624 * 12848 * * * 6474 13242 1010 10391 4175 8772 11997 7573 11419 5998 14052 9512 * * BCRC 20519 * * * * 1779 3399 6626 * 4524 2632 * * 4176 2935 7576 9517 2369 1010 6475 6880 5057 5958 * * * BCRC 20512 3397 3514 * * 2588 * * * * 4178 * * * * * 2987 4068 6620 * * 8770 2849 10391 11994 5048 12845 14058 6468 8538 5965 6880 1008 24

  25. Strain-to strain reproducibility of Candida albicans (Reference strains from CBS) ＊: reproducible m/z * * CBS 5990 * 2586 * * 3396 * 11417 6621 * * * * 13239 8564 13446 6467 6875 8765 4053 1011 13738 * * * CBS 2730 * 6473 3517 * 1011 6880 * * 3400 11876 * 12013 6207 8574 8774 10398 * * * CBS 6589 * 6624 * * 3516 2587 * 6469 * 3398 13251 * 1011 * * 8781 8565 10394 12003 11416 * * CBS 2718 * 2585 * * * * 6618 * * * * 6466 4065 6871 8764 11984 13235 8538 10375 * 1011 3513 * * CBS 6431 * 2586 * * 6876 * * * * * * 1011 4066 6622 * 3515 11399 11986 13240 8543 6470 10383 8768 25

  26. Strain-to-strain reproducibility of different Candida species ＊: reproducible m/z Candida tropicalis High strain-to-strain reproducibility BCRC 20560 * * * 8569 * 8812 7571 BCRC 21436 * 3492 3019 * 3018 2843 1011 7211 4498 2492 * 2495 3564 1011 6920 * 4503 7213 Candida glabrata CBS 2175 * * * * * 6589 * 3310 3314 1011 2148 10778 5389 * * 6933 * * 10777 * 6585 * * 1011 5843 * 6929 * 4167 5392 8864 8868 10926 2972 10922 2970 6218 4164 7666 7662 4818 9606 2147 6221 11095 5839 9607 11101 CBS 860 * * * * * * * * * * * * * * * 26

  27. Cluster analysis of mass spectral patterns of different strains of Candida species Discrimination to the species level attributed to small variations of both qualitative m/z values and quantitative relative intensities 19 tested samples 27 27

  28. Artificial tears for simulation and human tears/contact lens solution Human tear/contact lens solution from healthy volunteer Artificial ears for simulation Glass capillary tube Lysozyme (chicken egg) + 1 H+ Human lysozyme + 1 H+ 14296 14696 Schirmer test strips Contact lens solution Left eye Lysozyme + 2 H+ 7148 Contact lens solution Right eye Section C(II)_Paper ID : 230 Applications to food industry, environmental monitoring, and healthcare

  29. Tears and contact lens solution from patients with corneal ulcer Immersion liquids of non-washed worn contact lenses (without proteolytic enzyme) Tears collected by glass capillary tube Left eye Patient A Patient D Infected eye * * Abnormal marker Human lysozyme (1 H+) 14678 Human lysozyme (2 H+) 7339 Right eye Patient D * 14680 Patient B Infected eye Human lysozyme (1 H+) 14695 Left eye Patient E * Human lysozyme (2 H+) 14694 7339 * Patient C Infected eye * Right eye Patient E * 14695 Section C(II)_Paper ID : 230 Applications to food industry, environmental monitoring, and healthcare

  30. Common pathogens of patients with infectious corneal ulcer Early stage of cultivation (1-3 days) at 35 oC *Identification marker S. aureus P. aeruginosa TSAculture TSA culture 7582* 9093* 9626* 3876* 6886* 13955* Chocolate agar culture Chocolate agar culture 3881* 9092* 7581* 6887* 9634* 13956* Blood agar culture Blood agar culture 9093* 6887* 7582* 3882* 2311* 9631* 13956* Section C(II)_Paper ID : 230 Applications to food industry, environmental monitoring, and healthcare

  31. SwissProt database search by Mascot software

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