1 Proteomics Diagnosis of infectious disease and pathogens based on protein biomarkers of MALDI-TOF mass spectrometry Yong-Li Pan, Dec. 28, 2011 (Biomaterial Chemistry Course) Invasive candidiasis ( 念 念念念 ) Invasive aspergillosis ( 念念念 ) Genomics; Functional genomics Metabolomics Oligonucleotide array cDNA microarray ESI mass spectrometry MALDI mass spectrometry Normal Raman spectroscopy SERS Raman spectroscopy (Riboflavin, Pigment, etc.) Discovery/Development of Biomarker panels by Systems Biology 1
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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. - PowerPoint PPT Presentation
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1
Proteomics
Diagnosis of infectious disease and pathogens based on protein biomarkers of MALDI-TOF mass spectrometryYong-Li Pan, Dec. 28, 2011 (Biomaterial
ESI mass spectrometryMALDI mass spectrometry Normal Raman spectroscopy
SERS Raman spectroscopy
(Riboflavin, Pigment, etc.)
Discovery/Development of Biomarker panels by Systems Biology
1
Corneal ulcerationInfectious corneal ulcer
Infectious corneal ulcer is more centrally located, more discharge and greater vision-threatening than noninfectious.
Noninfectious corneal ulcer
A typical presentation of noninfectious corneal ulcer is more peripheral distribution, a clear zone between lesion and adjacent limbus.
However, sometimes they are difficult to differentiate in several cases without typical presentation.
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Species identification of lethal Aspergillus by detecting single conidiospores base on Raman spectroscopy
Stage I of A. fumigatus Stage II of A. fumigatus
2nd day
On SDA plate, 30~35 ℃Sporulation
1st day
Single spores acquired before the significant sporulation on the whole agar plate
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 photothermal mateial/device 4
cardiovascular disease, etc.)• Monitoring treatment• Detection of recurrence• Prognosis• Personalized medicine 個人化醫療
(pharmacogenomics and proteomics/molecular diagnostics)
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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 marker7
Discovery/development and validation of biomarkers by biosensing technology
Identification by conventional methods based on culture for checking
Workflow of developing reliable methods for rapidly screening lethal fungal species based on DNA microarray, Raman spectral and MALDI-TOF mass biosensing technologies
Establishment of reliable diagnostic criteria
Diagnostic performance evaluation
Optimization of rapid microbial detection methods
Statistical calculation and interpretation of experimental dataDiscussions
Characterization of different species
Optimization of rapid and cheap microbial preparation methods
Collection of target specimens according to purpose
Application in clinical microbial laboratories
Poor diagnostic ability
Excellent diagnostic ability and reliability
(short-term culture)
n=5~8 (3 SD)
n=46 (3 SD)
Validation of biomarkers
Detecting valid biomarkers by probes immobilized on biosensors
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Detection of Tear Proteins Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Drift tube (Vacuum system)
Ion
Det
ecto
r(ti
me
to m
/z)
Acceleration
Coc
ryst
aliz
atio
nof
pr
otei
n an
d m
atrix
+
+
Soft ionization Flight (Constant velocity)
+ + ++m1, v1m2, v2m3, v3
(m3>m2>m1; v1>v2>v3)(Energy: zeV)
Lens
A B
DC
Isolation/CultivationCocrystalization with matrix
Soft ionization
Contact lens solution
Bottom layer methodTear fluidThe early stage of keratitis
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Different ion sources for mass spectrometry
• 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
Nobel prize in chemistry (Koichi Tanaka, 田中耕一 , and J.B. Fenn, 芬恩 )
First soft ionization technique for high mass molecules (1987)
First MALDI TOF MS of filament fungal spores (2000)
MALDI TOF MS applied in pathogenic fungi
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
First MALDI TOF MS of whole fungal cell (2001)
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Application of MALDI TOF MS in rapid detection of bacterial isolation