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

1

Proteomics

Diagnosis of infectious disease and pathogens based on protein biomarkers of MALDI-TOF mass spectrometryYong-Li Pan, Dec. 28, 2011 (Biomaterial

Chemistry Course)Invasive candidiasis (念珠菌病 ) Invasive aspergillosis (麴菌病

)

Genomics; Functional genomics Metabolomics

0.0

0.5

1.0

1.54x10

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 16000 18000m/z

0.00

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0.50

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1.00

1.254x10

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 16000 18000m/z

Oligonucleotide arraycDNA microarray

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.

3

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

Molecular types of biomarkers

• Proteins: proteomics, mass spectrometry

• mRNA expression profiling: cDNA microarray, qPCR

• Ribosomal DNA or SNP (single nucleotide polymorphisms)

• Small molecules: metabonomics

5

Clinical application of 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)

6

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

8

ʳ * A16-1800a\0_A16\1\1SLin, "Baseline subt."

0

2

4

6

4x10

Intens. [a.u.]

ʳ

* C18-1800a\0_C18\1\1SLin, "Baseline subt."

0

2

4

6

84x10

Intens. [a.u.]

ʳ

* 7-P aeruginosa 10944-blood culture\0_B7\1\1SLin, "Baseline subt."

0

1

2

3

4x10

Intens. [a.u.]

5000 10000 15000 20000 25000 30000 35000m/z

600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800

Raman shift (cm-1)

abcdefgh

Proteomics/mass spectrometry

Biochemical component/Raman spectroscopy

Cell physiology/dielectrophresis

9

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

10

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

12

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

(1000~>10000 m/z)

氧化

還原

小分子電場

多電荷

表面張力

－

＋ Ionization

Spray

毛細管液體

(陽極 )

(陰極 )

13

MALDI: Matrix Assisted Laser Desorption Ionization

hn

Laser

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+.

AH+

+20 kV

Variable Ground Grid Grid

Sample plate

Sandler Mass Spectrometry User’s Group, UCSF, US

大分子

LaserIonization

Desorption

電場

Cocrystalization

固體

14

Time-of-flight mass analyzer

+

+

+

+

Ion SourceDrift region (flight tube)

dete

ctor

V

• Ions are formed in pulses.• Small ions reach the detector before large ones.• Measures the time for ions to reach the detector.

zeV2

mv 2

zeV2

mv 2


ESIMALDI

無電場 ( 等速飛行 )電場加速

15

Voyager-DE STR MALDI-TOF MS

Camera

Laser

Sample plate

Pumping Pumping

Timed ion selector Reflector

Linear detector

Extractiongrids

ReflectordetectorAttenuator

Prism

Collision cell


16

Bruker Daltonics MALDI-TOF mass spectrometry

Desorption and ionization by UV Laser (355 nm ND-YAG Laser)

Co-crystalization of matrix and protein

Steel target plateAutoflex, Bruker

Autoflex, Bruker

17

History of MALDI TOF MS applied in medical microbiology

Piseth Seng, et. al., Clinical Infectious Diseases 2009; 49:543–51

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)

18

Application of MALDI TOF MS in rapid detection of bacterial isolation

Traditional methods

MALDI TOF MS

Isolation from blood culturePiseth Seng, et. al., Clinical Infectious Diseases 2009; 49:543–51

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

http://pubs.niaaa.nih.gov/publications/arh26-3/images/israel1.gif

Separated, degraded peptide or lysed microorganisms

ESI or MALDI

TOF-MS or FT-MS

Mass spectral patternAmino acid sequencing

Ion detection

ʳ * Candida parapsilosis_BCRC-21544\0_K5\1\1Lin, "Baseline subt."

0200040006000

Intens. [

a.u.]

ʳ

* Candida lusitaniae_CBS-7270\0_C18\1\1Lin, "Baseline subt."

0200040006000

Intens. [

a.u.]

ʳ

* Candida glabrata_CBS-2175\0_C20\1\1Lin, "Baseline subt."

0

2000

4000

Intens. [

a.u.]

ʳ

* Candida krusei_BCRC-21720\0_C16\1\1Lin, "Baseline subt."

0200040006000

Intens. [

a.u.]

ʳ

* Candida albicans_BCRC-20519\0_C3\1\1Lin, "Baseline subt."

0

2000

4000

Intens. [

a.u.]

ʳ

* Candida tropicalis_BCRC-560\0_A14\1\1Lin, "Baseline subt."

0200040006000

Intens. [

a.u.]

5000 10000 15000 20000 25000 30000m/z

20http://www.peptide2.com/peptide/Wikipedia_Mass_Spectrometry_files/Ms_block_schematic.gif

21

ʳ * Candida parapsilosis_BCRC-21544\0_K5\1\1Lin, "Baseline subt."

0200040006000

Inten

s. [a.

u.]ʳ

* Candida lusitaniae_CBS-7270\0_C18\1\1Lin, "Baseline subt."

0200040006000

Inten

s. [a.

u.]

ʳ

* Candida glabrata_CBS-2175\0_C20\1\1Lin, "Baseline subt."

0

2000

4000

Inten

s. [a.

u.]

ʳ

* Candida krusei_BCRC-21720\0_C16\1\1Lin, "Baseline subt."

0200040006000

Inten

s. [a.

u.]

ʳ

* Candida albicans_BCRC-20519\0_C3\1\1Lin, "Baseline subt."

0

2000

4000

Inten

s. [a.

u.]

ʳ

* Candida tropicalis_BCRC-560\0_A14\1\1Lin, "Baseline subt."

0200040006000

Inten

s. [a.

u.]

5000 10000 15000 20000 25000 30000m/z

Homogenization in 5% formic acid and 35% ACN

Disrupt, lyse cells and extract proteins

100 for 5 mins℃Wet heating inactivation

Bio-safety LabWash, centrifuge,

freeze drying

Dried dropletSinapic acid cocrystalization

UV Laser (337 nm)

Beavis, R. C.; Chait, B. T. Rapid Commun. Mass Spectrom. 1989, 3, 432-435.

Common Standard Lab

3. Discriminatory ability analysis(cluster analysis and peak comparison)

Safe transportation and experiment

21

2. MALDI TOF MS experiment

1. Microorganism inactivation

Living yeast Dead yeast

Extracted proteinSmall amount of pellet

Isolated colonies

Sample Preparation and Measurement Condition - I

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)

0

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0.000.25

0.500.75

1.001.25

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Inten

s. [a.

u.]

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0

1000

2000

3000

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5000

Inten

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2000

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4000

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s. [a.

u.]

2000 4000 6000 8000 10000 12000 14000 16000 18000m/z

(a)

(b)

(c)

(d)

(e)

A. fumigatus (BCRC 32149)

A. niger (BCRC 30201)

A. flavus (BCRC 30006)

A. terreus (BCRC 30135)

A. versicolor (BCRC 31488T)

8322

5787

5818

5900

11028

(5942)

7925(8321)

2187

5941 (8335)2836

1565

4146 5773

(11190)93676927

(5798)

(8340)

(1565)

7219

136361116794306967

(1565)

5968

(1565)

8338

m/z

Inte

nsity

(a.

u.)

2000 4000 6000 8000 10000 12000 16000140000

1000200030004000

0

500

10001500

2000

x104 4000 6000 8000 10000 12000 14000 160002000

4000 6000 8000 10000 12000 14000 160002000

4000 6000 8000 10000 12000 14000 160002000

4000 6000 8000 10000 12000 14000 160002000

0.000.25

0.750.50

1.001.25

10002000

0

300040005000

10002000

0

300040005000

m/z

x104

0.0

0.5

1.0

1.54x10

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 16000 18000m/z

0.0

0.5

1.0

1.5

4x10

Inten

s. [a.u

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0.00

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0.50

0.75

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1.254x10

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s. [a.u

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0.0

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0500

10001500200025003000

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 16000 18000m/z

(a)

(b)

(c)

(d)

(e)

A. fumigatus (BCRC 32149) 14226

A. niger (BCRC 3201)

A. flavus (BCRC 30006)

A. terreus (BCRC 30135)

A. versicolor (BCRC 31488T)

12250

2836

5943 11259

10287 11942

13630

4143(4513)

7897

(5944)10835

672214402

7091

(14251)

1565

8232

4423 5974 6816(10545)

(5940)

96103950

11216

m/z4000 6000 8000 10000 12000 14000 16000 18000

2000

02000

50010001500

25003000

x104

x104

x104

4000 6000 8000 10000 12000 14000 16000 180002000

4000 6000 8000 10000 12000 14000 16000 180002000

4000 6000 8000 10000 12000 14000 16000 180002000

4000 6000 8000 10000 12000 14000 16000 180002000

0.000.250.50

1.001.25

0.75

0.0

0.5

1.5

1.0

0.0

0.5

1.5

1.0

0.00.20.40.6

0.8

m/z

2323

MALDI-TOF MS of living and wet-heating inactivated C. albicans

02000400060008000

Inten

s. [a.u

.]2000 4000 6000 8000 10000 12000 14000 m/z

02000400060008000

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 m/z

0

2000

4000

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 m/z

0.000.250.500.75

4x10

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 m/z

BCRC 20512

0200040006000

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 m/z

BCRC 21538

02000400060008000

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 m/z

BCRC 22063

6470

13262119881141210380876485596872

6616

1327587708567

6476 6879

6624 1199710391 11391

6476

6623

6878

1326987708566

1199410390 11413

BCRC 20513

13242

2588

66241010 4175 10391

662645241010

2632BCRC 20519

41766475

5057 5958

29351779

2369

11997877275736474

1751

BCRC 20518

13240

2588

6875662048211008

28514175 6346

2990 33988542

87701139995107449

140521141995125998

3399

951775766880

*** *

* *

*

13745

12848

*

*

*

*

(m/z)

Homogenization with 5% formic acid and 35% ACN

Living intact yeast cells

Inactivated yeast cells

*

*

*

*

*

*

*

*

*

*

*

*

*

*

**

*

*

*

*

*

*

*

*

*

*

*

**

*

*

*

*

*

*

*

*

*

*

*

*

＊ : Specific reproducible m/z in living and inactivated yeast cells (more than 2 strains)

23

Heat-extracted proteins may be removed by washing

2424

Strain-to strain reproducibility of Candida albicans (Reference strains from BCRC)

02000400060008000

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 m/z

BCRC 20513

13242

2588

66241010 4175 10391

0

2000

4000

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 m/z

662645241010

2632BCRC 20519

0.00.51.0

4x10

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 m/z

BCRC 20512

1008

2588

6620

41766475

5057 5958

29351779

2369

10391 11994 12845 140588770

853864686880

40682987

3514

11997877275736474

1751

02000400060008000

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 m/z

BCRC 20518

13240

2588

6875662048211008

28514175 6346

2990 33988542

87701139995107449

140521141995125998

3399

59655048

41783397

2849

951775766880

*

*

*

*

*

** *

* **

*

*

13745

12848

＊ : Specific reproducible m/z in more than 2 strains of the same Canddia species (small variation range)

* *

*

**

*

*

*

*

**

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

**

*

**

*

*

*

* *

2525

0.0

0.5

1.04x10

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 m/z

0

2000

4000

Inten

s. [a.u

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2000 4000 6000 8000 10000 12000 14000 m/z

0

2000

4000

Inten

s. [a.u

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2000 4000 6000 8000 10000 12000 14000 m/z

0.0

0.5

1.04x10

Inten

s. [a.u

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2000 4000 6000 8000 10000 12000 14000 m/z

CBS 5990

13239

01000200030004000

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 m/z

CBS 27306880

CBS 6589

CBS 2718

CBS 6431

13240

687625861011 3515 6622

8543 10383

1011

2585

35134065

66186871

2586

6621

68751011

3396

6467

11876 1201310398

6473

6207

35171011 34008574 8774

13251

120031141610394

662464692587

1011 878185653398

3516*

*

*

*

*

*

*

*

*

*

*

**

*

**

*

*

*

**

*

*

*

*

**

*

1344613738

11417

876585644053

11986113994066

64668538 8764 10375 11984 13235

6470 8768

Strain-to strain reproducibility of Candida albicans (Reference strains from CBS)

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

* **

*

*

*

*

*

*

＊ : reproducible m/z

2626

Strain-to-strain reproducibility of different Candida speciesCandida tropicalis

Candida glabrata

BCRC 20560

0100020003000

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 m/z

BCRC 21436

7213

69201011

28433564

30182495

4503

0200040006000

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 m/z

1011 72113019 3492

44982492

020004000

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 m/z

CBS 2175

10116589

69338868

9606

10778

2972

3314

4167 5392 7666 1092611095

2148

6221

5843

0200040006000

Inten

s. [a.u

.]

2000 4000 6000 8000 10000 12000 14000 m/z

10777

9607

886476626929

65855389

48181011

2147 2970 41645839

6218

3310

10922

11101

CBS 860

High strain-to-strain reproducibility

*

*

*

*

*

*

*

*

*

**

*

**

* *

*

**

**

*

*

**

*

*

* *

*

*

*

*

*

*

* *

*

＊ : reproducible m/z

7571

85698812

27270 50 100 150 200

0

2

4

6

8

10

12

14

16

18

20

Variance Weighted Distance Between Cluster Centers

Candida tropicalis_BCRC-21436

Candida tropicalis_BCRC-20560

Candida albicans_BCRC-20512

Candida albicans_CBS-2718








Candida krusei_BCRC-21321

Candida krusei_BCRC-21720

Candida glabrata_CBS-860

Candida glabrata_CBS-2175

Candida lusitaniae_BCRC-20326

Candida lusitaniae_CBS-7270

Candida parapsilosis_BCRC-21253

Candida parapsilosis_BCRC-21544

Dendrogram of Data with Preprocessing: 2nd Derivative (order: 2, window: 15 pt) + Normalize (1-Norm, Area = 1) + Autoscale

Cluster analysis of mass spectral patterns of different strains of Candida species

19 tested samples 27

Discrimination to the species level attributed to small variations of both qualitative m/z values and quantitative relative intensities

ʳ * tear tube 32x after 1d-L12-1800shots-200kDa\0_L12\1\1SLin, "Baseline subt."

0

1

25x10

Inte

ns. [

a.u.

]

ʳ

* tear from paper 4x 1800 shots\0_I20\1\1SLin, "Baseline subt."

0.0

0.5

1.05x10

Inte

ns. [

a.u.

]

ʳ

* Jung Hung Wang N20-OS-1800a\0_N20\1\1SLin, "Baseline subt."

0

1

4x10

Inte

ns. [

a.u.

]

ʳ

* Jung Hung Wang N21-OD-1800a\0_N21\1\1SLin, "Baseline subt."

0.0

0.5

1.0

4x10

Inte

ns. [

a.u.

]

2000 4000 6000 8000 10000 12000 14000 16000m/z

ʳ * tear 1x with SA-std protocol-two layer dried 52 per power-3300 adding\0_A8\1\1SLin, "Basel...

0

1

2

3

4

4x10

Inte

ns. [

a.u.

]

2000 4000 6000 8000 10000 12000 14000 16000 18000m/z

Artificial tears for simulation and human tears/contact lens solution

Artificial ears for simulationHuman tear/contact lens solution from

healthy volunteer

Section C(II)_Paper ID : 230 Applications to food industry, environmental monitoring, and healthcare

Left eye

Right eye

Schirmer test strips

Glass capillary tube



14296

7148

Lysozyme (chicken egg) + 1 H+

Lysozyme + 2 H+

Human lysozyme + 1 H+

14696

Tears and contact lens solution from patients with corneal ulcer

ʳ * corneal ulcer sample 4x-C3-1800\0_C3\1\1SLin, "Baseline subt."

0.0

0.5

1.0

1.55x10

Inte

ns. [

a.u.

]

ʳ

* 6314968-OD-T82-1800a-16x\0_O5\1\1SLin, "Baseline subt."

0

2

4

64x10

Inte

ns. [

a.u.

]

ʳ

* 60564348-OS-T81-1800a-2x\0_P20\2\1SLin, "Baseline subt."

0

2

4

6

84x10

Inte

ns. [

a.u.

]

2000 4000 6000 8000 10000 12000 14000 16000 18000m/z

Tears collected by glass capillary tube Immersion liquids of non-washed worn contact lenses (without proteolytic enzyme)


ʳ * C17-CL 002-left-2ul\0_C17\1\1SLin, "Baseline subt."

0.0

0.5

1.0

4x10

Intens. [a.u.]

ʳ

* C18-CLB 002-right-2ul\0_C18\1\1SLin, "Baseline subt."

0.00.5

1.0

1.5

4x10

Intens. [a.u.]

ʳ

* G14-CLB 001-left 2ul\0_G14\1\1SLin, "Baseline subt."

0.0

0.5

5x10

Intens. [a.u.]

ʳ

* E15-CLB 001-right 2ul-after cali\0_E15\1\1SLin, "Baseline subt."

0

2

4

4x10

Intens. [a.u.]

4000 6000 8000 10000 12000 14000 16000 18000m/z

Left eye

Right eye

Left eye

Right eye

Patient A

Patient B

Patient C

Patient D

Patient D

Patient E

Patient E

Infected eye

Infected eye

Infected eye

14678

7339

Human lysozyme (1 H+)


14695

7339



Abnormal marker*

*

*

*

*

*

*

14695

14694

14680

ʳ * A16-1800a\0_A16\1\1SLin, "Baseline subt."

0

2

4

6

4x10

Intens. [a.u.]

ʳ

* C18-1800a\0_C18\1\1SLin, "Baseline subt."

0

2

4

6

84x10

Intens. [a.u.]

ʳ

* 7-P aeruginosa 10944-blood culture\0_B7\1\1SLin, "Baseline subt."

0

1

2

3

4x10Intens. [a.u.]

5000 10000 15000 20000 25000 30000 35000m/z

ʳ * 4-S aureus 14957-TSA\0_B4\1\1SLin, "Baseline subt."

0.0

0.5

1.0

4x10

Intens. [a.u.]

ʳ

* 5-S aureus 14957-chocolate\0_B5\1\1SLin, "Baseline subt."

0.0

0.5

1.0

5x10

Intens. [a.u.]

ʳ

* 6-S aureus 14957-blood culture\0_B6\1\1SLin, "Baseline subt."

0

2

4

6

4x10

Intens. [a.u.]

4000 6000 8000 10000 12000 14000 16000 18000 20000m/z

Common pathogens of patients with infectious corneal ulcer


P. aeruginosaS. aureus TSA culture TSA culture

Blood agar culture

Chocolate agar culture Chocolate agar culture

Blood agar culture

Early stage of cultivation (1-3 days) at 35 oC

9093*7582*

13956*

6887*

3882*9631*2311*

6887*

6886*

9634*

9626*

3881*

3876*

9092*

9093*

13956*

13955*

7581*

7582*

*Identification marker

SwissProt database search by Mascot software

31

Documents

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