IntroductionRecent research has proposed rapid and robust identification of intact microorganisms using matrix assisted laser desorption/ ionization time-of-flight mass spectrometry and bioinformatics [1]. Previous work has relied primarily on desorption and detection of protein biomarkers weighing less than 20 kilo Daltons. Analysis of prokaryotic genomes predicts fewer proteins with higher masses per organism and, thus, the potential to provide more definitive microorganism identifications. However, higher mass proteins have not yet been readily accessible by MALDI and widely evaluated for rapid detection of bacteria. They are difficult to desorb because of suppression by other components of the lysed cell; they are detected with less sensitivity by most commercial ion detectors. This poster reports a procedure using acid-cleavable detergent and microwave to facilitate desorption of higher molecular weight protein biomarkers from lysed whole cells. This approach has been evaluated with Escherichia coli (K-12), Salmonella typhimurium, Bacillus anthracis Sterne, and Bacillus subtilis (168).

A microwave and detergent procedure to detect high molecular mass proteins from vegetative bacteria by MALDI-TOF MS

Elizabeth Patton1; Nathan Edwards2; Berk Oktem3; and Catherine Fenselau1

1 Chemistry and Biochemistry and 2Center for Bioinformatics, University of Maryland, College Park, MD; 3 Middle Atlantic Mass Spectrometry Lab, Johns Hopkins School of Medicine, Baltimore, MD

Literature cited1. Fenselau, C.; Demirev, P. A. Characterization

of intact microorganisms by MALDI mass spectrometry. Mass Spectrom. Rev. 2001, 20, 157-171.

2. Norris, J. L.; Porter, N. A.; Caprioli, R. M. Mass spectrometry of intracellular and membrane proteins using cleavable detergents. Analytical Chemistry 2003, 75, 6642-6647.

3. Wessel, D.; Flugge, U. I. A Method for the Quantitative Recovery of Protein in Dilute-Solution in the Presence of Detergents and Lipids. Analytical Biochemistry 1984, 138, 141-143.

Experimental methods and materials

Intact Cells Disrupt cell membrane and solubilize proteins with acid cleavable detergent

Clean up protein with a Folch extraction [3] Mix protein with saturated sinapinic acid (SA) Lower pH and microwave

Sample applied on topof dried layer of saturated SA crystals on slide

MALDI-TOF instruments:Kratos Axima CFR+ andComet Macromizer

20kDa 100kD

Search Rapid MicroorganismIdentification DataBase for best match

Zwitterionic 6-PPS Detergent [2]

N+O

O

CH3

CH3

CH3 S

O

O

O -

• Enhances the solubility of hydrophobic proteins

• Acid cleavable by microwaving at pH 1.4

• Eliminates detergent signal interference

Solvent System [3]

Salts

protein fraction at the interface

Lipids

C22H39NO5S 

CHCl3

CH3OH

H2O

Objectives1. To evaluate accessibility by MALDI to

higher mass proteins in intact bacteria 2. To evaluate the suitability of higher

mass proteins to provide identifications based on genomic database searching

Results

0

10

20

30

40

50

60

70

80

90

100

%Int.

20000 25000 30000 35000 40000 45000 50000 55000 60000

Mass/Charge

3520

535

418

2299

1

2022

6

2590

5

2231

3

2465

4

3561

4

2088

7

2666

5

4328

5

3238

8

3341

6

2851

4

2561

7

2422

7

3442

1

3034

8

3119

1

3827

5

4409

7

3738

9

4579

3

3914

7

4787

3

4128

7

4268

2

5051

0

5210

6

4662

4

5615

9

5516

5

5733

5

5949

6

0

10

20

30

40

50

60

70

80

90

100

%Int.

20000 25000 30000 35000 40000 45000 50000 55000 60000

Mass/Charge

3554

035

751

2301

0

2024

7

3595

0

2232

5

2465

8

2592

0

4328

9

2668

7

3116

1

2842

4

2975

5

3466

4

3257

8

3792

5

3686

7

3348

7

4553

3

0

10

20

30

40

50

60

70

80

90

100

%Int.

20000 25000 30000 35000 40000 45000 50000 55000 60000

Mass/Charge

2070

3

4133

6

2276

0

5471

3

2241

8

2021

2

3268

2

2003

2

2302

6

2740

4

2174

1

3371

0

2425

0

2576

4

4664

0

3288

3

2772

5

2902

3

2491

7

3525

2

3121

2

3415

3

3046

8

4354

2

3820

0

3978

6

3615

6

3752

5

3686

2

4284

0

4647

9

4428

6

4779

7

5065

3

4919

9

5976

0

5888

8

0

10

20

30

40

50

60

70

80

90

100

%Int.

20000 25000 30000 35000 40000 45000 50000 55000 60000

Mass/Charge

2190

5

2111

621

317

2400

8

2029

3

2162

8

2297

2

3627

3

4411

3

2469

3

3022

6

2551

7

2662

4

Escherichia coli

Salmonella typhimurium

Bacillus subtilis 168

Bacillus anthracis Sterne

Escherichia coli

1555

3

1771

4

3531

6

3751

538

049

3842

0

4333

4

Salmonella typhimurium

3549

3

3767

0

4324

3

5091

8

Salmonella typhimurium

7797

9

9140

6

1036

82

Bacillus subtilis 16816

386412

6474

1126

6711

6000

1008

38

5479

3

4634

9

1274

45

8396

5

8908

2

4300

0

3608

2

23972

2106321785

Bacillus anthracis Sterne

Reproducibility

ConclusionThis procedure afforded the desorption and detection of several proteins from intact species in molecular mass ranges above 20kDa. However, good spectra with peaks in the 20-50kDa range aren't enough for microorganism identification. To make high-mass biomarkers suitable requires one or more of the following advances, all of which increase the specificity of a spectrum peak with respect to its species/organism.a) a significant boost in mass accuracy,b) an understanding of which proteins, or protein subset, have abundant peaks in these spectra,c) obtaining peaks in a mass range above 80kDa.

0

20

40

60

80

100

%Int.

20000 25000 30000 35000 40000 45000 50000 55000 60000

Mass/Charge

3520

535

418

2299

1

2022

6

2590

5

2465

4

2119

822

140

2666

5

4328

5

3238

8

3341

6

2851

4

3442

1

3034

831

191

3827

5

4409

7

3738

9

4579

3

3914

7

4787

3

4128

7

5051

0

5210

6

4662

4

5615

955

165

5733

5

5949

6

Comparison of Axima spectra of four speciesComparison of Axima spectra of four species

Comparison of Comet Macromizer spectra of four speciesComparison of Comet Macromizer spectra of four speciesThe cryodetector is more sensitive to higher masses because the signal is independent of mass and, thus, impact velocity.

Spectra are reproducible from spot to spot.Spectra are reproducible from spot to spot.

Escherichia coli

This simulation illustrates that high-mass, in the This simulation illustrates that high-mass, in the 20-50KDa range, is insufficient for 20-50KDa range, is insufficient for microorganism identification with current microorganism identification with current technologies. Ribosomal proteins from the technologies. Ribosomal proteins from the RMIDB database were selected and then a RMIDB database were selected and then a random error, based on the instrument accuracy random error, based on the instrument accuracy (x-axis), was applied to their exact theoretical (x-axis), was applied to their exact theoretical masses; the RMIDB was used to "look-up" the masses; the RMIDB was used to "look-up" the spectrum (number of peaks detected on y-axis) spectrum (number of peaks detected on y-axis) and check the e-value (z-axis) of the correct and check the e-value (z-axis) of the correct answer.answer.

3 58 10

13 1520

5

10

15

2030

50

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

chance match

probability

peaks

ppm