Identification of Bacteria Using Phylogenetic ... · Clostridium perfringens (epsilon toxin) 6. Coxiella burnetti (Q fever) 7. Escherichia coli (diarrheagenic) 8. Listeria monocytogenes

Identification of Bacteria Using Phylogenetic Relationships Revealed by MS/MS Sequencing

of Tryptic Peptides Derived from Cellular Proteins

Jacek P. DworzanskiGeo-Centers, Inc.,

Aberdeen Proving Ground, MD 21010-0068

Samir Deshpande1; Rui Chen2; A. Peter Snyder3;Liang Li2 and Charles H. Wick3

1Science and Technology Corporation, Edgewood, MD 21040; 2Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2; 3 U.S. Army Edgewood Chemical Biological Center,

Aberdeen Proving Ground, MD 21010-5424

2004 Joint Service Scientific Conference on Chemical & Biological Defense Research

15-17 November 2004; Hunt Valley, Maryland

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4. TITLE AND SUBTITLE Identification of Bacteria Using Phylogenetic Relationships Revealed byMS/MS Sequencing of Tryptic Peptides Derived from Cellular Proteins

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13. SUPPLEMENTARY NOTES See also ADM001849, 2004 Scientific Conference on Chemical and Biological Defense Research. Held inHunt Valley, Maryland on 15-17 November 2004 . , The original document contains color images.

14. ABSTRACT

15. SUBJECT TERMS

16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT

UU

18. NUMBEROF PAGES

27

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b. ABSTRACT unclassified

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Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18

Category B

1. Brucella species (brucellosis)2. Burkhoderia pseudomallei 3. Burkholderia mallei (glanders) 4. Campylobacter jejuni5. Clostridium perfringens (epsilon

toxin)6. Coxiella burnetti (Q fever)7. Escherichia coli (diarrheagenic)8. Listeria monocytogenes9. Rickettsia prowazekii (typhus fever)10. Salmonella11. Shigella species12. Staphylococcus aureus (enterotoxin

B)13. Vibrios (pathogenic)14. Yersinia enterocolitica

National Institute of Allergy and Infectious DiseasesNational Institute of Health

Category A, B & C Priority Bacterial PathogensCategory A

1. Clostridium botulinum2. Bacillus anthracis (anthrax) 3. Francisella tularensis (tularemia)4. Yersinia pestis

Category C

1. Mycobacterium tuberculosis(multiple drug resistant)

2. Rickettsias (other)

Genomes of all above organisms have been sequenced

Prokaryotic Ongoing Genome Projects: 489 {528}Archaeal: 28 {27} Bacterial: 461 {499}

5/12/2004 versus {11/12/2004}

Number of Fully Sequenced Genomes of Eubacteria: 145 {178} (From 1995)Number of Fully Sequenced Genomes During Last 12 Months: 55 {68}

Bacillus anthracis A1055 (Group C) TIGRNIAIDBacillus anthracis Ames Ancestor TIGRNIAID / NSFBacillus anthracis Ames Florida TIGRNSFBacillus anthracis Austrailia 94 (GT55, Group A3a) TIGRNIAIDBacillus anthracis CNEVA-9066 (GT79 Group B2) TIGRNIAIDBacillus anthracis Kruger B (GT87 Group B1) TIGRNIAIDBacillus anthracis Vollum (GT77 Group A4) TIGRNIAIDBacillus anthracis Western N. America (GT3 Group A1a)TIGRNIAIDBacillus anthracis STN DOE/JGIBacillus anthracis ZK DOE/JGI

Example of Ongoing Genome Projects

First, some terminology…Taxonomy - the science of naming and classifying organisms;

Classification - placement of an organism within a scheme relating different groups of organisms;

Identification - the determination of whether an organism should be placed within a group of organisms known to fit within some classification scheme;(the practical use of classification criteria)

Phylogenetics - focuses on evolutionary relationships betweenorganisms or genes/proteins

Phylogenetic ApproachThe ideal means of identifying and classifying bacteria would be to compare each gene sequence in a given strain with the gene sequences for every known species.

Taxonomy of Bacteria(Linnaean System)

Bacteria Firmicutes Bacilli BacillalesBacillaceae Bacillus Bacillus subtilis

Bacteria Proteobacteria γ-ProteobacteriaEnterobacteriales EnterobacteriaceaeEscherichia Escherichia coli

KingdomPhylumClassOrderFamilyGenusSpecies

Examples: Bacillus subtilis & Escherichia coli

Universal Phylogenetic Tree of Bacteria Based on SSU rRNA Sequences

Aquificae

Termotogae

Planctomycetes

ActinobacteriaFirmicutes

CyanobacteriaFusobacteria

Proteobacteria

SpirochaetesChlamydiae

Deinococcus-Thermus

Bacteroidetes/Chlorobi

0.1 base substitution per nucleotide

Archaea, Eucarya

Reepresentatives of Marked PhylogeneticDivisions (Phyla) of Bacteria Are Included in the Database

Reepresentatives of Marked PhylogeneticDivisions (Phyla) of Bacteria Are Included in the Database

Approach: (1) Bacterial Sample Processing

Peptides

Lysis Digestion(trypsin)

ProteinsBacteria

Peptides

Lysis Digestion(trypsin)

ProteinsBacteria

Flow of Genetic Information

mRNA

DNA CCTGAGCCAACTATTGATGAA

PEPTIDE

CCUGAGCCAACUAUUGAUGAA

Protein

transcription

translation

Flow of Genetic Information

mRNA

DNA CCTGAGCCAACTATTGATGAACCTGAGCCAACTATTGATGAA

PEPTIDEPEPTIDE

CCUGAGCCAACUAUUGAUGAACCUGAGCCAACUAUUGAUGAA

Protein

transcription

translation The relation between the sequence of bases in DNA and the sequence of amino acids in a protein

The relation between the sequence of bases in DNA and the sequence of amino acids in a protein

Mass SpectrometryMass SpectrometryElectrospray Ionization

PowerSupply

+ _

++

++

++

++++

+

++

+ + +

--

-

--

--

--

-

-

---

-+

+

++

++

++++

+

++

+ + +

--

-

--

--

--

-

-

---

-

Electrospray Ionization

PowerSupply

+ _

++++++

PEPTIDE

m1m6 m4 m3 m2m7 m5 m1m1m6m6 m4m4 m3m3 m2m2m7m7 m5m5

FragmentIons Out

++

++

++++

++++++ ++ ++++ ++ ++ ++ ++++

++++

++++

++++

++

++++ ++++ ++++++++ ++++ ++++ ++++ ++++++++

++++++++

++++++++

++++++++

++++

Peptide IonsIn

Approach: (2) Tandem Mass Spectrometry of Peptide Ions

PEPTID

PEPTIDR

m7

P #1

PEPTIDKP

PE

PEP

PEPT

PEPTI

Mass/Chargem6m5m4m 3m2m1

Inte

nsity

PEPTIDE

P

PE

PEP

PEPT

PEPTI

PEPTID

Mass/Chargem6m5m4m 3m2 m7m1

Inte

nsity

PEPTIDE

P

PE

PEP

PEPT

PEPTI

PEPTID

PEPTIDE

Mass/Chargem6m5m4m 3m2 m7m1

Inte

nsity

PEPTIDE P #3 P #2

MS/MS Spectra of Peptide Ions

P #1

P #2

P #3

Unknown Bacterium

Approach: (3) Sequencing of Peptide Ions

B. anthracis V. choleraeY. pestis

P #1

P #2

P #3 P #3

Database

Matching

P #1

P #2

P #3

Unknown Bacterium

Identified agentY. pestis

Approach: (4) Matching of Identified Tryptic Peptides to Theoretical Peptides of Database Bacterial Proteomes

X1X2X3…K

X1X2X3…R

TrypticPeptides

X1X2X3…K

X1X2X3…R

Virtual Array of Peptide Sequences

1 2 3 n = 170…..

••••••

ORF m

ORF 3

ORF 2

ORF 1

••••

••

Bacterial Proteomes

Tran

slat

ed P

rote

in S

eque

nces

m = 500 to 7,500

Total Number of Protein Coding Sequences: 500,000

Matching

Classification and Identification of Unknown Bacterium

…..X1X2X3…K s1X1X2X3…R s2

…..…..…..…..…..…..…..

…..

…..

…..…..…..

X1X2X3…K s3

X1X2X3…R sm

…..

u b2 b3 bn

Matrix of Assignments

Sequ

ence

s

…..

…..Bacterial Proteomes

…..

b1Tryptic peptides

…..X1X2X3…K s1X1X2X3…R s2

…..…..…..…..…..…..…..

…..

…..

…..…..…..

X1X2X3…K s3

X1X2X3…R sm

…..

u b2 b3 bn

Matrix of Assignments

Sequ

ence

s

…..

…..Bacterial Proteomes

…..

b1Tryptic peptides

Cluster Analysis

u b2 b3 b4 b5b1u b2 b3 b4 b5b1

Principal ComponentAnalysis

F1F2

F3

F1F2

F3

F1F2

F3

SIM

-Sco

re

b1b2 b3 bn…..

100

Affinity Histogram

Screen Capture Image Displaying Raw LC-MS/MS Data

L. lactis

Discriminant Analysis ParametersSEQUEST Output Data

AssignmentMatrix

Bacterium Number

SIM

-Sco

re

Filters

Screen Capture Image Displaying Accepted Peptide Assignments(P = 90%)

L. lactis

Screen Capture Image Displaying Accepted Peptide AssignmentsAfter Removal of Degenerate Peptide Sequences

L. lactis

Phylum1 2 3 4 5 6 7 8 9 10 11 12

SIM

-Sco

re

0

20

40

60

80

100

Phylum1 2 3 4 5 6 7 8 9 10 11 12

ID-S

core

0

20

40

60

80

100

Prot

eoba

cter

ia

Firmicutes

F2

FirmicutesPhylum level

Class1 2 3

SIM

-Sco

re

0

20

40

60

80

100

Class1 2 3

ID-S

core

0

20

40

60

80

100

F2

Bacilli

Clo

str id

ia

Mol

licut

es

BacilliClass level

Order1 2

SIM

-Sco

re

0

20

40

60

80

100

Order

1 2

ID-S

core

0

20

40

60

80

100

F2

Bacillales

Lact

obac

illal

es

BacillalesOrder level

Family1 2 3

SIM

-Sco

re

0

20

40

60

80

100

Family1 2 3

ID-S

core

0

20

40

60

80

100

F2

Ente

roco

ccac

eae

Lact

obac

illac

eae

Stre

ptoc

occa

ceae

Stre

ptoc

occa

ceae

Family level

Genus1 2

SIM

-Sco

re

0

20

40

60

80

100

Genus1 2

ID-S

core

0

20

40

60

80

100

F2

Lact

ococ

cus

Stre

ptoc

occu

s

Lact

ococ

cus

Genus level

Species1 2 3 4 5 6

SIM

-Sco

re

0

20

40

60

80

100

Species1 2 3 4 5 6

ID-S

core

0

20

40

60

80

100

F2

L. lactis

S. m

utan

sS.

pne

umon

iae

R6

S. p

neum

onia

e R

T4S.

pyo

gene

sS.

aga

lact

iae

L. lactis

Species level

Identification of Bacteria Using Phylogenetic Relationships Revealed

by MS/MS Sequencing of Tryptic Peptides

(Lactococcus lactis)

Analysis of Bacterial Mixture[E. coli (K-12) and B. subtilis (2:1), w:w]

Phylum (Division)1 2 3 4 5 6 7 8 9 10 11 12

ID-S

core

0

10

20

30

40

50

60Phylum Level

Firmicutes

Proteobacteria

Spirochaetes

Class1 2 3

ID-S

core

0

20

40

60

80

100

BacilliFirmicutes

Class1 2 3 4

ID-S

core

0

20

40

60

80

100

γ-Proteo-bacteria

Proteobacteria

Species1 2 3 4

ID-S

core

0

20

40

60

80

100

B. subtilisBacillus

Strain1 2 3 4

ID-S

core

0

20

40

60

80

100

E. coli K-12Escherichia

BacillalesBacillaceae

Enterobacteriales Enterobacteriaceae

Identification of Bacteria Using Phylogenetic Relationships Revealed

by MS/MS Sequencing of Tryptic Peptides

(Bacillus cereus)Phylum (Division)

1 2 3 4 5 6 7 8 9 10 11 12

SIM

Sco

re

0

20

40

60

80

100

Phylum (Division)

1 2 3 4 5 6 7 8 9 10 11 12

ID S

core

0

20

40

60

80

100

Firmicutes

Prot

eoba

cter

ia

B act

e roi

dete

s /C h

l or o

bi G

rou p

Firmicutes

F2

Phylum level

Phylum (Division)

1 2 3 4 5 6 7 8 9 10 11 12

SIM

Sco

re

0

20

40

60

80

100

Phylum (Division)

1 2 3 4 5 6 7 8 9 10 11 12

ID S

core

0

20

40

60

80

100

Firmicutes

Prot

eoba

cter

ia

B act

e roi

dete

s /C h

l or o

bi G

rou p

Firmicutes

F2

Phylum level

Firmicutes

Classes

1 2 3

Gen

omic

Sim

ilarit

ies

Scor

e

0

20

40

60

80

100

Classes

1 2 3

ID S

core

0

20

40

60

80

100Bacilli

Clo

str id

ia

Mol

licut

es

Bacilli

F2

Class level

Order

1 2

Gen

omic

Sim

ilarit

ies

Scor

e

0

20

40

60

80

100

Order

1 2

ID S

core

0

20

40

60

80

100

Bacillales

Lact

obac

illal

es

Bacillales

F2

Order level

Family

1 2 3

ID S

core

0

20

40

60

80

100

Family

1 2 3

Gen

omic

Sim

ilarit

ies

Scor

e

0

20

40

60

80

100

Bacillaceae

List

eria

ceae

Stap

hylo

cocc

acea

e

Bacillaceae

F2

Family level

Species

1 2 3 4

Gen

omic

Sim

ilarit

ies

Scor

e

0

20

40

60

80

100

Species

1 2 3 4

ID S

core

0

20

40

60

80

100

B.h

alo d

u ra n

s

B. cereusgroup

B. s

ubtil

is

O. i

hey e

nsi s

B. cereusgroup

F2

Species level

Species1 2 3 4 5 6 7 8 9 10

Gen

omic

Sim

ilarit

ies

Sco

re

0

20

40

60

80

100

Species1 2 3 4 5 6 7 8 9 10

ID S

core

0

20

40

60

80

100

F2

aa a a

cc t

h s i

B. CereusATCC 14579

Subspecies level (B. cereus group)

-0.2

0.0

0.2

0.4

0.6

-0.15

-0.10

-0.05

0.00

-0.4

-0.2

0.0

PC 3

PC 1

PC 2

UnknownB. cereusATCC 14789

B. cereus Group B. subtilis/other Bacilli

Principal Component Analysisof Peptide Assignments

Representation of the database Bacillaceae species and unknown organismin the principal component space (PC 1, PC 2, PC 3) reflecting 80 % of the total information included in the assignment matrix of 125 amino acid peptide sequences to bacterial proteomes.

B. c

ereu

sgr

oup

0 20 40 60 80 100 120Linkage Distance

B. subtilis

B. licheniformis

O. iheyensis

B. halodurans

B. cereus ATCC10987

B. thuringiensis

B. cereus ZK

B. anthracis A2012

B. anthracis Sterne

B. anthracis Ames ancestorB. anthracis Ames

B. cereus ATCC14579Unknown

Error

Baci

llace

ae

B. c

ereu

sgr

oup

0 20 40 60 80 100 120Linkage Distance

B. subtilis

B. licheniformis

O. iheyensis

B. halodurans

B. cereus ATCC10987

B. thuringiensis

B. cereus ZK

B. anthracis A2012

B. anthracis Sterne

B. anthracis Ames ancestorB. anthracis Ames

B. cereus ATCC14579Unknown

Error

Baci

llace

ae

Cluster Analysis of Peptide Assignments

Hierarchical clustering of Bacillaceae species in 125-dimensional space of peptide sequences.(Complete linkage; squared Euclidean distances)

Differences in Proteome Composition Between an Unknown Sample and Database Bacteria

Pept

ide

Num

ber

0102030405060708090

100110120

B. anth

racis

A2012

B. anth

racis

Ames

B. anth

racis

Ames an

cesto

r

B. anth

racis

Sterne

B. cere

us A

TCC 10

987

B. cere

us ZK

B. thur

ingien

sis

B. cere

us A

TCC 14

789

ESYAAVQADTASK

VGSPQPGDLVFFQGTYK

LVSLAEQQLGGYQK

EYEVPITAAQADQIVLLMK

STDTLGAQILGNTMEGLYR

TNTPMLLQVLEDEVFK

TGDAALGSISNILLR

GDTLTAVDNDLSAWFWDEK

DVNEHTLEEEELPVNIEAYKIEDALNSTR

Blue Line(—) absent; Yellow (—)present

Unknown sample: correctly identified as B. cereus ATCC 14789

45

47

107

0 200 400 600 800 1000 1200 1400 1600

100

50

0

m/z

Rel

ativ

e A

bund

ance

0 200 400 600 800 1000 1200 1400 1600

100

50

0

m/z

Rel

ativ

e A

bund

ance

b2b3

b5

b4

b7b6

b13b12

b11b10

b9

b8

y2

y3

y5

y4

y6

y7

y8

y13

y12y11

y10

y9

b2b3

b5

b4

b7b6

b13b12

b11b10

b9

b8

y2

y3

y5

y4

y6

y7

y8

y13

y12y11

y10

y9

107

L V S L A E Q Q L G G Y Q Kb2 b3 b5b4 b7b6 b13b12b11b10b9b8

y2y3y5 y4y6y7y8y13 y12 y11 y10 y9

213.3 300.4 413.5 484.6 613.7 741.9 870.0 983.1 1040.2 1097.3 1260.4 1388.6

1421.6 1322.4 1235.4 1122.2 1051.1 922.0 793.9 665.7 552.6 495.5 438.5 275.3

Product Ion Mass Spectrum of a Peptide IonAmino Acid Sequence Information Obtained in Less than 1 second

Discriminative Power of DNA and Protein Sequences

…..L V S L A E Q Q L G G Y Q KAmino Acid

Sequence From the MS/MS Spectrum

Accession LVSLAEQQLGG[ ]YQK 1430022721 52 ...........[ ]... 65 Bacillus cereus ATCC 14579 21402693 52 ...........[ ]F.. 65 Bacillus anthracis str. A2012 42783829 52 ...........[.]F.. 65 Bacillus cereus ATCC 10987 21401004 56 ...........[G]VTR 69 Bacillus anthracis str. A2012 3688809 54 ..QM....M..[ ]... 67 Bacillus firmus 15613701 53 ..AM.......[G]F.. 67 Bacillus halodurans 15615764 54 ..AM....M..[ ]F.Q 67 Bacillus halodurans 134232 56 ..Q........[G]RSK 66 Bacillus megaterium 134239 56 ..Q........[G]RF 69 Bacillus megaterium 134223 56 ..Q........[G]RF 69 Bacillus megaterium 21399863 54 ..AM.......[G].TR 64 Bacillus anthracis str.A2012 30020123 56 ..AM.......[G].TR 70 Bacillus cereus ATCC 14579 30021203 56 ..AM.......[ ]RANR 70 Bacillus cereus ATCC 14579 42782199 56 ..AM.......[ ]RANR 70 Bacillus cereus ATCC 10987 21401007 56 ..AM.......[ ]RANR 70 Bacillus anthracis str. A2012 21398813 54 ..AM...S...[ ]FH. 67 Bacillus anthracis str. A2012 134230 57 .....Q.....[G]TSF 70 Thermoactinomyces sacchari

Smal

l, A

cid-

Solu

ble

Spor

ePr

otei

ns

…..L V S L A E Q Q L G G F Q KAmino Acid Sequences of

Matching Peptides Found in the Database

SASP-21402693

B.cereus.ATTC 14579……GAT CAA AGT GAT CGA CTC GTT GTT AAT CCG CCA ATG GTT TTTB.anthracis A2012 ……….GAT CAA AGT GAT CGA CTC GTT GTT AAT CCG CCA AAG GTT TTT

DNA Sequences

Peptide # Sequence Protein

8 IEDALNSTR 60 kDa chaperonin GROEL 12 DVNEHTLEEEELPVNIEAYK Hypothetical protein BC0479

Putative transcriptional regulator19 ESYAAVQADTASKGDTLTAVDNDLSAWFWDEK Spore coat-associated protein N KQPNFDDSSNFAK Hypothetical protein BA 3347 [Bacillus anthracis Ames]

47 TGDAALGSISNILLR Flagellin 59 TNTPMLLQVLEDEVFK Propionyl-CoA carboxylase biotin-containing subunit 73 STDTLGAQILGNTMEGLYR Oligopeptide-binding protein oppA 93 EYEVPITAAQADQIVLLMK IG hypothetical 17696

107 LVSLAEQQLGGYQK Small acid-soluble spore protein 119 VGSPQPGDLVFFQGTYK N-acetylmuramoyl-L-alanine amidase

2945

Selected Peptide Sequences Discriminating Between an Unknown and Database Bacteria

47

45

Genes Encoding Identified Proteins

Location on the chromosome of Bacillus cereus ATCC 14579 (Mbp)1 2 3 4 5

Pro

tein

leng

th (

num

ber

of c

odon

s)

-600

-400

-200

0

200

400

600

0

(-) DNA Strand

(+) DNA Strand

• Identification of pure cultures as well as mixtures of microorganisms.

CONCLUSIONS

The results demonstrate that mass spectrometry-based proteomics approach allows for :

• High confidence level classification and identificationof bacteria based on genome traceable, proteomic similaritiesand differences between an analyzed microorganism andreference bacteria;

Thank you !!!Thank you !!!