1 January 25th, 2006 Innovative Solutions for Protein Biomarker Discovery and Validation Indications in green = Live content Indications in black = Edit

1 January 25th, 2006

Innovative Solutions for Protein Biomarker Discovery and Validation

© 2006 Applera Corperation and MDS, Inc.

Courtesy of PPD, Inc.

Indications in green = Live content

Indications in black = Edit in master

Indications in white = Locked elementsTemplate release: Date

Presentation title:

28 pt. Arial Regular, light orange

Presenter’s name and title:

20 pt. Arial Regular, white

Applied Biosystems logo must not be moved, added to, or altered in any way.

Background shouldnot be modified.

Courtesy of PPD, Inc.

Biomarker Discovery using iTRAQTM

Reagents

Brian Williamson, Ph.D.

January 2006




Expanding Expression Analysis Technology

• ICAT® reagents in 2001

– First innovation in tagging technologies for LC-based workflows– Protein-based labeling strategy enabling identification of medium to low

abundant proteins from complex samples– Duplex relative quantitation (compare only 2 different samples)– Quant in MS enabling expression dependent experiments

• Challenge for the technology was to address the following: – Protein coverage (labeling other than Cysteine)– Peptide-based vs. Protein-based labeling– PTMs – Multiplexing beyond two samples (duplex)




Amine specific

PRG

Peptide Reactive Group

Charged Neutral loss

Isobaric Tag(Total mass = 145)

Reporter Balance PRG

Gives strong signature ion in MS/MS Gives good b- and y-ion series Maintains charge state Maintains ionization efficiency of peptideSignature ion masses lie in quiet region

Reporter(Mass = 114 thru 117)

Balance changes in concert with reporter mass to maintain total mass of 145Neutral loss in MS/MS

Balance(Mass = 31 thru 28)

Isobaric Tag(Total mass = 145)

= MS/MS Fragmentation Site

Isobaric TagTotal mass = 145

Reporter Group mass114 –117 (Retains Charge)

Balance GroupMass 31-28 (Neutral loss)

Amine specific peptidereactive group (NHS)

N

N

O

O

N

O

O

Multiplexed protein quantitation in saccharomyces cerevisiae using amine-reactive isobaric tagging reagents Ross, PL., et al, Mol Cell Proteomics 2004 3: 1154-1169.

iTRAQ™ Reagent Design




1347.0 1349.6 1352.2 1354.8 1357.4 1360.0

Mass (m/z)

1352.84

-Reporter-Balance-Peptide INTACT- 4 samples identical m/z

114

115

116

117

111.0 112.8 114.6 116.4 118.2

Mass (m/z)

114

116

115

117

- Reporter ions DIFFERENT

MS

Mix-N H114 31

-N H115 30

-N H116 29

-N H117 28

9.0 292.8 576.6 860.4 1144.2 1428.0

Mass (m/z)

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sity y8

P

A b2

y1

0

q,H

72.1 b4

b1

45.1

b7

LT 112.

1

y3

b1

0

y5

b9

y2 b6

74.1 13

52.8

y6

142.

1

39.0 y4 b8

y9 y1

1

- Peptide fragments EQUAL

MS/MS b

y

b

yb

yb

y

+

+

+

+

-PRG114 31

-PRG115 30

-PRG116 29

-PRG117 28

S1

S2

S3

S4

Par

alle

l D

enat

ure

& D

iges

t

Isobaric Tagging - General Method (4-Plex)




111.0 112.8 114.6 116.4 118.2 120.0

Mass (m/z)

5186.0

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sity

Xrn

1

wt

Up

f1

SP

T =

0

T =

60

T =

30

T =

120

Tes

t

C2

C1

C3

Potentials of Multiplexing

Duplicates and triplicates of experimentTime course studiesReproducibility of sample prepNormal versus diseased versus drug treatedRelative and absolute quant with synthetic peptides




Spotfire K-means Clustering of Protein-level Ratios

G1L S PM G1L S PM G1L S PM




9.0 292.8 576.6 860.4 1144.2 1428.0

Mass (m/z)

8396.7

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sity

y8

P

A b2

y1

0

q,H

72.1 b4

b1

45.1

b7

LT 112.

1

y3

b1

0

y5

b9

y2 b6

74.1 13

52.8

y6

142.

1

39.0 y4 b8

y9 y1

1

869 871 873 875 877 879

Mass (m/z)

y8

757 759 761 763 765 767

Mass (m/z)

b7

*-TPHPALTEAK-*

111.0 112.8 114.6 116.4 118.2 120.0

Mass (m/z)

114.

1

116.

1

115.

1

117.

1

MS/MS Spectra of a Singly-charged Peptide




MS/MS Spectra of a Doubly-charged Peptide

y7b2Reporter region

*VLVDTDYK*




Summed Ion Intensity (~75,000 Spectra)

Reporter Group Placement: Selection of ‘Quiet Region’




Control

Example: Time course labeling

Test 1 Test 2 Test 3

Trypsin Digestion

MIX

LC MS/MS Analysis

Simplified Workflow: (One extra step)

SCXID and

MS/MS

Label with iTRAQ™ Reagents114 115 116 117

1 hr, RT,Single addition

Single 2D LC analysis for combined samples (4-plex)

Quant



© 2006 Applera Corperation and MDS, Inc.Data courtesy of Michael Siu, York University, Toronto

Discovery and Identification of Protein Markers in Endometrial Carcinoma

• Over 5% of all cancers in women

• Challenge in Screening• Pap test

• low sensitivity

• No biochemical markers available to monitor or screen disease

• Invasive techniques available in advanced stages of disease

• Yet high risk groups are recognized• Diabetic, hypertensive, Tamoxifen treatment




Workflows

1. Protein Profiling

SELDI Protein Reader

SELDI-QSTAR XL MS Determine Accurate Mass

2. Off-Line Purification of Putative Marker

Re-confirm MW of Marker

Digest and Identify with oMALDI QSTAR XL MS Analysis

3. ICAT® Reagent Label

Quantitate and Identify Markers not seen in 2 using ESI QSTAR XL MS System

4. iTRAQ™ Reagent Label

ID by SELDI




3 Malignant Cases2 Non-Malignant Cases

Distinct Protein Peak In Malignant CasesLow in Non-Malignant Cases

Higher Resolution on QSTAR XL Determines Mass to be 10843Da

Off-Line Protein Separation, Concentration and Identification




SDS-PAGE of SEC Fraction containing the 10843Da Protein

MS/MS Spectrum of Precursor 1529.82 on oMALDI QSTAR

3 Unique Peptides Identify Chaperonin 10




Differential Expression using iTRAQ™ Reagent ApproachMultiplexing allows 2 normal samples (proliferative and secretory) to be compared to 2 diseased samples

Trypsinize and Label with iTRAQ® Reagent

Normal Endometrium Cancerous Endometrium

114

SCX Fractionation

Nano-LC/Mass Spectrometry using QSTAR® XL MS System

115 116 117

Combine

Parallel Digestion and Labeling

Proliferative Secretory




Differential Expression using iTRAQ™ Reagent Approach

OverExpression of Chaperonin 10Non-Cysteine containing Protein

Quantitatio

n

100 200 300 400 500 600 700 800 900m/z, amu

0

5

10

15

20

25

30

35

40

45

50

54

b2

b3

b4 b5

b6 b7

y7 y6

y5

y4

y3

y2 y1

114 115 116 117m/z, amu

Normal

Normal

Cancer Cancer

*VLQATVVAVGSGS*K* iTRAQ Labeled Residue




Differential Expression using iTRAQ™ Reagent Approach

OverExpression of Chaperonin 10

Several novel markers were also identified and are currently under investigation

4 Peptides in total identify and quantify Chaperonin 10

Validated by Western Blotting and Immunohistochemical Staining




•Tagging Approaches lead to the identification and quantitation of markers such as protein kinases, signal transduction proteins and structural proteins

•Greater coverage than non-tagged approaches in a shorter time

•iTRAQ™ Reagent Labeling and Nano LC/MS/MS

•Simplest technique

•Identification and Quantitation of potential markers is automatic

Conclusions




Discovery of Putative Biomarkers for Anaplastic Oligodendrogliomas in

Human Brain Tissue using iTRAQ™ Reagents and the 4800 MALDI

TOF/TOF™ Analyzer




•Aggressive form of brain tumors

•Account for 5% of brain tumors in the US

•Treated by surgery followed by radiation therapy

•Current detailed study of these tumors is currently being investigated using pathological, proteomic and genomic data to gain a greater understanding of this disease.

What are Anaplastic Oligodendrogliomas?




Identify putative protein markers that:

1.Differentiate the tumor tissue from normal brain tissue

2.Differentiate different tumor types

using the 4800 MALDI TOF/TOF™ Analyzer and iTRAQ™ Reagents.

Research Objectives




WorkflowHuman glioma 　 (MRI)

Tumor Normal

Normal

1 Individual

Normal

1 Individual

Glioma

Insensitive to Chemotherapy

Glioma

Sensitive to Chemotherapy

Label with 117

Process according to iTRAQ™ Reagent Protocol

Label with 116Label with 115Label with 114 Combine

Parallel Digestion and Labeling

SCX Fractionation

RP 1D LC MALDI

MS/MS Analysis using 4800 MALDI TOF/TOF™

Analyzer




Simultaneous ID and QuantitationEnolase Gamma- Neuronal Protein

MS/MS for ID

BOTH tumor types down-regulated compared to normal

DATNVGDEGGFAPNILENSEALELVK




Simultaneous ID and QuantitationCaldesmon

BOTH tumor types up-regulated compared to normal

MS/MS Spectrum for consistency

YEIEETETVTK




Simultaneous ID and QuantitationPutative Markers Between Tumor Types

Glial fibrillary acidic protein (GFAP)up-regulated compared to normal and Sensitive tumor type

Annexin A1 up-regulated compared to normal and Insensitive tumor type

Peptide LADVYQAELR

Peptide GVDEATIIDILTK




Superior QuanTIS™ Precursor Selection for Robust Quantitation

Co-Eluting Precursors in MS (differ by 5Da)

1323

1328





Precursor Ion Selection- 1323 m/zProtein 114 115 116 117

Syntaxin 1B2 Peptide 1323 0.43 0.44 0.07 0.06

Protein average 0.34 0.39 0.14 0.15

From Syntaxin 1B2





Precursor Ion Selection- 1328 m/zProtein 114 115 116 117

3-hydroxyacyl-CoA dehydrogenase

Peptide 1328 0.13 0.25 0.26 0.36

Protein average 0.16 0.23 0.22 0.39

From 3-Hydroxyacyl-CoA Dehydrogenase




Protein No. Peptides

Alpha-soluble NSF attachment protein

SNAA_HUMAN 3

Beta-soluble NSF attachment protein

SNAB_HUMAN 6

Synaptogyrin 1 SNG1_HUMAN 3

Myelin basic protein (MBP) MBP_HUMAN 18

Neurofilament triplet M protein NFM_HUMAN 15

Neurofilament triplet L protein NFL_HUMAN 11

Enolase Gamma ENOG_HUMAN 14

Syntaxin binding protein 1 STB1_HUMAN 27

Synapsin I (Brain protein 4.1) SYN1_HUMAN 18

Brain acid soluble protein 1 BASP_HUMAN 16

Myelin proteolipid protein (PLP) MYPR_HUMAN 13

Myelin-oligodendrocyte glycoprotein precucursor

MOG_HUMAN 4

Synaptotagmin I SYT1_HUMAN 7

Alpha-synuclein SYUA_HUMAN 3

Visinin-like protein 1 (VILIP) VIS1_HUMAN 6

Alpha-internexin (Alpha-Inx) AINX_HUMAN 12

Neuromodulin NEUM_HUMAN 8

Synaptosomal-associated protein 25

SN25_HUMAN 10

Syntaxin 1B2 ST1C_HUMAN 6

Proteins identified as down regulated in the tumor samples compared to normal brain tissue




Summary•4594 Peptides confidently identified matching to 1513 proteins

•Reduced to 484 proteins (proteins with >3 peptides)

•Data visualization using Spotfire Decision site:

Cluster Profile/Distribution

1 Proteins down-regulated in tumor samples

2 Proteins that show no change in expression between normal and tumor tissue

3 Proteins up-regulated in tumor samples

4 Proteins up-regulated in the second tumor sample only

0

50

100

150

200N

o. p

rote

ins

1 2 3 4

Cluster Number

K-means Cluster Analysis




Conclusion

MALDI TOF/TOF™ Analyzer with iTRAQ™ Reagents enabled:

•Single experiment for 2 controls (biological variation) and 2 tumor types (insensitive and sensitive to chemotherapy)

•Accurate/robust quantitation with the new improved technology using QuanTIS™ precursor ion selector

•Several proteins were found to be up-regulated in one tumor, these included Glial fibrillary acidic protein which is a known marker for this type of tumor and Annexin A1 which was up-regulated in the insensitive tumor.

•Studies are underway to investigate these potential marker proteins in more detail, possibly moving to early stage validation on a larger sample set. The protein expression data will also be compared to gene expression data to determine correlation.




Biomarker Discovery with a Multiplex Quantitative

Proteomics Platform in Human Cerebral Spinal Fluid of Patients with Various Neurodegenerative

Diseases

Fadi Abdi, Ph.D.

October 14th 2005




DLB

label each with 117

Direct spotting onto MALDI

target

MS and MS/MS

Analysis

4800 TOFTOF Analyzer

Organic Fractionation

Normal AD PD

Parallel reduction, alkylation, digestion for each fraction

Strong Cation Fractionation

Combine all Fraction 1s, Fraction 2s and Fraction 3s

RP LC-MALDI for each fraction

Organic Fractionation Organic Fractionation Organic Fractionation

1 2 3 1 2 3

label each with 114

label each with 115

label each with 116

1 2 3

1 2 3 1 2 3 1 2 3 1 2 3

1 2 3 1 2 3

• Five ml CSF from each patient was combined into a pooled AD, PD, DLB and control samples (10/group; 50 ml total) • Each pooled CSF sample underwent discontinuous gradient extractions with acetonitrile as descried previously

• Three fractions were generated from each batch of pooled CSF: pellet following 1.5 volumes acetonitrile, pellet following 3.0 volumes acetonitrile, and supernatant following 3.0 volumes ACN.

• 100 μg protein from each corresponding fraction was digested with trypsin and then labeled with one of the four iTRAQTM reagents




Down regulated > 50%Up regulated > 50%

Alzheimer’s Disease

Parkinson’s Disease

Dementia w/ Lewy Body

75

48

33

19

11

6

Results• Protein identification was based on meeting the criteria of having at least one

peptide whose individual score was above the 95% confidence threshold (p < 0.05) and also identified as the top-ranked matching sequence for that spectrum

• For more stringency to protein identification we only considered proteins with at least two peptides with individual score of 99% confidence threshold (p <0.01) or higher. Using this criteria we were able to identify 798 unique proteins.




0 50 100 150 200 250 300 350 400

Proteins Identified

Unclassified

Signal Transduction

Protein Metabolism

Neural Activity

Lipid and Fatty Acid Metabolism

Ion Transport

Immunity and Defense

Carbohydrate Metabolism

Amino Acid Metabolism

Some of the biological processes of the identified proteins




0 10 20 30 40 50 60 70

Unclassified

Signal Transduction

Protein Metabolism

Neural Activity


Ion Transport



Unchanged

Up Regulated

Down regulated

Dementia w/ Lewy Body Disease

Proteins Identified

0 10 20 30 40 50 60 70

Unclassified

Signal Transduction

Protein Metabolism

Neural Activity


Ion Transport



Parkinson’s Disease

Proteins Identified0 10 20 30 40 50 60 70

Unclassified

Signal Transduction

Protein Metabolism

Neural Activity


Ion Transport



Alzheimer’s Disease

Proteins Identified

Panes A, B, and C show the number of modulated proteins identified and the biological processes from each diseased sample

A B

C




Conclusions• This approach identified more than 1,000 proteins in human CSF • The majority of identified proteins were involved in cell immunity and

defense, cell signaling, and synaptic transmissions.• Among >1,000 proteins identified, numerous proteins appeared to be

unique to patients with AD, PD and DLB. • Proteins from the Apolipoproteins family were identified in all three

diseases and were modulated when compared to healthy individuals.• Several proteins involved in neural activities and synaptic

transmissions were also identified and seem to be modulated in all three diseases.

• Several of these putative biomarkers have been validated using specific antibodies against CSF samples from individual patients.

Documents

1 January 25th, 2006 Innovative Solutions for Protein Biomarker Discovery and Validation Indications in green = Live content Indications in black = Edit