single-nucleotide polymorphism and mutation analysis … · single-nucleotide polymorphism and...

Single-Nucleotide Polymorphism and Mutation

Analysis – and Its Impact on Personalized Medicine

Broadcast Date: Tuesday, December 11, 2012

Time: 11 AM ET

Sponsored by


Analysis – and Its Impact on Personalized

Medicine



Your Moderator

Tamlyn Oliver Managing Editor

Genetic Engineering & Biotechnology News



Paul M. Thomas, Ph.D. Research Assistant Professor

Department of Molecular Biosciences & Associate Director

Proteomics Center of Excellence

Northwestern University

Paul Thomas

Assoc. Director, Proteomics Center of Excellence


UNDERSTANDING

BIOLOGICAL COMPLEXITY

WITH TOP DOWN

PROTEOMICS

DNA

Gene Family,

coding SNPs

Alternative

splicing

mRNA Protein

Covalent

Modification

FROM ONE GENE, MANY PROTEOFORMS: A

MAJOR THEME IN CONTEMPORARY

PROTEOMICS

PROTEIN VARIABILITY IN HIGHER PROTEOMES: THE AGE OF PROTEIN ISOFORMS

Sources of

variability • Alternative Splices

• Endogenous

Cleavages

• Alternative

Promoter Usage

Isoform: Large changes to amino acid sequence

PROTEIN VARIABILITY IN HIGHER PROTEOMES: THE AGE OF PROTEIN ISOFORMS AND PROTEOFORMS

Proteoform: The complete set of modifications on an

amino acid sequence

Correct Gene Family

Correct Gene

Correct Protein Form

SHOTGUN ANNOTATION OF

DIVERSE PROTEIN FORMS

TECHNICAL HYPOTHESIS

Measurement of intact proteins (isoforms and

proteoforms) will provide strong correlations between

mass spectrometric data and complex phenotypes.

TOP DOWN VS. BOTTOM UP APPROACHES

TO DNA-PREDICTED PROTEIN SEQUENCE

ANALYSIS

DEVELOPMENT OF A GENERAL MEASUREMENT

PLATFORM FOR INTACT, MODIFIED PROTEINS

DEVELOPMENT OF A GENERAL MEASUREMENT

PLATFORM FOR INTACT, MODIFIED PROTEINS

2. Automated

Instrumentation

1. “Front End” Separations

3. “Back end” data

processing and

informatics

Top Down Proteomics Work Flow

Sample cleanup

(SDS removal)

GELFREE – MW

based separation

Online nano-LC-MS/MS:

12 T LTQ-FTMS

–

+

200 kDa

10 kDa

Cell lysate

or nuclei

(Optional) isoelectric

focusing

+ _

+ Anode – Cathode

Membrane Trap

SDS-PAGE

Column

Collection

Chamber

Intact protein separation based on molecular mass:

Time

(60-90 min)

MW

GEL ELUTED LIQUID FRACTION

ENTRAPMENT ELECTROPHORESIS (GELFREE)

J.C. Tran; A.A. Doucette, Anal. Chem. 2008, 80, 1568-1573

Mass Specs for Top

Down

•High Resolution MS and MS/MS

•Low ppm mass accuracy

8008

13C0

724722720

RP = 5000

Large Molecule Isotopic Distributions

ResolvingPower (RP):

122121120

= 1000m

m

80168000

RP = 30,000

Small Molecule

(120 Da)

Small Peptide

(720 Da)

Small Protein

(8000 Da)

13C1

13C3

13C5

13C2

13C1

13C0

13C0

“monoisotopic”

peak

“chemist’s

average”

“most abundant”

isotope peak

ADVANTAGES OF FT-MS

Resolution/Resolving Power up to 106, typically 105

isotopes

Mass Accuracy 10 ppm or better

TO

P D

OW

N,

19

95

Top Down Proteomics Work Flow

Sample cleanup

(SDS removal)

GELFREE – MW

based separation

Online nano-LC-MS/MS:

12 T LTQ-FTMS

–

+

200 kDa

10 kDa

Cell lysate

or nuclei

(Optional) isoelectric

focusing

+ _

Selected Ion Chromatogram

Protein Separation on nanoLC-FTMS

17279.0 Da

10

15

20 (kDa)

15928.5 Da 18414.6 Da

45 proteins detected

UniProt entries for Homo sapiens,

Release 2011-04

Features Recognized

Reduce Redundancy at the Gene and Protein

Level

Allow 213 PTMs/SNPs possible per

sequence

PROPER PRESENTATION OF HUMAN PROTEIN

DATABASES FOR MASS SPECTRAL SEARCHING

50,190 Unique

Base Sequences

20225 Entries

8.5 Million

Proteoforms

(5 GB)

• Initial Methionine

• Peptide Cleavage Events

• Known Modifications

• Sequence Variants

• Conflicts

The Process of “Shotgun Annotation”

(Kelleher Group, JACS, 2004) Allows automated detection of combinatorial

modifications

Proteomics

to the

rescue!

EXAMPLES OF

BIOLOGICAL

COMPLEXITY

HETEROZYGOTE QUANTITATION

(EXPENSIVE PCR)

Objective: Identify proteins associated with coronary

artery disease

Technique: Dif ferential Top Down Mass Spectrometry

Top Down Question: Can a specific proteoform be

associated with a biological phenotype?

STUDY DESIGN

RESULTS

ApoC-III ApoC-III

OH

Unmodified

ApoC-III

Linear Branched

SENESCENCE

CHEMOTHERAPY-INDUCED ACCELERATED

SENESCENCE

Roberson, R. S et al. Cancer research 2005, 65, 2795-803.

Wang, Q. Wu, P. C. et al. International journal of cancer. 2011, 128, 1546-58.

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

6.00E+07

7.00E+07

8.00E+07

9.00E+07

1.00E+08

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 9 Day 11 Day 14 Day 17

Ce

ll C

ou

nt

(ce

lls/m

L)

25 nM camptothecin DMSO

Treatment: 25nM camptothecin

for 24 hours

Control

Senescent Escape

HMGA1 ISOFORMS

HMGA1b:

HMGA1a:

Control

Senescent

Escape

Acetylation

Phosphorylation

Methylation

HMGA2A

HIERARCHY OF

PHOSPHORYLATION

Senescent

840 845 850 855 860 865 870 875 880m/z

1Pi

3Pi

4Pi

5PiS104, S100/S101, S44, T40

Acetylation

Phosphorylation

PROTEOMICS CENTER OF EXCELLENCE

NORTHWESTERN UNIVERSITY

http://pce.northwestern.edu

CONSORTIUM FOR TOP DOWN PROTEOMICS

(CASCAÍS, PORTUGAL)

http://www.topdownproteomics.org

SANIBEL CONFERENCE ON

TOP DOWN MASS SPECTROMETRY

http://www.asms.org/conferences/sanibel-conference



Pierre Thibault, Ph.D. Principal Investigator

Proteomics and Bioanalytical Mass Spectrometry Research Unit, IRIC,

University of Montreal

High performance MS offers new perspectives on the molecular definition of the MHC I immunopeptidome for personalized medicine

Pierre Thibault

Institute for Research in Immunology and Cancer

Department of Chemistry, Université de Montréal

36

http://www.umontreal.ca/

Outline of the presentation

What is the immunopeptidome and can MS determine its composition and changes?

How dynamic is the immunopeptidome?

Is the immunopeptidome useful for immunotherapy?

Minor histocompatibility antigens for cancer immunotherapy

IRIC/UdeM 37

Antigen presentation and processing

IRIC/UdeM 38

Antigen processing and presentation (Classical view)

MHC class I

Nucleus

Golgi

ER

Plasma membrane

CD8 TcR

MHC I-peptide complex

Secretory vesicles

Virus and endogenous antigens

Tap

Tapasin

Amino peptidase

Peptidases

Peptide epitope

Ubiquitin chain

Proteasome

MHC class II

Nucleus

Golgi

ER

CD4 TcR

MHC II-peptide complex

exogenous antigen

Invariant Chain (Ii)

Endocytic vacuole

Lysosome

CLIP

What is the immunopeptidome?

IRIC/UdeM 39

Peptides presented by MHC molecules of APC.

Binding specificity defined by MHC molecule

High diversity: > 5000 different peptides/cell

Wide abundance: 101-103 molecules/cell

Unrelated to protein abundance

Shaped by translation, proteasome degradation, cytosolic and ER proteases, MHC loading complex in ER, MHC binding affinity.

Evolved to deal with viral infections, but play important functions as odorant-based mate selection, tumor immunosurveillance and tissue rejection.

Diversity of MHC I peptides

IRIC/UdeM 40

Mouse model typically present H2b alleles:

H2Kb H2Db Qa2b

MHC motif viewer Immunogenetics 60, 759 (2008) 8-11 mers peptide

Binding in MHC I groove

Human leukocyte antigens (HLA) are highly polymorphic and are represented by HLA-A, HLA-B and HLA-C. Most frequent HLA alleles in Caucasian population are:

A*01:01 (15.1 %)

A*02:01 (27.2 %)

A*03:01 (12.6 %)

B*07:02 (11.1 %)

B*44:02 (11.7 %)

B*08:01 (10.9 %)

Allele frequency: Human Immunology 62, 1009 (2001); http://www.allelefrequencies.net/

Immunopeptidome challenges

IRIC/UdeM 41

Diversity of motifs

Low abundance (1-104 molecules/cell)

100 fmoles require ~ 108 -109 cells

Isolation of MHC I peptides

Identification of MHC I peptides by MS/MS and DB searching strategies

Identification of peptide variants (polymorphism, modifications, etc…)

Monitoring changes in peptide abundance

Validation of immunological relevance

Isolation of MHC I peptides

IRIC/UdeM

IP of MHC I peptide complex

Soluble HLA expressed by transfected cells

Elution of MHC I peptides from cells

Mild acid elution

Science, 255, 2669 (1992) Eur. J. Immunol., 30, 1172 (2000)

Availability of haplotype-specific mAb

Peptide recovery

Purification on 5kDa membrane

Cell transfection

sHLA secreted

IP isolation

Peptide purification

Generic approach applicable to primary cells

Minimization of cell lysis and contaminants

Acid release

Peptide purification

Immunogenetics 45, 379 (1997) Eur. J. Immunol. 32, 213 (2002)

J. Immunother. Emphasis Tumor Immunol. 14 : 94 (1993)

42

Identification of MHC I peptides

IRIC/UdeM 43

LTQ Orbitrap XL MS

Murine immunopeptidome

IRIC/UdeM 44

Volcano plot distribution of peptides from EL4 thymic cells

682 peptide clusters only found in WT (FC >5, p-values <0.05) ~ 1/3 of peptide population

H2Db H2Kb Qa2

Allele distribution

Binding affinity

Top 1%

Top 5%

Top 10%

Fortier et al. J. Exp. Med, 205, 595 (2008)

IRIC/UdeM 45

The immunopeptidome conceals a tissue-specific signature

z score = (intensity G Th mean intensity G T1 … T n )/SD G T1 … T n , where G Th is any gene on the microarray from the thymus tissue and T1 … T n represents the aggregate measure of all tissues.

IRIC/UdeM 47

average intensity T0

aver

age

inte

nsi

tyT48

unchanged

overexpressed (FC > 3)

significantly differentially

expressed (FC >5)

104

108

107

105

106

104 105 106 107 108

-1

0

1

2

3

0 6 12 24 48

Rapamycin (h)

log 2(

fold

chan

ge)

RICTOR proteinRICTOR MHC I peptideMat1 proteinMat1 MHC I peptide

Mol. Syst. Biol. 7, 533 (2011)

Dynamic changes of the immunopeptidome

Up

regu

lati

on

Rictor

Mat1

IRIC/UdeM 48

Identification of neoplastic antigens

IRIC/UdeM 48

• 196 peptides MHC I identified (8% total population) • 21 peptides MHC I over-expressed in neoplastic thymic

cells

X-linked lymphocyte regulated protein 3

Cyclin-dependent kinase inhibitor 1B

Fortier et al. J. Exp. Med, 205, 595 (2008)

IRIC/UdeM 49

TAA candidate selection

p-values (EL4/Thy) Source proteins Sequence

0.0203 85 ( Xlr3a) X-linked lymphocyte-regulated protein 3A VAAANREVL

0.0396 27 (RCC2) Regulator of chromosome condensation 2 AAYRNLGQNL

0.0359 17 (Pfdn5) Prefoldin 5 SMYVPGKL

0.0044 11 (Tmod1) Tropomodulin-1 SSIVNKEGL

0.0215 10 (Sgk) serum/glucocorticoid regulated kinase STLTYSRM

0.0222 9 (Dnmt1) DNA methyltransferase LSLENGTHTL

0.0183 5 (Pa2g4) Proliferation-associated protein 2G4 AQFKFTVL

0.0151 3 (Eif3s10) Eukaryotic translation initiation factor 3 subunit 10 QSIEFSRL

0.0327 3 (Cxcr4) Chemokine receptor 4 VVFQFQHI

0.0032 -3 (Cdkn1b) Cyclin-dependent kinase inhibitor 1B FGPVNHEEL

0.0186 -3 (RhoH) Rho-related GTP-binding protein YSVANHNSFL

0.0158 -6 (Rmnd5a) Required for meiotic nuclear division 5 homolog A WAVSNREML

0.0206 -10 (Ddx5) DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 NQAINPKLLQL

0.0082 -12 (Mns1) Meiosis-specific nuclear structural protein 1 KIIEFANI

0.0007 -22 (Pik3ap1) Phosphoinositide-3-kinase adaptor protein 1 YGLKNLTAL

IRIC/UdeM 50

Cancer vaccines and antitumor T cells

Dendritic cell

Naïve T-cell

Antigenic peptides

Direct binding

Activation & proliferation

Tumor cell

Processing and presentation

Tumor cell lysis & Cytokine release

Cytotoxic T lymphocyte

IRIC/UdeM 51

0 20 40 60 80 100 0

10

20

30

40

50

60

70

80

90

100

DC control

DC + STLTYSRM

DC + VAAANREVL Surv

ival

(%

)

Day post inj. EL-4

Immunopeptidome provides valuable TAAs

• Vaccinated mice infected with neoplastic thymic cells survived whereas non vaccinated mice died in less than 35 days post-injection

Immunization 106 DC (D-1, D-7)

Inj. 5 x 104 cell. EL-4

n = 10 mice per group

Minor antigens and cancer immunotherapy

IRIC/UdeM 52

• 5th most frequent cancer in human (140,000 new cases in the USA in 2009)

• The most effective treatment: Immunotherapy GVL (allogenic hematopoietic stem cells transplantation) though it remains a rudimentary form of leukemia immunotherapy

• Limitations : development of autoimmune response or graft vs. host disease, GVHD (occurrence 60%)

Leukemia

Vaccination with TAAs provides modest response rates in humans. Injection of allogeneicT lymphocytes, used for the treatment of hematopoietic malignancies, has met with a remarkable GVL effect with minimal GVHD due mainly to the recognition of MiHAs.

Identification and characterization of new minor antigens (MiHA) is key to develop an effective immunotherapy approach to leukemia

Minor antigen-targeted immunotherapy

42-yr old patient with GVHD

Nature medicine, 11, 1222 (2005)

Nature of minor antigens

IRIC/UdeM 53

Gene A Recipient Gene A Donor

... G C A A G A … … C G T T C T …

... G C A A G A … … C G T T C T …

... G C A C G A … … C G T G C T …

... G C A A G A … … C G T T C T …

MHC

MiHA

Recipient APC Donor APC

MHC

MiHA

Recipient APC

MHC

peptide TCR

MHC mismatched MHC matched

Donor T cell

Recipient APC

Donor T cell

Targeted elimination of leukemic cells

MHC

peptide

Some MHC-I peptides are polymorphic; they are present in some persons, but in other MHC-matched subjects they are absent or present a slightly different amino acid sequence.

Identification of minor antigens

IRIC/UdeM 54

GCA AGA GAT AAT CTG DNA sequence

Ala Arg Asp Asn Leu reference protein DB

GCA CGA GAT AAT CTG

Ala Arg Asp Asn Leu

synonymous SNP

GCA AAA GAT AAT CTG

Ala Lys Asp Asn Leu

non-synonymous SNP

variant matched in protein DB

variant not matched in protein DB

SNP within MHC I peptide

SNP in flanking region of MHC I peptide

Unassigned MS/MS Δ abundance (allele frequency)

Assigned MS/MS Δ abundance (processing)

Consequences of SNP on MHC I peptide

Less than 50 MiHAs identified over the past 15 years Spencer et al., Current Opinion in Organ Transplantation 2010

Challenges in the identification of MiHAs

IRIC/UdeM 55

SNPs occur every 100 to 300 bases along the 3-billion-base human genome. MiHAs estimated to represent < 1 % of MHC I peptide population

Require high throughput MS/MS sequencing

Low abundance (1-104 molecules/cell) High sensitivity MS analyses

Polymorphic variants de novo sequencing or individualized DB

Accurate fragment ion measurements

Correlation with transcriptomics

Distribution and frequency

Immunogenicity

MiHA discovery platform

IRIC/UdeM 56

Can

did

ate

sele

ctio

n

DB

sea

rch

LC

-MS/

MS

Ion profiling (label-free quantitative proteomics)

Peptide sequencing

MH

C I

pep

tid

e is

ola

tio

n

Orbitrap Elite MS

IRIC/UdeM 57

117824 high res. MSMS spectra HLA-matched patient B cells (M & R) 2D-LC-MS/MS (n=4 biol. Rep.) Orbitrap Elite MS

Mascot DB Search (Individualized DB)

26241 MS/MS assigned

MHC filter • Peptides (8-11 mers) • HLA motifs • Binding score < 750 nM • FDR 5%

3422 unique MHC I peptides

• 3303 common peptides (96.5%) • 86 peptides differentially abundant Fold change > 3, p-values <0.05 (2.5 %) • 33 peptides unique to one patient (1.0 %)

Overview of MHC I work flow and strategy for the identification of MiHAs

M R

0

1

2

3

4

5

6

-5 -4 -3 -2 -1 0 1 2 3 4 5

-Lo

g 10 (

p-v

alu

e)

Log2 (fold change)

IRIC/UdeM 58

RT: 0.00 - 70.00 SM: 15G

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70

Time (min)

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

240000

260000

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

240000

260000

Intensity

NL:2.77E5

m/z= 447.26-447.27 MS R_R1_061010_2

NL:2.77E5

m/z= 447.26-447.27 MS m_r1_061010_2

3.9 x 105

AVGPHLTAK

19.8

1.8 x 106

19.8

100 200 300 400 500 600 700 800 900

m/z

0

25

50

75

100

Rel

. In

t. (

%)

y5 y6

y8

y7

y4

y2

y6+2

y7+2

b8

y8+2

143.12

723.41 362.21

171.11 333.70

411.74 319.20 569.34 432.28 666.39 506.27

218.15 822.48

747.41 595.32

m/z 447.26 2+

m/z 447.26 2+

A T L H P G V

Time (min)

147.11

Tumor necrosis factor a-induced protein 8-like protein 1

• Differentially abundant peptides (> 3 fold change, p-values <0.05) represent ~ 2.5 % of all identified MHC I peptides across patients. • Out of 3422 MHC I peptides, 33 (1 %) are detected in only one patient

Fragment mass accuracy < 5 ppm

Example of differentially abundant MHC I peptides

Identification of MiHA from known SNPs

IRIC/UdeM 59

DNA - M

AGG => Arg (R) GGG => Gly (G)

A/G

A/G

DNA - R

Forward

Reverse

Forward

Reverse

A/G

A/G

0 5 10 15 20 25 30 35 40 45 50 55 60 65Time(min)

0

10

20

30

40

50

60

70

80

90

10039.6

1x105

0 5 10 15 20 25 30 35 40 45 50 55 60 65Time(min)

0

10

20

30

40

50

60

70

80

90

100

NL:1E51x105

WT in M

WT in R

m/z 638.36 2+

Variant in R

0 5 10 15 20 25 30 35 40 45 50 55 60 65 700

10

20

30

40

50

60

70

80

90

10043.3

2x106

Time(min)

Variant in M

m/z 588.81 2+

IRIC/UdeM 60

Identification of new MiHA

Reference DB Predicted

IC50

MHC allele Variant peptides Predicted

IC50

MHC allele

QELDRVFQKL 541 B*4403 QELDGVFQKL 809 B*4403

SPGERATL 325 B*0801 SPGESATL 2621 C*1601

SRALRLTAF 279 B*0801 STALRLTAF 306 C*1601

AMYDKGPFRSG 2122 C*1601 AMYDKGPFRSK 12 A*0301

RVSLPTSPG 7208 C*1601 RVSLPTSPR 235 A*0301

TANALKILL 2157 C*1601 TANALNILL 592 C*1601

Examples of identified MiHAs

• AA variant has minimal effect on HLA binding for position other than anchor site.

• SNP can result in significant changes in HLA binding and MHC allele distribution when the corresponding AA is located within an anchor site.

Summary

IRIC/UdeM 62

• Thermo Scientific™ Orbitrap Elite™ MS provides enhanced sensitivity, resolution and throughput for the identification of MHC I peptides and the discovery of MiHAs.

• de novo sequencing using high resolution MS/MS and Peaks facilitated the identification of new minor antigens that can be correlated by targeted sequencing (Sanger) and/or individualized DB search.

• Quantitative profiling of more than 3400 MHC I peptides in HLA-matched siblings of which 0.3% correspond to MiHAs.

Perspectives

• Identification of TAAs and MiHAs opens up new perspectives in the development of antigen-specific immunotherapy for more effective GVL.

• Opportunity for broader application in the development of T cell-based cancer immunotherapy.

Acknowledgements

IRIC/UdeM 63

Collaborators

Claude Perreault (PI: IRIC)

Etienne Gagnon, Danielle DeVerteuille,

Diana Granados, Marie-Pier Hardy,

Sylvie Brochu, Celine Laumont

Tariq Douada

Sébastien Lemieux (PI: IRIC-Bioinfo)

Grégory Voisin, Jean-Philippe Laverdure

Genomics (Pierre Chagnon)

Wafaa Yahyaoui

Missing: Marie-Helene Fortier Alexandre Bramouillé Wafaa Yahyaoui Tara Muratore-Schroeder Antoine Zieger

Danielle Caron

Christelle Pomiès Nebiyu Abshiru

Christina Bell Gaëlle Bridon

Chantal Durette Pierre Thibault

Olivier Caron-Lizotte Mathieu Courcelles

Eric Bonneil Fréderic Lamoliatte

Evgeny Kanshin Dev Sriranganadane

Absent: Roshan Elisabeth

http://www.ncic.cancer.ca/ncic/internet/home/0,,84658243___langId-en,00.html

Thank you and Q&A

IRIC/UdeM 64

http://www.umontreal.ca/



Single-Nucleotide Polymorphism and Mutation Analysis –

and Its Impact on Personalized Medicine

Q&A



Your Moderator

Tamlyn Oliver Managing Editor

Genetic Engineering & Biotechnology News



Paul M. Thomas, Ph.D. Research Assistant Professor

Department of Molecular Biosciences & Associate Director

Proteomics Center of Excellence




Pierre Thibault, Ph.D. Principal Investigator

Proteomics and Bioanalytical Mass Spectrometry Research Unit, IRIC,

University of Montreal



Thank You For Attending

Single-Nucleotide Polymorphism and Mutation Analysis –

and Its Impact on Personalized Medicine

Broadcast Date: Tuesday, December 11, 2012

Time: 11 AM ET

Sponsored by

single-nucleotide polymorphism and mutation analysis … · single-nucleotide polymorphism and...

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