l17 disease state profiling biomarker discovery jul8 final.ppt notes for ms short... · 1...

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Bioanalytical Instrumentation --Mass Spectrometry

Lecture 17: Disease State Profiling and Biomarker Discovery

Lingjun Li, University of Wisconsin-Madison

BME 595/CHEM 590

July 8, 2011

Tsinghua University, Beijing, China

Outline

• Introduction

• Challenges and approaches to biomarker discovery

• Tissue-based biomarker discovery

• Biofluid-based biomarker discovery

• Targeted MS for protein biomarker verification and validation

• Applications to various diseases

Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li

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Biomarker : a quantifiable characteristic associated with an expressed trait such as a normal biological state, a pathological process, or a pharmacological response to disease treatment


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Current FDA Approved Cancer Biomarkers

5 Glycoproteins, 1 Carbohydrate, 15 Proteins, 2 DNA

Ludwig and Weinstein, Nat Rev Cancer 5, 845-856 (2005).


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Overview of sample types employed in clinical studies to identify and evaluate disease target proteins respective to their discovery, verification, and validation in the biomarker development pipeline

J. Proteome Res., 2011, 10 (1), pp 5–16

Typical workflow for MS-based biomarker discovery and validation

Hawkridge & Muddiman, Annu. Rev. Anal. Chem. 2, 265-77 (2009).


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Hawkridge & Muddiman, Annu. Rev. Anal. Chem. 2, 265-77 (2009).

Hypothetical levels and data output of 4 proteins for both a healthy and a diseased individual as a function of time.

Inherent variability of plasma proteins in both healthy and diseased individuals

Street & Dear, Br J Clin Pharmacol. 69, 367-78 (2010).

Practical considerations when investigating the urinary proteome


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Dynamic plasma protein concentration range and the three main plasma protein categories are shown as reported by Anderson et al. Red dots indicate proteins identified by the HUPO plasma proteome project (PPP) and yellow dots represent currently used biomarkers in the clinic. Suitable minimal range of detection for biomarker targeting in plasma is shown with dotted lines.


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• Post-mortem tissue based assays for the presence of protease resistant prion protein.• Immunohistochemistry – Gold Standard

Schaller, et al. Acta Neuropathologica 1999, 98, 7.

• An ante-mortem test is urgently needed!

Current Diagnostics


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Blood = Great Challenge!

Hypothesis: The pathophysiological changes associated with prion infection will result in an altered protein profile of the blood plasma.

Mol Cell Proteomics2003 2: 1096-1103

Lectins with broad selectivities:• Con A (Concanavalin): mannosyl and glucosyl residues

• WGA (Wheat Germ Agglutinin): N‐acetyl‐glucosamine (GlcNAc) and sialic

acid

Comparative Glycoproteomics: Flowchart

Plasma/CSF

Immunoaffinitydepletion

LAC

Trypsin digestion

2D HPLC

RPLCMALDI FTMS

ESI MS/MS

Peptide profiles

Isotopic labeling

Wei, X and Li, L. Brief Funct Genomic Proteomic, 2009, 8, 104

Lectin Affinity Chromatography


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LC‐MS/MS of the fractions collected from high pH RPLC

2D RP‐RP Separation

Number of Proteins Identified

•A total of 708 proteins identified and relatively quantified•False Discovery Rate (FDR) = 3%

Wei, X. Herbst, A. Aiken, J. and Li, L. J Proteome Res, 2011 Submitted

Number of Unique Peptides


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Glycoproteins of Low Abundance

MS/MS spectra from tryptic peptides of representative glycoproteins.

(A) Epidermal Growth Factor Receptor400ng/mL;

(B) Angiotensin‐Converting Enzyme 450ng/mL;

(C) CD44, 81ng/mL.

Sensitivity of Detection:

CD44 (81 ng/mL)

(81ng/mL x 1uL) / 43kDa = 1.9 fmole

•Reaction can be completed in 5 min

•Labels N-terminus and Lys

•Overall 28 or 32 Da shift per incorporated label

•4 Da difference between “heavy” and “light” per label

Quantitation via Isotopic Labeling

Fu, Q and Li, Li. Anal. Chem., 2005, 77, 7783


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Expected Ratio (D:H) 1:1 2:1 5:1 10:1

HIATNAVLFFGR 0.91 2.12 3.94 5.41

YPILPEYLQCVK 1.12 1.73 4.51 5.30

GGLEPINFQTAADQAR 1.24 1.90 4.57 5.30

DILNQITKPNDVYSFSLASR 1.13 1.82 5.19 6.58

Mean 1.10 1.89 4.55 5.74

Error% 10% 5.5% 9.0% 43%

SD 0.14 0.17 0.51 0.58

Standard

(D-labeled) 1 : 2 : 5 : 10

Standard(H-labeled)

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Wei, X. Herbst, A. Schmidt, J. Aiken, J and Li, Li. LCGC North America, 2009, 27, 154

Relative Quantitation via Isotopic Labeling

Selected Proteins Showing Consistent Trends

Apolipoprotein E

Wei, X. Herbst, A. Aiken, J. and Li, L. J Proteome Res, 2011 Jun 3;10(6):2687-2702.


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XICInfected : XICControl

SCInfected: SCControl

Haptoglobin 3.8 3.2

Hemoglobin alpha chain 3.8 3.8

Isotopic Labeling (XIC) VS. Spectral Counting (SC)

LKYVMLPVADQDK TYFPIIFDVSIIGSAQVK

Wei, X. Herbst, A. Aiken, J. and Li, L. J Prot Res, 2011, 10, 2687Liu, H.,Sadygov, R.G., Yates, J.R. Anal. Chem., 2004, 76, 4193

SC Hypothesis:

Protein Abundance

MS/MS sampling rate

Serum Amyloid P‐Component (SAP)

• A secreted glycoprotein at low concentration in body fluids

• Associated with amyloid deposits in neurodegenerative diseases

• New therapeutic target for Alzheimer’s Disease

Wei, X. Herbst, A. Aiken, J. and Li, L. J Proteome Res, 2011 10, 2687-2702

Validating SAP with Western blot


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Summary – Prion Disease Biomarker

An effective analytical platform has been developedLectin Affinity Chromatography

Multidimensional Separation

LC-MS/MS

708 proteins were identified and relatively quantifiedIsotopic Labeling and Spectral Counting

53 increased, 58 decreased more than 2 folds

A list of potential candidates were generated

Selected Reaction Monitoring (SRM)/ Multiple Reaction Monitoring (MRM)

SRM – Targeted detection and quantification of selected proteotypic peptides (PTPs) w/ known fragmentation properties in a complex sample matrix. The purpose of PTPs is to serve as surrogates for the candidate protein. Two criteria for PTPs: (1) unique amino acid sequence for the candidate protein; (2) easily detectable by MS.

Aebersold et al., Curr. Opin Chem Biol 2009, 13: 518-525.


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Key information for the generation of SRM assays:• Selection of the target protein (candidate list)• PTPs have to be identified for each target protein• The best transitions for each PTP and their optimal instrument

parameters need to be determined

Targeted MS approach for protein biomarker verification

Meng and Veenstra, J Prot. 2011, In press

Absolute quantitation using MRM-MS in combination with isotope-labeled internal standard

Compare the peak area ratios between the endogenous peptides and those obtained from spiked stable isotope-labeled internal standard.


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Aebersold et al., Curr. Opin Chem Biol 2009, 13: 518-525.

Comparison of different strategies for the verification of protein biomarkers in plasma

MS Quantitation by Stable Isotope Standards for the Use with Capture by Anti-Peptide Antibodies (SISCAPA)

Anderson NL et al., J Proteome Res 2004, 3, 235-244.

In the SISCAPA method, anti-peptide antibodies immobilized on 100 nanoliter affinity columns are used to enrich specific peptides along with spiked stable-isotope-labeled internal standards of the same sequence. Upon elution from the anti-peptide antibody supports, electrospray mass spectrometry is used to quantitate the peptides (natural and labeled).

The SISCAPA-SRM method showed improved sensitivity and throughput w/o further depletion or fractionation steps. LOQ in the low ng/ml range.


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Schematic Diagram for the SISCAPA Method for Peptide Quantitation


Selection and Synthesis of Peptides for SISCAPA Technology


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Pros and Cons for the SISCAPA Method

Pros:• SISCAPA–SRM offers improved sensitivity• High-throughput sample processing is achievable because of an

automated magnetic bead-based approach and the potential to multiplex the number of peptides measured in one analysis.

Cons:• For each peptide an antibody has to be generated, which

increases the lead time and the costs for developing the SRM assay.

• Each combination of antipeptide antibodies multiplexed in a single enrichment has to be tested for interference from cross reactivity of the antibodies.

Performance profiles of targeted protein approaches for biomarker measurements in terms of limit of detection, analyte and sample throughput. These characteristics are schematically depicted for SRM-based measurements and compared to ELISA, the gold standard platform in the clinic.

Targeted Protein Approaches for Biomarker Validation

Surinova et al., J. Proteome Res., 2011, 10 (1), pp 5–16


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Impact of SRM in the verification of biomarker candidates. SRM assays for the candidate proteins are generated using synthetic peptide libraries andstored in a publicly accessible database. The optimized SRM assays are then used to determine the detectability of the candidate proteins in a subsetof plasma samples. Isotopically labeled standard peptides for the detectable candidates allow accurate quantification of the target proteins over largecohorts of plasma samples. Scheduled SRM measurements based on the elution time of the peptide permit multiplexing of hundreds of candidates ina single analysis.

Imaging MS of a Mouse Brain Tumor

Chaurand et al., Anal. Chem. 76, 87A-93A (2004).


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Work flow of an IMS experiment.

Seeley E H , Caprioli R M PNAS 2008;105:18126-18131

Coronal rat brain images of four different proteins showing very different distributions throughout the brain.



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IMS allows for distinction of different molecular species of β-amyloid plaques in an Alzheimer's disease model.


H&E-stained section and mass spectral images of a human breast carcinoma section.



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Overall Strategy for protein ID

Ducret and coworkers, MCP 5, 1876-1886 (2006).

Groseclose MR et al. J Mass Spectrom 2007, 42:254–262

On-Tissue Digestion Protocol


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MS/MS spectra of ion at m/z1460.90 acquired directly from the tissue section following digestion and matrix coverage.

Groseclose MR et al. J Mass Spectrom 2007, 42:254–262

Images of tryptic peptides generated from the digestion of the 14.2-kDa isoform of myelin basic protein

Cazares et al., Anal Bioanal Chem May 4, 2011, published on-line

Workflow of MALDI IMS for Biomarker Discovery


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Comparison of MALDI-IMS data in frozen and universal molecular fixative (UMFix)-processed tissue in a pair of matched prostate tissue samples


MALDI-IMS of prostate biopsy cores. Top panel: Biopsy cores preserved in UMFix;Bottom panel:MALDI-IMS map of the m/z 4355 MEKK2 in a serial section of the biopsy cores.Pca: prostate cancer, PIA: Prostatic Inflammatory Atrophy


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Schemes for data collection and sequence ID for endogenous peptides and proteins

In Situ Trypsin Digestion of Prostate Tissue



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Targeted MALDI-IMS for the analysis of selected biomolecules

Conjugation of a triarylmethyl (trityl) mass tag to an antibody molecule. Covalent attachment of the trityl mass tag to the primary amine of an antibody is facilitated by the N-hydroxysuccinimide ester group on the trityl molecule.


Targeted MALDI-IMS for prostate-specific antigen (PSA)


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Targeted carbohydrate affinity labeling of sialyl Lewis X antigen expression in renal tissue


Summary Points

MALDI-based tissue imaging shows potential for biomarker discovery and drug distribution profiling

The SRM technology bridges the gap between the generation of candidate lists and verification in plasma


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Future Issues

• The multitude of MS-based biomarker discovery platforms has made differentiating analytical from biological variability between labs very difficult, if not impossible.

• Careful use and systematic development of prefractionation methods for plasma have been and will continue to be essential for MS-based biomarker-discovery efforts.

• Synthesizing stable isotope-labeled internal standards at the protein level with site- and structure-specific PTMs will be critical for quantitative SRM/MRM analysis and validation.

• The entry of IMS into a discovery platform would benefit from the ability to multiplex quantitation, integration of pathology and MS data.


l17 disease state profiling biomarker discovery jul8 final.ppt notes for ms short... · 1...

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