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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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15
• 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)
1
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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Anderson NL et al., J Proteome Res 2004, 3, 235-244.
Schematic Diagram for the SISCAPA Method for Peptide Quantitation
Anderson NL et al., J Proteome Res 2004, 3, 235-244.
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.
Seeley E H , Caprioli R M PNAS 2008;105:18126-18131
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IMS allows for distinction of different molecular species of β-amyloid plaques in an Alzheimer's disease model.
Seeley E H , Caprioli R M PNAS 2008;105:18126-18131
H&E-stained section and mass spectral images of a human breast carcinoma section.
Seeley E H , Caprioli R M PNAS 2008;105:18126-18131
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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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Cazares et al., Anal Bioanal Chem May 4, 2011, published on-line
Comparison of MALDI-IMS data in frozen and universal molecular fixative (UMFix)-processed tissue in a pair of matched prostate tissue samples
Cazares et al., Anal Bioanal Chem May 4, 2011, published on-line
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
Cazares et al., Anal Bioanal Chem May 4, 2011, published on-line
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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.
Cazares et al., Anal Bioanal Chem May 4, 2011, published on-line
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
Cazares et al., Anal Bioanal Chem May 4, 2011, published on-line
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.
Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li