Serendipity in the Blood: Mass spectrometry in the discovery of clinical biomarkers

AFMR SymposiumBoston, 4/24/13Mary F Lopez, DirectorBRIMS Biomarker Research Initiatives in Mass Spectrometry

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BRIMS is focused on Clinical Proteomics

MISSIONTo develop and promote comprehensive, MS-based workflows from sample preparation to bioinformatic analysis for robust

biomarker discovery and clinical validation.

To achieve its mission, BRIMS pursues collaborations with key opinion leaders engaged in clinical biomarker research to access valuable biological content with the aim of establishing Thermo Fisher workflows and platforms as the industry standard.

Biomarker Translational Center BT• Partnership with Thermo Clinical Diagnostics Division for Biomarker Discovery

and Translation to Diagnostics Informatics Center of Excellence ICE

• Development of informatics systems that will facilitate success in mass spectrometry-based Clinical Proteomics and Translational Research

www.facebook.com/brims.center

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Clinical Proteomics Research CenterThe Clinical Proteomics Research Center explores neurovascular pathophysiology using a translational approach - with the goal of developing prognostic tools to guide clinical decision-making.

MingMing Ning, MDAssistant Professor in Neurology,Harvard Medical School

Associate in Neurology,Massachusetts General Hospital

Director, Clinical Proteomics Research Center

Collaboration: BRIMS and Mass General Hospital

Discovery and verification of cerebrovascular and stroke biomarkers

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Atrial septum

•The prevalence of PFOs in the general population is around 25%, but it is doubled in cryptogenic (unknown cause) stroke patients. These patients are often young and “healthy”. •If there is a clot traveling into the right side of the heart, it can cross the PFO, enter the left atrium, and travel out of the heart and to the brain causing a stroke.•This suggests a causal relationship between PFO and cryptogenic stroke.•Supported by NIH/NINDS (Dr Tom Jacobs), MGH Cardio-Neurology Division evaluates patients with PFO related stroke and the therapeutic efficacy of surgical PFO closure and other stroke treatment.•Venous blood samples from stroke patients with PFO were taken before and at 12 month follow up after PFO closure.

•Biomarkers for PFO-related stroke could be clinically useful.

PFO and Stroke

When the atrial septum does not close properly, it is called a patent foramen ovale or PFO.

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Question:

How do the protein profiles in stroke patients differ before and after surgical closure of PFO?

Discovery and verification of cerebrovascular and stroke biomarkers

Number ofpatients

Sample type Patient

4 PFO pre OP Stroke4 Patient matched PFO

post OP, 12 moStroke

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Last year we found that albumin decreases after stroke and PFO closure

Albumin 9 peptides

PreClosure 12mo Follow up

Ratio = 0.843 +/- 0.293

PFO closure

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Could albumin-bound proteins be important in PFO?

Albumin is known to play a role in cardiovascular disease and stroke*.Do albumin-bound protein profiles in stroke patients differ before and after closure of PFO?If so, could this suggest a role for albumin in the deactivation of thrombolytic or vasoactive substances?

*Biochim Biophys Acta. 2013 Apr 17. pii: S0304-4165(13)00140-2. doi: 10.1016/j.bbagen.2013.04.012. [Epub ahead of print]Novel Insights into the Pleiotrophic Effects of Human Serum Albumin in Health and Disease.Ha CE, Bhagavan NV.SourceDepartment of Native Hawaiian Health. Electronic address: chungeun@hawaii.edu.

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Workflow for mass Spectrometry-based analysis of albumin-bound proteins

Analyze with quantitative, high resolution LC-MS/MS

Extract with anti-albumin Ab

Clinical samples

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Enrichment of albumin-binding proteins using MSIA (Mass Spectrometric Immunoassay)

Sample extraction and elution (automated)

Reduce, alkylate and digest with trypsin

Dispense eluent into a microtiter plate, and neutralize

LC-MS analysis

•Anti-albumin Ab is covalently bound to a monolithic microcolumn (1mm) in a pipette tip

•The sample volume (saturating for albumin) is repeatedly drawn up and down through the column to allow binding to Ab

•Bound sample is rinsed and eluted for analysis

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MSIA enrichment is more selective than other immunoenrichment techniques such as magnetic beads

IGF1 peptide MSIA tips

IGF1 peptide Magnetic beads

Example: High resolution LC-MS of IGF1 peptide enriched from plasma using MSIA tips vs magnetic beads demonstrates improved signal-to-noise

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What about specificity?

Negative control:

• Anti-betalactoglobulin (a non-human protein) was coupled to MSIA tips

• All samples were extracted in parallel with anti-albumin tips

• Proteins demonstrating significant binding to anti-betalactoglobulin tips were flagged and excluded from analysis

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How do we analyze the data?

MS

RT: 43.74 - 44.64

43.8 43.9 44.0 44.1 44.2 44.3 44.4 44.5 44.6Time (min)

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Rel

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bund

ance

44.06829.89

44.20829.88

44.13829.88

44.24829.89

44.31829.89

44.03829.89

44.38829.89

44.42829.89

44.46829.8943.92

829.89 44.50829.8943.89

829.8943.75

832.48 44.58829.89

44.251079.56

44.44595.32

44.10800.18

43.971218.34

44.14800.13 44.40

496.4844.52578.95

44.57741.76

43.841079.53

NL: 3.36E7Base Peak F: FTMS + p NSI Full ms [350.00-1500.00] MS MMingValdez20-Plate1-110

NL: 1.93E6Base Peak F: ITMS + c NSI d Full ms2 MS MMingValdez20-Plate1-110

RAW files(MS1 and MS2)

Proteome Discoverer SW Pinpoint SW

Create a spectral database of high confidence (1%FDR) peptides to obtain quantitative information

Database search algorithm for peptide and protein identification

Result:

• Area under the peak provides quantification

• Alignment of isotope peaks confirms identity

• Lack of interfering peaks confirms specificity

Ion chromatogram

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Albumin-bound proteins results overview

Albumin coverage = 83%Albumin-bound proteins

Number of peptides

143 18 2

12 >2total = 163

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apolipoprotein A-II preproprotein [Homo sapiens] apolipoprotein C-I precursor [Homo sapiens] apolipoprotein D precursor [Homo sapiens] C4b-binding protein alpha chain precursor [Homo sapiens] hemoglobin subunit delta [Homo sapiens] histidine-rich glycoprotein precursor [Homo sapiens] kininogen-1 isoform 2 precursor [Homo sapiens] apolipoprotein A-I preproprotein [Homo sapiens] apolipoprotein C-III precursor [Homo sapiens] apolipoprotein E precursor [Homo sapiens] hemopexin precursor [Homo sapiens] serum paraoxonase/arylesterase 1 precursor [Homo sapiens] apolipoprotein L1 isoform a precursor [Homo sapiens] apolipoprotein C-II precursor [Homo sapiens] plectin isoform 1e [Homo sapiens] complement C1s subcomponent precursor [Homo sapiens] complement C1r subcomponent precursor [Homo sapiens] vitronectin precursor [Homo sapiens] alpha-2-HS-glycoprotein preproprotein [Homo sapiens] leucine-rich repeat neuronal protein 4 precursor [Homo sapiens] vitamin K-dependent protein S preproprotein [Homo sapiens] immunoglobulin lambda-like polypeptide 5 isoform 1 [Homo sapiens]

Results: Quantified albumin-bound vs all proteins on PreClosure and FollowUp samples

• 22 proteins were common to both anti-albumin extracted and unextracted PreClosure and FollowUp samples

• 141 proteins were unique to anti-albumin extracted samples.

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PreClosure/FollowUp abundance ratio of albumin-bound proteins

• All extracted proteins were normalized to the total albumin concentration in each sample

•21 proteins were represented by >1 peptide

0.00 0.50 1.00 1.50 2.00 2.50

serum paraoxonase/arylesterase 1 …

apolipoprotein A-I preproprotein [Homo …

clusterin preproprotein [Homo sapiens]

apolipoprotein C-III precursor [Homo sapiens]

apolipoprotein E precursor [Homo sapiens]

complement C1r subcomponent precursor …

vitronectin precursor [Homo sapiens]

histidine-rich glycoprotein precursor [Homo …

kininogen-1 isoform 2 precursor [Homo …

apolipoprotein A-II preproprotein [Homo …

apolipoprotein D precursor [Homo sapiens]

microtubule-associated protein 2 isoform 1 …

alpha-1-antitrypsin precursor [Homo sapiens]

immunoglobulin lambda-like polypeptide 5 …

fibrinogen beta chain isoform 1 …

fibrinogen gamma chain isoform gamma-A …

serum albumin preproprotein [Homo sapiens]

C4b-binding protein alpha chain precursor …

fibrinogen alpha chain isoform alpha-E …

bifunctional heparan sulfate N-…

hemoglobin subunit alpha [Homo sapiens]

Ratio FollowUp/PreClosure

Ratio FollowUp/PreClosure

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Ingenuity Pathways Analysis (IPA)

Top Canonical Pathways

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Summary

• Total albumin levels are slightly decreased 12 months after stroke-related PFO closure• We have developed a workflow that is semi-automated, specific and sensitive for the analysis of albumin-bound proteins• Application of the workflow to matched PFO stroke patient PreClosure and FollowUp samples identified 128 quantifiable albumin-bound proteins• Our analysis demonstrated differential expression of albumin-bound proteins 12 months after stroke related PFO closure• Major IPA pathways (Pvalue < 0.01) included coagulation, prothrombin extrinsic and intrinsic and atherosclerosis signaling• These data are consistent with a possible role for albumin in the deactivation of thrombolytic or vasoactive substances in PFO.

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Acknowledgements

MGHMingMing NingEng LoFerdinand BuonannoSherry Chou

BRIMS TeamMary LopezDavid SarracinoBryan KrastinsJennifer SuttonMaryann VogelsangAmol PrakashScott PetermanGregory ByramGouri Vadali

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