1. Serendipity in the Blood: Mass spectrometry in the discovery of clinical biomarkers AFMR Symposium Boston, 4/24/13 Mary F Lopez, Director BRIMS Biomarker Research Initiatives in Mass Spectrometry

2. 2 BRIMS is focused on Clinical Proteomics MISSION To 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

3. 3 Clinical Proteomics Research Center The 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, MD Assistant 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

4. 4 proprietary & confidential 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.

5. 5 Proprietary & Confidential 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

6. 6 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

7. 7 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. Source Department of Native Hawaiian Health. Electronic address: chungeun@hawaii.edu.

8. 8 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

9. 9 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 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

10. 10 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

11. 11 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

12. 12 How do we analyze the data? MS 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

13. 13 Albumin-bound proteins results overview Albumin coverage = 83%

14. 14 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.

15. 15 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

16. 16 Ingenuity Pathways Analysis (IPA) Top Canonical Pathways

17. 17 Proprietary & Confidential 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.

18. 18 Acknowledgements MGH MingMing Ning Eng Lo Ferdinand Buonanno Sherry Chou BRIMS Team Mary Lopez David Sarracino Bryan Krastins Jennifer Sutton Maryann Vogelsang Amol Prakash Scott Peterman Gregory Byram Gouri Vadali Follow us!