2. Fourier Transform Mass Spectrometry Identification of Disease Biomarker Proteins 1 P. Pevsner, 1 G. Kruppa, 1 T. Remsen, 2 F. Naftolin, 1 P. Kessler and 1 A. Stern 1 Department of Pharmacology New York University School of Medicine, New York, NY, USA 2 Dept of Obstetrics and Gynecology, New York University School of Medicine, New York, NY, USA paul.pevsner@nyumc.org

3. Protein Biomarkers Colorectal tumors In vitro fertilization embryos Retinoic acid treated breast cancer cells Stroke

4. Colorectal tumors gi|119592539 hCG1787564 [Homo sapiens] Mass: 57590 gi|119592490 hCG2040674 [Homo sapiens] Mass: 108178

5. In vitro fertilization high potential embryos gamma-aminobutyric- acid receptor subunit tetatricopeptide repeat protein 9

6. Retinoic acid treated breast cancer cells GI 113413194, PREDICTED: similar to Prostate, ovary, testis expressed protein on chromosome 2 [Homo sapiens]

7. Stroke gi|19923891| 4 kinesin-like 8 isoform, mw 35081 gi|119624552| 4 kinesin light chain 4 isoform CRA_b, mw 68102

8. Background and Significance The probability of cure is directly related to the stage of the cancer at diagnosis and surgical resection. Identifying patients with micro metastatic Colorectal cancer is the second-leading cause of cancer death in the USA. disease who have been shown to benefit from adjuvant therapy is presently difficult due to a lack of reliable biomarkers.

9. Method Development Protein Extraction Trypsin Digest

10. High Pressure Extraction All tissues have a pressure profile High pressure protein yield High pressure duration of trypsin digest from 12 hours to < 45 minutes

11. Barocycler Pressure Biosciences, South Easton, MA Pulse tubesBarocycler

12. MALDI (MATRIX ASSISTED LASER DESORPTION IONIZATION MASS SPECTROMETRY) MALDI IMAGING (IMS)

13. Tissue Preparation Cryoprotection to prevent freezer artifact Cryosection in ice to avoid polymers from embedding material (OCT) Cryosections 1µ thick Histology IMS Protein extraction and trypsin digest

14. Histology Hematoxylin and Eosin stain

15. IMS Place cryosections on conductive plates Wash section with ethanol followed by xylene Add protein calibrants Sublimation of Sinapic Acid Matrix onto plate avoiding solvents BioMap software reconstruction

16. Protein extraction and Trypsin digest 60 second high pressure extraction of tissue section-solvent / buffer Lypholization of Sample 45 minute trypsin digest Nanoflow LCMS

19. MS Spectrum

20. Type of search : Peptide Mass Fingerprint Enzyme : Trypsin Fixed modifications : Carbamidomethyl (C) Mass values : Monoisotopic Protein Mass : Unrestricted Peptide Mass Tolerance : ± 0.3 Da Peptide Charge State : 1+ Max Missed Cleavages : 1 Number of queries : 23 gi|119592539gi|119592539 Mass: 57590 Score: 71 Expect: 0.015 Queries matched: 9 hCG1787564 [Homo sapiens] 12 - 23 R.DVAIEFSQEEWK.C 80 - 92 R.HHIGDFSFQEIEK.D 212 - 222 R.EKSFQCIESGK.A 223 - 232 K.AFNYSSLLRK.H 263 - 272 R.RCHTGEKPYK.C 340 - 352 K.SYLACHRSIHTGK.K 403 - 412 R.RLHTGEKPYK.C 404 - 412 R.LHTGEKPYK.C 460 - 474 R.LHSGEKPCKCGECDK.A

21. Colorectal Cancer High Pressure tissue extraction Shortens Sample Preparation Increases protein yield Decreases trypsin digest time IMS may alter the current paradigm of tumor diagnosis and staging.

22. Collegues and Students Paul Kessler Tiffany Remsen David Vecchione Alex Kogos Gary Kruppa Fred Naftolin Arnold Stern Jorge Ghiso Jonathan Melamed

23. Challenges of FTMS Sample Prep ESI-ECD/ETD MALDI