Fourier Transform Mass Spectrometry Identification of Disease Biomarker

Proteins1P. Pevsner, 1G. Kruppa, 1T. Remsen, 2F. Naftolin, 1P. Kessler

and 1A. Stern

1Department of Pharmacology New York University School of Medicine, New York, NY,

USA

2Dept of Obstetrics and Gynecology, New York University School of Medicine, New York, NY, USA

paul.pevsner@nyumc.org

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

Colorectal tumors• gi|119592539

hCG1787564 [Homo sapiens] Mass: 57590

• gi|119592490 hCG2040674 [Homo

sapiens] Mass: 108178

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

Retinoic acid treated breast cancer cells

GI 113413194, PREDICTED: similar to Prostate, ovary, testis expressed protein on chromosome 2 [Homo sapiens]

Stroke

• gi|19923891| 4 kinesin-like 8 isoform, mw 35081

•gi|119624552| 4 kinesin light chain 4 isoform CRA_b, mw 68102

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.

Method Development

•Protein Extraction•Trypsin Digest

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

Barocycler Pressure Biosciences,

South Easton, MA

Pulse tubesBarocycler

MALDI (MATRIX ASSISTED LASER DESORPTION IONIZATION MASS SPECTROMETRY)MALDI IMAGING

(IMS)

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

Histology

Hematoxylin and

Eosin stain

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

Protein extraction and

Trypsin digest•60 second high pressure extraction of tissue section-solvent / buffer

•Lypholization of Sample•45 minute trypsin digest•Nanoflow LCMS

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%Int.

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Mass/Charge1[c].F6

1529.75{r5401}1800.93{r6804}

1790.90{r6515}

1804.88{r6799}1586.79{r6213}

1480.80{r3574} 1598.77{r6983} 1672.83{r6399}1794.88{r4616}1517.73{r6028} 1653.00{r3500}

1769.00{r3595}1511.79{r3030}

MS Spectrum

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|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

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.

Collegues and StudentsPaul KesslerTiffany RemsenDavid VecchioneAlex KogosGary KruppaFred NaftolinArnold SternJorge GhisoJonathan Melamed

Challenges of FTMS• Sample Prep• ESI-ECD/ETD• MALDI