Fourier Transform Mass Spectrometry Identification of Disease Biomarker Proteins 1 P. Pevsner, 1 G....
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Transcript of Fourier Transform Mass Spectrometry Identification of Disease Biomarker Proteins 1 P. Pevsner, 1 G....
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
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.
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
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
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.
1500 1550 1600 1650 1700 1750 1800
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