Sample Spotting:
The acidified digest was spotted directly onto a thin layer of matrix on a stainless steel target plate. The matrix consisted of the following:
Four parts of a saturated solution of a-cyano-4-hydroxycinnamic acid (CCA) in acetone was mixed with one part of a 1:1 mixture of acetone:isopropanol
containing 10 mg/ml nitrocellulose.
Standards:
Digest are externally calibrated against a calibration curve of the following peptides to yield data with mass accuracies of better than 50 ppm.
| Monoisotopic Mass [M+H]+ |
Peptide (catalogue number) |
| 1046.5423 |
Angiotensin II (Sigma A9525) |
| 1296.6900 |
Angiotensin I (Sigma A9650) |
| 1347.7359 |
Substance P (Sigma S6883) |
| 1619.8229 |
Bombesin (Sigma B4272) |
| 2093.0900 |
Adrenocorticotropic Hormone Clip 1-17 (Sigma A2407) |
| 2465.2027 |
Adrenocorticotropic Hormone Clip 18-39 (Sigma A0673) |
| 3147.4700 |
Somatostatin (Sigma S6135) |
Where improved accuracy is required the occurrence in the sample spectrum of the autolysis peaks (842.509, 2211.104) of porcin trypsin (modified
sequencing grade Promega catalogue no. V5111) allows internal calibration to better than 30 ppm.
Mass Spectrometry:
Analysis of peptide digest was carried out on a Reflex III MALDI-ToF mass spectrometer (Bruker UK Ltd., Coventry). A nitrogen laser was used to
desorb/ionise the matrix/analyte material, and ions were detected in positive ion reflectron mode.
For further information about MALDI-ToF MS and Interpretation of Peptide Mass Fingerprint Spectra, see the instrumentation section of this website.
Database Searching:
The calibrated spectra for each sample were searched against a weekly updated copy of the SPtrEMBL database, using an in-house version (v1.9) of
the MASCOT search tool.
For further information about MASCOT visit: http://www.matrixscience.com
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