The tryptic digest of lysozyme containing aspartyl hydrazide was frationated by RP-HPLC (conditions described below) and analyzed using a 4800 MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Framingham, MA). The instrument was calibrated externally daily with a calibration mixture from the Sequazyme peptide mass standard kit (P2-3143-00, Applied Biosystems, Framingham, MA). Digested samples were dried by speed-vac then re-dissolved in 0.05% trifluoroacetic acid (TFA)/50% acetonitrile in water. An aliquot of 0.6 μL was spotted on the plate and co-crystallized with an equal volume of a saturated solution of α-cyano-4-hydroxycinnamic acid (CHCA) dissolved in the same solvent. For tandom mass spectrometry experiments, the precursor mass chosen was 1818.02 m/z (1817.92 m/z calculated) corresponding to hydrazine-modified tryptic fragment 97-112 (KIVSZG ZG
Sequazyme peptide mass standard kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Sequazyme peptide mass standard kit is a laboratory equipment product designed to provide peptide mass standards for calibrating and validating mass spectrometry instruments. The kit contains a set of well-characterized peptides with known molecular weights, which can be used to ensure the accuracy and precision of mass measurements during peptide analysis.
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2 protocols using sequazyme peptide mass standard kit
1
Lysozyme Tryptic Digest Analysis by MALDI-TOF/TOF
2
MALDI-TOF Protein Identification Protocol
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SDS-PAGE gels were stained with Coomassie Brilliant Blue solution (Amresco). For MALDI-TOF analysis, the stained target bands were carefully cut from the gel, destained, trypsinized, and dried. Before the sample/matrix solution dried, 0.5 µl (∼500 femtomoles of each calibrant) of calibration mixture from the Sequazyme peptide Mass standard kit (Applied Biosystems, Framingham, MA, USA) was added on top of each spot to provide internal calibrants. Once the spots on the target plate dried, the plate was introduced into the Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems) for analysis. The resulting mass spectra were calibrated by a two-point internal calibration with one of the trypsin autolysis peaks in the calibration mixture solution. The protein database was searched using the MASCOT program.
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