( involves Met-specific chemical ...)Identification of Low-Level Oxidation Products in Calmodulin Using NanoSpray ,,, Ion Trap Mass Spectrometry

involves Met-specific chemical cleavage (by cyanogen bromide, CNBr) or proteolytic digestion followed by mass spectrometry.

Goal

In this report, we describe the direct analysis of a CaM tryptic digest using a technique that shows:

1) The ability of MSn to quickly and easily identify MetSO-containing peptides.

2) The method for sequencing these peptides and determining the exact site of oxidation. (This is especially useful for peptides containing Met residues)

3) Complete characterization of oxidized CaM in a single experiment using less than 1 pmol of material.


Sequence and tryptic mapping of vertebrate CaM. The arrows indicate theoretical cleavage sites for trypsin, while the letters indicate tryptic fragments observed in oxidized CaM.

Experimental Conditions

Sample Preparation: A 40-M solution of High Purity Bovine Brain Calmodulin (Calbiochem) was oxidized in air-saturated sodium phosphate buffer (20 mM, pH 7.4), containing 100-mM KCl at room temperature and purified by HPLC on a HyPURITY C8 guard column (2 2 mm) (Thermo Hypersil-Keystone) equilibrated with 0.1% (v/v) aqueous formic acid and eluted with 80% AcN/20% H2O/0.1% formic acid (v/v). CaM samples were collected, dried under vacuum, re-dissolved in 50-mM Tris-HCl buffer (pH 8.2), and digested with sequencing-grade bovine pancreas trypsin for 12 hours at 37 C with a CaM:trypsin ratio of 100:1.

Sample Analysis: The CaM digest was separated on a 150 0.18 mm HyPURITY C8 column (The
'"/>

Source:


Page: All 1 2 3 4 5 6
Related biology technology :

1. Identification of Differentially Expressed Gene Products with the CastAway System*
2. An Optimal DNA Microarray Substrate for the Identification of Fetal and Somatic Liver Stem Cells of the Rat
3. Detection and Identification of Phosphorylation Sites in Proteins Using LC/MS/MS with Neutral Fragment Loss Mapping
4. Protect RNA and Improve Target Cell Identification
5. Identification of Disease Causing Mutations in Phenylketonuria by Denaturing Gradient Gel Electrophoresis Using the DCode System
6. Identification of Nonspecific Products Using Melt-Curve Analysis on the iCycler iQ Detection System, Rev A
7. Identification of cells in S phase using the Cell Proliferation Fluorescence Kit and IN Cell Analyzer 1000
8. Identification of Insecticides and Herbicides by LC/MS/MS Using the API 2000 LC/MS/MS System
9. Identification of Phase I and Phase II Metabolites of Buspirone on the Q TRAP LC/MS/MS System
10. ESR1 Gene Expression in Oncology and Metabolic Diseases Using the ASCENTATM System Target Validation and Identification of Novel Disease Indications
11. High-Throughput System Generates Ultra-Pure PCR Products Suitable for All Applications