Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

386 Török et al.
3. The reaction can then be terminated by gel-filtration on a Sephadex PD10 column
equilibrated in H 2O. Excess insoluble and soluble reagent is removed on
addition of 2.5 mL of the reaction mixture to the column and elution of 3.5 mL
with H 2O (see Note 1).
4. Singly labeled FL-calmodulin (calmodulin labeled with 5-DTAF on Lys 75) is
resolved from unlabeled calmodulin and doubly labeled FL-calmodulin
(calmodulin labeled with 5-DTAF on Lys 75 and Lys 148) using HPLC with a Vydac
reverse-phase C 18 column (10 × 250 mm). For analytical purposes, an aliquot
(10 µL) is chromatographed at a flow rate of 2.5 mL/min with a linear gradient
from 70% solvent A — 30% solvent B to 30% solvent A — 70% solvent B over
40 min. Absorption is measured at 215 nm and fluorescence is monitored at
450 nm (excitation) and 526 nm (emission). See ref. 6 for HPLC methodology.
5. The HPLC analysis shown in Fig. 1, should show three main absorption peaks in
the following order:
a. Unlabeled calmodulin — absorption peak with no associated fluorescence
b. Singly labeled calmodulin — absorption peak with associated fluorescent peak.
c. Doubly labeled calmodulin — absorption peak with associated fluorescent
peak.
6. The preparative procedure follows the aforementioned method where singly
labeled FL-calmodulin is purified in several batches. The UV-absorbing peaks
corresponding to calmodulin, singly labeled FL-calmodulin, and doubly labeled
FL-calmodulin are collected, pooled, and freeze dried.
7. The freeze-dried product is desalted by dissolving it in Tris-HCl, pH 7.5 buffer
and passing the solution through an H 2O-equilibrated PD10 column. Again,
2.5 mL of the solution is applied and 3.5 mL is eluted with H 2O. This solution is
then freeze dried.
8. Protein molecular weights were determined by electrospray ionization mass spectrometry
(7,8) on a Platform single-quadrupole mass spectrometer (Micromass,
UK). Proteins were desalted prior to analysis using a 2 mm × 2 cm column
(Upchurch Scientific, Oak Harbor, WA) slurry packed with poros R2 (Perseptive
Biosystems, Framingham, MA) and fitted across ports 1 and 4 of a Rheodyne
7000 valve. 100–200 pmol of protein diluted in 10% acetonitrile, 0.1% formic
acid buffer were loaded onto the column via port 5 and were desalted with
250–1000 µL of the same buffer depending on the initial salt concentration. A
130-A syringe pump (Perkin Elmer) running 70% acetonitrile, 0.1% formic acid
at 10 µL/min was connected to port 2. After desalting of the protein, the Rheodyne
was switched to connect ports 1–2 and 3–4 (with port 4 connected to the mass
spectrometer) and thus protein was eluted off the column into the mass spectrometer.
The mass spectrometer was operated at an electrospray voltage of 3.5 kV, a
cone voltage of 30 V, and was calibrated using myoglobin. Electrospray mass
spectrometry of singly and doubly labeled FL-calmodulin gave a series of peaks
that correspond to protein molecules with varying net charges z.
Figure 2A shows a mass-to-charge ratio m/z of each of the major peaks, the average
mass is 17254.7 (± 5) Da. This mass represents calmodulin (16791.4 Da) with