3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P30 SOLATION OF Cu-METALLOPROTEINS
IN Mus MusCulus bRAIN ExTRACTS by
REVERSE PhASE-hPLC COuPLED TO ICP-MS
M. GOnZáLEZ-FERnánDEZ, T. GARCíA-BARRERA,
J. LóPEZ-BAREA, C. PUEYO and J. L. GóMEZ-ARIZA
Dpto. Química y Ciencias de los Materiales. Faculatad de
Ciencias Experimentales. Universidad de Huelva, Campus
de El Carmen, 21007 (Huelva), Spain,
macarena.gonzalez@dqcm.uhu.es
Introduction
Orthogonal chromatography systems coupled to ICP-
MS and organic mass spectrometry has been widely used for
metal-biomolecules characterization in biological samples
connected with in environmental issues. The use of these
analytical approaches provides very interesting information
to deep insight on toxicological concern of metals in the
environment. The use of heteroatoms can be used as markers
that simplify the traditional proteomic approaches 1,2 .
In a previous study, several organs of Mus musculus
mice were extracted and analyzed by size exclusion chromatography
coupled to UV and inductively coupled plasma
mass spectrometry (SEC-UV-ICP-MS) 3 . Some differences
were found in the molecular mass distribution patterns of elements
in the studied organs as in the case of a Cu-containing
fraction that was only present in the brain. This fraction was
collected, lyophilized and separated by reversed-phase (RP)
high performance liquid chromatography (HPLC) following
the Cu with the ICP-MS.
2D-PAGE study of the extracts was performed in
parallel, for the identification of overall proteins, after tryptic
digestion of the spots. Comparison of metallomics and proteomics
results was performed and correlated with Mus musculus
genome information from data base.
Experimental
S t a n d a r d S o l u t i o n s a n d R e a g e n t s
Methanol (Teknokroma, Barcelona, Spain) was of LC
gradient grade. Double de-ionized water (18.2 MΩ CM)
obtained from a Milli-Q water system (Millipore, Bedford,
MA, USA) was used throughout. All the reagents used were
of the highest available purity.
The buffer solution was prepared by dissolving 100 mM
of ammonium acetate (Merck, Darmstadt, Germany) in water
containing 1 % (v/v) methanol (buffer A) and in methanol
(buffer B), adjusting the pH to 7.4. Suprapur acetic acid (100%
m/m) and ammonia (25% m/m) used for pH adjustment of
the mobile phases were purcharsed from Merck 4 .
I n s t r u m e n t a t i o n
HPLC was performed with an Agilent 1100 Series
(Waldbronn, Germany). Reversed-phase HPLC was performed
with a Spherisorb ODS 2 column (250 mm × 4.6
mm, 5 μm particle size: type PEEK) (Waters, Massachusetts,
USA). The reversed-phase HPLC column was directly con-
s639
nected to the nebulizer of the ICP-MS instrument via PEEK
tubing.
Elemental detection was performed using a model
Agilent 7500ce ICP-MS instrument (Waldbronn, Germany)
Table I
Instrumental conditions for (RP-HPLC) and (ICP-MS)
Instrumental operating conditions
Reverse phase-HPLC conditions
Columns Spherisorb ODS 2 (250 mm × 4.6 mm, 5 μm)
Mobile phase Concentration gradient methanol (pH 7.4)
A: 100 mM nH 4 Ac in 1% methanol
B: 100 mM nH 4 Ac in methanol
Time [min] Buffer [%]
0 0
5 30
15 30
25 0
Flow rate 0.4 ml min -1
Injection volume 50 μl
ICP-MS conditions
Forward power 1,500 W
Plasma gas flow rate 15.0 dm –3 min –1
Auxiliary gas flow rate 1.00 dm –3 min –1
Carrier gas flow rate 0.86 dm –3 min –1
Sampling depth 6.5 mm
Sampling and skimmer cones Platinum
Dwell time 0.3 s per isotope
Isotopes monitored 63 Cu
P r o c e d u r e s
Animals and sample preparation
Mus musculus (inbred BALB/c strain) mice were from
Charles River Laboratory (Spain). Mice of 7 weeks of age
were fed ad libitum with feed conventional pellets. This feed
contained 11.9 % moisture, 16.1 % crude protein, 3.1 %
crude oil, 60 % n-free extract (including starch, sugars,
crude fibre, etc.) and 5.1 % total minerals. Cu concentrations
included in the feed were as follows (the limits recommended
for this element is indicated in parentheses): 17 mg kg –1
Cu (10–35 mg kg –1 ).
Mice were individually killed by cervical dislocation
and dissected. Individual organs were excised, weighed in
Eppendorf vials, cleaned with 0.9% naCl solution, frozen in
liquid n 2 and stored at –80 °C until they were used for extract
preparation. Mice were handled according to the norms stipulated
by the European Community. The investigation was performed
after approval by the Ethical Committee of the University
of Córdoba (Spain). Entire organs of each type (lungs,
livers, spleens, kidneys, brains, testicles, hearts and muscles)
from 20 different animals were pooled. The weight of the
brain in the pool was 3.265 g. After that a solution (3 ml g –1 )
was added containing the following: 50 mM Tris-HCl buffer
solution at pH 8, 1 mM DTT, 1 mM PMSF and protease inhibitors
(100 μl ml –1 ). Later, benzonase was added (500 U ml –1 )