The Bonded-Phase
Support
Metallomic Studies with
PAGE-LA-ICPMS
First, proteins are separated in their
native (metal-containing) state by using
native PAGE. This separates the
proteins based on molecular weight. We
push the protein towards metal
complexation by using a metal-loaded
buffer during separation (Le Chatelier’s
Principle).
Zn Signal
Histidine
Fa
Anode
M+
M+
ProteinA
ProteinA
The membranes are
ablated with a laser and
the metals are detected
by ICPMS.
Internal Standards for ICPTOF-MS
ETV-ICP-MS for Isobars and
Isotopes
Second Vaporization Stage
First Vaporization Stage
Graphical Illustration of %RSD
Eapplied
Auxiliary
Electrode
2
1.8
3-electrode
potentiostat
1.6
Reference
Electrode
1.4
1.2
U238/Hf178
1
U238/As75
0.8
0.6
0.2
Metal
Recovery
Stream
Clean Effluent
Stream
An electrical
potential is used
to change the
binding
characteristics of
the column.
0
6
7
8
9
10
11
12
13
14
15
Torch Position (mm)
2.0E+07
By definition, a small change
in the ratio between two
elements as a condition
changes, is indicative of a
good analyte-IS pair. The
%RSD of these ratios is used
as a quantitative measure of
internal standard
compatibility.
1.5E+07
1.0E+07
5.0E+06
0.0E+00
0
2
4
6
8
10
0
2
4
Time, s
6
8
10
6
8
10
Time, s
8.0E+06
6.0E+06
4.0E+06
2.0E+06
0.0E+00
0
2
4
6
8
10
0
2
4
Time, s
Time, s
One problem with ICP-MS is elements of the same nominal
mass (isobaric interference). ETV can be used to separate
some problematic elements based on their differing
volatilities. Rb and Sr can be separated to remove the isobar
at mass 87.
Determining the Relationship Between
%RSD and Chemical Properties
Mn+
+
M+ ProteinA
Creating Chemical-free
Remediation Systems
Mn+
hv
FRET
Fa
100000
40000
NH2
C O HN
Asparagine
NH2
Tryptophan
Glutamine
A FRET pair (a donor, Fd, and an
acceptor, Fa) lie on opposite ends
of a peptide chain. A metal ion
(Mn+) then comes into contact with
the peptide chain which has
specific binding characteristics.
Mn+
Cathode
60000
NH
CH2
Fd
hv
80000
NH2
Arginine
N
CH2
CH2
O
To ICP-MS
valve
Fd
120000
NH2
C
CH2
Though many labs rely on solution nebulization
for sample introduction, this is not always the best
technique. It can be problematic for some matrices
(e.g. salty solutions, organic solutions, and solids or
slurries). An alternative is electrothermal vaporization
(ETV). This uses a carbon tube to vaporize the sample
before introduction to the ICP-MS. Vaporization
temperatures of up to 3,000o C can be achieved in a
controlled manner. It can handle a wide variety of
sample types, and generally has higher sample
introduction efficiency than nebulizers.
0.4
Gel
-
C
+
CH2
Fluorescence resonance energy transfer (FRET) can be
used to determine various characteristics of metal
binding. FRET involves the transfer of energy between a
fluorescent donor and an acceptor molecule. The
efficiency of the energy transfer is dependent on the
distance between the molecules, which can be related to
their spectroscopic properties. This concept can be
utilized for a metal ion sensor.
Blotting
Membranes
Cathode
NH3+
Lysine
OGlutamate
OAspartate
Other Chelating Residues
Developing Fluorescencebased Sensors
Objective: Determine what metals are associated with
what proteins, and how strongly they are bound.
Next, the separated proteins/metals
are transferred to functionalized
quartz membranes through a
process known as electroblotting.
The metalloproteins are attracted to
the anode, while the positively
charged free metal ions are
attracted to the cathode.
NH
O
O
Intensity
M+
M+
OH
Tyrosine
Anion Binding
Residues
(CH2)4
C
C
R
(CH2)3
CH2
Intensity
M+
SH
Cysteine
When the metal binds, the
peptide will wrap around
the metal, decreasing the
two fluorophores’ distance
and causing the likelihood
of FRET to increase.
Free metal can be
bound and released
by exposing the
ligand to successive
reduction and
oxidation cycles.
Mn+
Mn+
Oxidation
238U/(IS)
Counter
Electrodes
Flow
Working
Electrodes
0
A
Mn+
Reduction
Mn+
20000
Mn+
Mn+
r = 0.0037
r = 0.81
60.0%
Mg 1:10
50.0%
B
Ti 10:1
40.0%
Ti 1:1
Δmass
238U/(IS)
Isotope Ratio Error
M+
COOH
CH
%RSD
M+
C
CH
%RSD
M+
H2 N
CH
CH2
H
Normalized Ratio
M+
Cation Binding Residues
Column
Ion-Exchange System
Amino Acid
Sample
For the past several years, one of the primary
focuses of our research group has been the
development of novel ion-exchange systems for the
purpose of metal remediation from aqueous systems.
Expanding on hints from Mother Nature, we chose to
explore the metal chelation abilities of proteins and, in
particular, their constituent amino acids. In order to
simplify these ion-exchange systems, short-chain
homopolymers consisting of repeating monomers of a
specified amino acid residue have been used. These
systems exhibit many of the characteristics for an ideal
ion-exchanger – strong binding; fast, efficient release
and structural stability. These biologically-based
systems also have the added benefit of being
environmentally friendly, unlike many traditional
exchange systems which require harsh extraction
agents.
ICP-MS is the cutting edge technology for atomic
spectrometry. It can offer part per trillion detection
limits, over 5 orders of magnitude of linear response,
and works for almost all elements in the periodic table.
It uses an inductively coupled plasma (~8,000 K) as the
ionization source. Our ICP-MS uses a time of flight
system for mass analysis.
ΔIP
30.0%
Ba 10:1
20.0%
Ba 1:1
10.0%
0.0%
2050
-10.0%
2100
2150
2200
2250
2300
2350
2400
-20.0%
-30.0%
-40.0%
Scale up of the
electrochemical reactor to
practical size requires
consideration of materials,
geometry, operating
conditions, and overall cost.
Detector Potential
Each point on the scatter plots illustrated in the example plot
above represents a ratio of 238U and one of approximately 100
IS considered. Analyte-to-IS mass separation typically offered
the strongest and most consistent relationship to %RSD for
all conditions.
The time of flight design is able to offer excellent isotope ratio
precision as a result of simultaneous ion extraction from the
plasma. However, difficulties have been encountered with ratio
accuracy. Factors that cause this and possible fixes are
actively being researched.
Distance
Questions? Email Isaac.
isaac.arnquist@mail.utexas.edu
Questions? Email Shelly.
slcasciato@mail.utexas.edu
2450
Questions? Email Ram.
ramk@mail.utexas.edu
Questions? Email Haley.
hjfinjo@mail.utexas.edu
Visit us! On the web: http://research.cm.utexas.edu/jholcombe/ In the Lab: Welch 3.240 and 3.238
Questions? Email Adam.
adamrowland@mail.utexas.edu