Agilent ICP-MS Journal - Agilent Technologies

Phosphorus in
Phosphorylated
Deoxyribonucleotides
using LC-ICP-MS
Daniel Profrock, Peter Leonhard
and Andreas Prange, GKSS Research
Centre Geesthacht, Germany.
Introduction
Phosphorus plays an important role in
cell biology for protein phosphorylation,
energy storage and transport at
the cellular level or as one essential
part of the ribose-deoxyribose
phosphorus backbone in the RNA
or DNA chain [1]. The measurement
of phosphorus would provide
information about the phosphorylation
state of a protein, which, in turn, has
a significant effect on different
metabolic pathways. Measuring
phosphorus can also be used for the
detection and quantification of RNA
or DNA due to the fixed stoichiometry
of this element in the above mentioned
macro molecules.
ICP-MS represents a highly sensitive
technique for the determination of
phosphorus in biological samples.
Moreover, the Agilent 7500c Octopole
Reaction System (ORS) ICP-MS
overcomes the limitation of
conventional systems by removing
the interferences caused by
polyatomic ions ( 14 N 16 O 1 H, 15 N 16 O)
on mass 31 P [2].
ICP-MS Experiments
The 7500c was optimized to minimize
the background of the interfering
ions on phosphorus while maintaining
good overall sensitivity.
Instrumental detection limits for
the simultaneous determination of
phosphorus and other trace elements
in an aqueous solution were
calculated.
Detection limits down to 125 ng/L
were achieved for phosphorus and
from 18 ng/L ( 55Mn) up to 49 ng/L
( 54Fe) for other trace elements
measured simultaneously. The
detection limits for all elements
measured are summarized in Table 1.
Isotope (mass) Detection limit
(ng/L)
P (31) 125
Cr (52) 21
Mn (55) 18
Fe (54) 49
Ni (58) 26
Co (59) 19
Cu (63) 25
Zn (66) 37
Cd (114) 32
Pb (208) 24
Table 1 Detection limits for phosphorus and some
selected simultaneously detected trace elements
in an aqueous, acidified solution, estimated
according to the method outlined in ref. 3.
HPLC-ICP-MS Experiments
Using UV detection at 254.4nm,
method optimization was undertaken
with a mixture of dUMP, dAMP,
dGMP, dCMP and cTMP. Based on
the results, a 15 mmol L21, pH 5.8
ammonium acetate buffer and 2.5%
methanol (v/v) were used for all
further experiments. The setup
provides baseline separation of the
five investigated compounds in ca. 12
min.
Figure 1a shows a chromatogram of
the element specific determination
of phosphorus in deoxynucleotides
obtained by HPLC-ORS-ICP-MS.
Single compound samples were used
for peak assignment. The chromatogram
shows one phosphorus- containing
(but not UV active) peak, which
remains unidentified. Detection
limits based on the compound and
on phosphorus were calculated for
each dNMP - see Table 2. An aliquot of
enzymatically digested calf thymus
DNA sample was also separated
under optimized HPLC conditions -
see Figure 1b. The four nucleotides
were baseline separated. Single
compound samples and mixtures of
commercially available deoxynucleotides
were used for peak identification.
Figure 1 Separation and element
specific detection of calf thymus
DNA digested with nuclease P1
analyzed with HPLC-ORS-ICP-MS.
(a) Mixture of dAMP, dCMP, dGMP
and dTMP (100 mg/L of each compound)
measured on the mass of 31
P for comparison of the retention
times. (b) Enzymatic digest of calf
thymus DNA with nuclease P1
measured on the mass of 31 P.
J. Anal. At. Spectrom., 2003, 18, 708-713 -
Reproduced by permission of The Royal
Society of Chemistry
The four peaks in the chromatogram
could be clearly assigned to dAMP,
dGMP, dCMP and dTMP by comparison
of the retention times. New unknown
peaks were also found during
chromatographic separation of
enzymatically digested DNA samples
which could not be identified by
comparison of the retention times.
Species DL species DL of P DL of P
(ug/L) in dNMP
(ug/L)
absolute (pg)
dAMP 48 5 50
dGMP 56 6 60
dCMP 42 4 40
dTMP 34 3 30
Table 2. Detection limit for phosphorus in
monophosphorylated deoxynucleotides obtained
with HPLC-ORS-ICP-MS
Conclusions
Using the Agilent 7500c ORS-ICP-MS
as a sophisticated detector for HPLC
has proved to be a suitable technique
for the separation and element specific
determination of phosphorylated
deoxynucleotides via the phosphorus
located in the sugar backbone of each
nucleotide. Polyatomic ions formed
in the plasma and the interface region
of the ICP-MS that interfere with the
determination of P at mass 31 were
minimized by the addition of helium
to the collision cell, allowing detection
limits down to 3 ug/L for dTMP.
Furthermore, used as a standalone
ICP-MS, the Agilent 7500c can be used
for the simultaneous determination of
phosphorus and other trace elements
in acidified aqueous solutions down
to the low-ppt level.
References
1 G. Loffler and P. E. Petrides, Biochemie
und Pathobiochemie, Springer, 1998, pp.
23-30.
2 N. Yamada, J. Takahashi and K. Sakata, J.
Anal. At. Spectrom., 2002, 17, 1213-1222.
3 D. R. Bandura, V. I. Baranov and S. C.
Tanner, Anal. Chem., 2002, 74, 1497-1502.
6 Agilent ICP-MS Journal January 2004 - Issue 18 www.agilent.com/chem/icpms