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A high-throughput method for Saccharomyces cerevisiae (yeast) ionomics


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Reliable and rapid analytical methods are the backbone for generating data and deciphering gene functions in the post-genomics era. We describe here a high-throughput method for the rapid profiling of fourteen elements in the 5153 strain gene deletion collection of Saccharomyces cerevisiae. Samples were grown and processed in standard 96-well plate format followed by inductively coupled plasma mass spectrometry (ICP-MS) analysis. Optical densities of the yeast were measured prior to ICP-MS analysis and used for the normalization of the data. The elemental profiling data are stored in an online database for later bioinformatics analysis. This method has the capacity to run 288 yeast samples per day on a single ICP-MS, and has allowed the quantification of the ionome in four replicate cultures of approximately 240 yeast deletion strains per week, along with appropriate wild-type and positive control strains. We identified 400 strains that were outliers from the overall deletion collection in at least one element out of the fourteen that were monitored.
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A high-throughput method for Saccharomyces cerevisiae (yeast) ionomics
John M. C. Danku,
Luke Gumaelius,
Ivan Baxter
and David E. Salt*
Received 29th February 2008, Accepted 22nd August 2008
First published as an Advance Article on the web 21st October 2008
DOI: 10.1039/b803529f
Reliable and rapid analytical methods are the backbone for generating data and deciphering gene
functions in the post-genomics era. We describe here a high-throughput method for the rapid profiling
of fourteen elements in the 5153 strain gene deletion collection of Saccharomyces cerevisiae. Samples
were grown and processed in standard 96-well plate format followed by inductively coupled plasma
mass spectrometry (ICP-MS) analysis. Optical densities of the yeast were measured prior to ICP-MS
analysis and used for the normalization of the data. The elemental profiling data are stored in an online
database for later bioinformatics analysis. This method has the capacity to run 288 yeast samples per
day on a single ICP-MS, and has allowed the quantification of the ionome in four replicate cultures of
approximately 240 yeast deletion strains per week, along with appropriate wild-type and positive
control strains. We identified 400 strains that were outliers from the overall deletion collection in at
least one element out of the fourteen that were monitored.
Ionomics is the ‘‘quantitative and simultaneous measurement of
the elemental composition of living organisms, and changes in
this composition in response to physiological stimuli, develop-
mental state and genetic modification’’.
The ionome is an
expansion on the previous concept of the ‘‘metallome’
include metalloids and non-metals.
Since the ionome is involved
in a host of biologically important phenomena such as osmo-
regulation, transport, signalling, enzymology and electrophysi-
an understanding of its genetic basis, and how it interacts
with other cellular systems is paramount. This in turn is predi-
cated on developing reliable and rapid analytical tools capable of
handling the large numbers of biological samples required for
such an analysis.
A recent study characterized the ionome of 4385 Saccharo-
myces cerevisiae (yeast) mutants strains (homozygous diploid
collection) which represent a subset of the complete yeast
knockout collection including only non-essential genes.
However, given the limited sample throughput of the method
used by Eide and co-workers only single cultures of the majority
of the 4385 yeast mutants were analyzed. Such a limited sampling
density for each mutant reduces significantly the resolution of the
yeast ionome study published by Eide and co-workers.
improve both the sampling density, and to complete analysis of
the full collection of yeast knockouts (5153 strains, MATa
haploid collection
) it is essential to streamline and optimize the
ionomic method, to allow the efficient processing of significantly
more samples without the loss of analytical precision.
We describe here a high-throughput elemental profiling
methodology for the analysis of the yeast ionome that employs
a 96-well plate format integrated into the process from sample
growth through to the final ICP-MS analysis. This method has
the capacity to analyze 288 yeast cultures per day on a single
ICP-MS. Application of this methodology has allowed the
analysis of the complete yeast gene deletion collection of 5153
strains with four replicate cultures per mutant strain. To enhance
downstream data normalization each 96-well plate also con-
tained the same set of four different control lines. Analysis of the
complete knockout collection took 6 months and represents the
processing of approximately 30 000 yeast cultures. This repre-
sents a 10-fold increase in throughput over the previous study
while maintaining a high level of precision and improved
analytical sensitivity. The availability of such a high-throughput
methodology now opens up the possibility of performing other
genome-wide ionomics analyses in yeast, including the screening
of the complete ORF (open reading frame) over expression
collection, and cDNA (complementary DNA) expression
libraries from heterologous genomes and metagenomes.
Experimental section
A quadrupole inductively coupled plasma mass spectrometry,
ICP-MS, (Elan DRC II, Perkin Elmer, Shelton, CT, USA)
coupled with an SC-2 autosampler and an Apex Q sample
introduction system (Elemental Scientific Inc., Omaha, NE,
USA) was used for analysis of the yeast cultures. A liquid
handling robot, MultiPROBE II PLUS HT EX, (Perkin Elmer)
was used to perform the initial yeast inoculation into 96-well
square deep-well plates. A shaking incubator (Shel Lab SI6,
Sheldon Mfg. Inc., Cornelius, OR, USA), which was custom
fitted to accommodate up to 15 deep-well plates (Metalhead
LLC, Salem, IN, USA), was employed for yeast cultivation.
Yeast optical densities (OD) were measured using an OpsysMR
plate reader (DYNEX Technologies, Chantilly, VA, USA).
Center for Plant Environmental Stress Physiology, 1165 Horticulture
Building, Purdue University, West Lafayette, IN 47907, USA. E-mail:
Bindley Bioscience Center, Discovery Park, Purdue University, West
Lafayette, IN 47907, USA
† Presented at the 2008 Winter Conference on Plasma Spectrochemistry,
Temecula, CA, USA, January 7–12, 2008.
This journal is ªThe Royal Society of Chemistry 2009 J. Anal. At. Spectrom., 2009, 24, 103–107 | 103
TECHNICAL NOTE | Journal of Analytical Atomic Spectrometry
Digestion of yeast samples was performed using a multi-block
heater (Lab Line Instruments, Melrose Park, IL, USA).
Custom made AcroPrep 96 PVDF (polyvinylidene fluoride) filter
membrane (0.45 mm, 350 mL) micro-well plates (Pall Life
Sciences, Ann Arbor, MI, USA) were used for processing yeast
samples. Clear View micro-plates were used for yeast optical
density measurements. 96-well square deep-well (2 mL) plates
(Axygen Scientific, Union City, CA, USA) were used both for
yeast growth and after processing for ICP-MS analysis (using the
SC-2 autosampler). Polypropylene lids (Axygen) were used to
cover the deep-well plates during sample storage and digestion,
while Axymat (Axygen) chemically resistant and flexible lids
were used to cover the plates during mixing. Adhesive breathable
sealing film (AeroSeal, Dot Scientific Inc., Burton, MI, USA)
was used to seal the deep-well plates during yeast growth. Three
multi-channel pipettors with volume ranges of 2 to 20 mL, 20 to
300 mL and 100 to 1200 mL were used to dispense yeast cultures,
reagents and solutions.
Standards and reagents
AR Select grade concentrated nitric acid from Mallinckrodt
(Phillipsburg, NJ, USA) was used for sample digestion. Single
element standard stock solutions for the calibration procedure
and for spiking yeast growth media were obtained from ULTRA
Scientific (Kingstown, RI, USA). Deionized water (18 MU) for
all dilutions was from a NANOpure Diamond (Barnstead
International, Dubuque, IA, USA) water purifier. Triton X-100
was obtained from Sigma (St. Louis, MO, USA), and was added
to both the processed samples and calibration standards to
enable smooth self aspiration up the micro-nebulizer. Sodium
chloride, sodium ethylenediaminetetraacetate (EDTA), meth-
anol and ethanol were purchased from Mallinckrodt (Phillips-
burg, NJ, USA). Synthetic defined minimal medium components
for yeast culture were from the following vendors: Yeast
Nitrogen Base with nitrogen (YNB) and CSM-Ura (complete
supplement mixture minus uracil), Sunrise Science Products
(obtained from MIDSCI, St. Louis, MO, USA); uracil from BIO
101 (Vista, CA, USA), and D-Glucose monohydrate from
Research Products International (Mt. Prospect, IL, USA).
Sample preparation
The yeast knock-out (YKO) collection used in this work was
from the yeast MATacollection generated from the BY4741
background—MATa his3O1 leu2O0 met15O0 ura3O0.
Most of the collection was obtained from Dr Tony Hazbun of
Purdue University and the rest purchased from Open Biosystems
(Huntsville, AL, USA). The stock YKO lines came in 96-well
micro-plates and were maintained at 80 C.
Growth of yeast was carried out in two stages. The first stage,
preliminary growth or pre-growth, involved bulking up from the
stock collection; the second, growth for analysis or simply
growth, was for both the ICP-MS analysis and the corresponding
optical density measurement. The yeast culture medium used
was synthetic defined minimal (YNB + CSM Ura + uracil)
medium. The minimal growth medium was supplemented with
the following elements for the second stage of growth: 2 ppb Co,
20 ppb Cd, 50 ppb Mo, 100 ppb Ni and 200 ppm Na. This was to
either compensate for the elements lacking (Co, Cd, Ni) in the
synthetic medium or else to increase the levels (Mo, Na) for
better ICP-MS detection. Note that the same lots or mix of lots,
of medium components were used throughout this work to
ensure consistent growth conditions. In all cases growth was
carried out in 96-well square deep-well plates.
For pre-growth 5 mL of starter yeast stock (inoculate) was
added to 500 mL of medium per well. The plate was sealed with
breathable sealing film and incubated at 30 C and 400 rpm for
48 h. Usually two and a half 96-well plates of the primary yeast
collection were utilized per week for analysis, along with back-
ground, wild-type and positive controls strains. During the
growth stage 20 mL of yeast inoculate was added to 750 mLof
supplemented medium per well. This stage required the use of
a liquid handling robot. A total of twelve 96 deep-well plates
were usually generated from the two and a half bulked pre-
growth yeast plates. Each plate had 20 yeast lines transferred
from a pre-growth plate with four replicates per line. These
covered 10 of the 12 columns of the plate (two lines per column)
with the background and wild-type, and the two positive control
lines occupying the other two columns. The plates were sealed as
before. Three plates per day were grown with the rest kept in the
refrigerator (4 C) and grown on successive days. The incubation
conditions in this case were 30 C and 400 rpm for 36 h. The yeast
cells are at the post-diauxic growth period before harvesting for
both of these growth conditions. The cells grow rather slowly
during this growth phase.
Eide and co-workers
harvested yeast
cells at a similar growth phase.
Sample processing for ICP-MS analysis, until the final step,
was performed in AcroPrep 96 PVDF filter membrane micro-
plates. The hydrophobic membrane of the plate was wetted with
methanol and then rinsed with deionized water. Yeast cultures
(200 mL well
) were transferred from the growth plates into filter
plates using multi-channel pipettes. The same amounts were
concurrently transferred into Clear View microtiter plates and
the optical densities measured with a plate reader. The cells in
the filter plates were washed and rinsed in situ, respectively, with
EDTA (1 mM, pH 8) and deionized water, using a vacuum
manifold. Four separate washes and rinses were performed
(350 mL well
each). The filter plates were dried (88 C for 2 h)
to restore the membranes hydrophobicity. Washed yeast cells
were digested directly inside the filter plates (100 mL well
acid, 88 C for 40–45 min) using a heating block. The yeast
digests were drawn through the filter and into 96 deep-well
collection plates containing Triton X-100 (0.025% v/v, 300 mL
) with Ga (6.67 ppb) internal standard solution. The final
dilution volume was 1.6 mL well
including Ga (5 ppb) internal
standard and Triton X-100 (0.005% v/v).
ICP-MS analysis
The processed yeast samples were run on an Elan DRC II ICP-
MS equipped with ESI SC-2 autosampler unit that could
accommodate 96 deep-well plates, and an Apex Q sample
introduction system. Triton X-100 (0.005% v/v) was also added
to the calibration standards as well as the wash solution to reduce
surface tension and enable smoother self aspiration of the PFA
104 | J. Anal. At. Spectrom., 2009, 24, 103–107 This journal is ªThe Royal Society of Chemistry 2009
micro-nebulizer. Fourteen elements (Na, Mg, P, S, K, Ca, Mn,
Fe, Co, Ni, Cu, Zn, Mo, Cd) were monitored in the yeast
A flowchart representation of the yeast ionomics high-
throughput workflow is shown in Fig. 1.
Results and discussion
The method reported here is a low cost, rapid, robust and
sensitive method for the elemental analysis of yeast, allowing for
the precise analysis of many thousands of samples with the
capacity to resolve small ionomic differences between samples.
Three 96-well plates of yeast cultures can be processed per day
using a single ICP-MS instrument. Currently, we analyze
approximately 240 yeast deletion strains per week, each in
replicates of four cultures, along with background, wild-type and
positive control strains. This translates into a throughput of
about 30 000 samples in 6 months. In comparison Eide and
analyzed approximately 10 000 samples in 24
months. Fourteen biologically essential or potentially toxic
elements (Na, Mg, P, S, K, Ca, Mn, Fe, Co, Ni, Cu, Zn, Mo, Cd)
are quantified in the yeast samples, and the elemental profiling
data stored in an online ionomics database, based on the Purdue
Ionomics Information Management System (PIIMS).
lithium, arsenic and selenium were also monitored during the
preliminary stages but later dropped due to low detection of these
elements in the yeast samples. Even after supplementation of the
culture medium with these elements there was not an appreciable
uptake by the yeast cells. ICP-MS is needed for the method
described here. However, ICP-OES (optical emission spectros-
copy) could also be used but would require minor modifications
of the protocol. For instance, a larger amount of yeast culture
would need to be digested to increase the elemental concentrations
in the sample, or a larger flow PFA micro-nebulizer could be
used with the Apex sample introduction system in order to
increase the amount of sample reaching the plasma. Assuming one
already has the necessary equipment and personnel in place, the
cost for sample analysis in the method reported here is under
US$200 per 96-well plate (or $2 per sample; 96 samples per
plate), making this a truly low cost, high throughput method.
In order to develop a high-throughput ionomic method that
not only can rapidly characterize many thousands of yeast
strains, but can do so in a robust, routine and analytically precise
manner, several factors have to be taken into account. Chief
among these is to use standardized instrumentation, equipment
and materials. The 96-well plate sample format was deemed
suitable for this purpose. It allows for parallel handling of large
numbers of yeast strains and can fit nicely into most liquid
handling robotic systems, and some specially configured ICP-MS
autosampler units. The method reported here was therefore
designed to allow the use of a 96-well plate format from yeast
cultivation through to ICP-MS analysis.
Yeast culture in a 96-well plate format requires the correct
amount of agitation, media volume and headspace to allow
adequate aeration for uniformity of yeast growth in all wells. The
initial conditions were adapted from work performed on bacte-
rial strains.
Preliminary experiments were performed to estab-
lish the ideal orbital shaking frequency of 400 rpm. The media
volumes used for the pre-growth and growth stages were selected
based on the amount needed to adequately carry out the yeast
culture processing, and the fact that there is some evaporative
loss during the cultivation. Evaporative loss was found to be
practically identical in all wells, and independent of position in
the incubator.
The custom ordered AcroPrep 96-well plate with poly-
vinylidene fluoride (PVDF) hydrophobic filter membrane was
chosen because the hydrophobic/hydrophilic nature of the
membrane can be switched by wetting with solvent, drying or
acid digestion. Once yeast cells had been dispensed into the wells,
this membrane property means that all the various sample
processing stages, including washing and digestion, can occur in
the same plate, minimizing the need for repeated transfers
between plates, reducing sample loss associated with transfers,
and the potential for contamination.
As part of this high-throughput yeast ionomic methodology,
we include 4 control yeast strains in each 96-well plate. These
control strains are the parental line for the knockout collection
YDL227c, which is derived from BY4741, along with two
previously identified yeast ionomic mutants
YLR396c (deletion
of VPS33 involved in vacuolar function) and YPR065w (deletion
of ROX1 involved in mineral nutrient homeostasis). YPR065w
and YLR396c, known to have elevated or depressed concentra-
tions of several elements (including Na, Mg, S, K, Mn, Co),
used as positive controls to confirm that for each 96-well plate
analyzed the analytical methodology is working correctly. The
ionomic profiles for these positive control lines are displayed in
Fig. 2A and B. Here the elements monitored by ICP-MS are
plotted against their z-scores, which represent the number of
standard deviations away from the mean of the control parental
line (YDL227c) grown in the same 96-well plate. Arabidopsis
Fig. 1 Yeast ionomics high-throughput method workflow. OD—optical
This journal is ªThe Royal Society of Chemistry 2009 J. Anal. At. Spectrom., 2009, 24, 103–107 | 105
thaliana shoot ionomic data was previously displayed in a similar
It is clear from this type of z-score plot that both
positive control lines, YLR396c and YPR065w, show major
differences in several elements when compared to the YDL227c
background. YLR396c has elevated levels of Mg, S, Co, Ni and
Cd, and reduced Na, K, Mn, Cu and Mo levels. Whereas
YPR065w has elevated levels of K, Co, Ni and Cd, and reduced
Na, Mg, Ca and Mo levels.
In any high-throughput analysis platform, where comparisons
need to be made between samples, analytical precision is critical.
A precise measurement is one that when made repeatedly on the
same sample will give the same answer. Similarly, repeated
ionomic analyses of two different yeast strains, if made in
a precise way, would be expected to produce two sets of similar
ionomic profiles. Such profiles, if analyzed using Principal
Component Analysis (PCA) would form two clear clusters of
points associated with each of the two yeast strains. Using this
approach we show that the four control yeast strains BY4741,
YDL227c, YLR396c and YPR065w, included in all of the
96-well plates analyzed in this screen, form discrete clusters based
on a PCA of their ionomic profiles (Fig. 3). Such clear clustering
of strains, based on their ionomic profile measured repeated
across 356 96-well plates, clearly establishes the high precision of
our method; from yeast cultivation through ICP-MS analysis.
In this screen the first 850 yeast deletion strains were run
with eight replicate cultures, and eight deletion strains per plate.
Statistical simulations based on this initial data set established
that the high precision of the method would allow the reduction
of the number of replicate yeast cultures needed in the analysis.
The difference in means between the eight replicates and
a randomly selected set of four replicates was calculated for each
element across the 850 lines. All of the elements except Ca had
a difference <10% for 95% of the strains, whereas the mean
difference for Ca of <22%. This was attributed to the low levels
of Ca in the wild-type yeast. Given the only minor differences in
mean differences between eight and four replicate cultures we
adopted four replicate cultures for the rest of the screen. This
reduced level of replication allowed an increase in the number of
yeast strains that could be analyzed per 96-well plate, producing
a significant increase in throughput without loss of sensitivity to
detect ionomic differences between yeast strains.
High sensitivity, or the ability to detect small ionomic differ-
ences between yeast strains, is another important parameter of
this methodology. A major factor limiting sensitivity is the
reproducibility or precision of the measuring device.
Here we
express the sensitivity as the average percent relative standard
deviation (%RSD) for each element, based on each line across the
356 96-well plates analyzed in the screen. That is, if the %RSD is
small then we would expect to be able to detect small differences
between yeast strains. Table 1 shows the %RSD for each line
analyzed in every 96-well plate run in this screen (n¼6888
individual samples) at different confidence levels. It is apparent
from Table 1 that most of the elements monitored have 95% of
their %RSD values < 12% (Mg, P, K, Mn, Co, Ni, Cu, Zn and
Cd). The corresponding values for Na, Mo, S and Fe are <
20%RSD. Again, Ca shows the highest value of 32%RSD, due to
its rather low levels in yeast. Overall, the %RSD values measured
across the complete screen are low, allowing detection of small
differences in the ionome of the yeast strains in the deletion
collection. The %RSDs obtained from yeast are generally lower
than those previously observed in a genomic-scale ionomic
Fig. 2 Ion profile data for yeast mutant lines. Standard deviations (s.d.)
from the mean for (A) positive control yeast, YLR396c, and (B) positive
control yeast, YPR065w. Mean and standard deviations are calculated
for each element from the background yeast, YDL227c (n¼4), and used
to calculate the number of standard deviations each strain is distant from
the mean background value for each element. Same scaling on ordinate
used to highlight differences in mutant magnitude.
Fig. 3 Principal component analysis of the yeast ionome of the four
yeast control strains included in the 356 96-well plates analyzed. Black—
BY4741, blue—YDL227c, red—YPR065w, light grey—YLR396c.
106 | J. Anal. At. Spectrom., 2009, 24, 103–107 This journal is ªThe Royal Society of Chemistry 2009
screen of A. thaliana,
likely due to the more reproducible
method of cultivation and sample analysis afforded by yeast.
A preliminary analysis of the ionomic data set from the yeast
gene deletion collection identified 400 strains that display
differences from the overall average of the complete deletion set,
with at least one element exceeding three z-scores (out of a total
of 4952 lines, excluding repeat lines). Eide et al.
identified 233
strains using the same criterion (out of 4385 yeast mutants).
In the present study, a low cost, robust, precise, sensitive and
high-throughput method for profiling the yeast ionome was
developed. The method involves a novel integration of 96-well
plate sample formats for yeast cultivation, sample preparation
and ICP-MS analysis, with the power of simultaneous determi-
nation of a broad range of elements using ICP-MS. This analysis
platform was coupled to an online ionomics database (Yeast-
PIIMS) for data management. The value of this system has
been established by using it to profile fourteen elements across
the entire set of 5153 mutant strains in the yeast gene deletion
collection, representing the analysis of approximately 30 000
yeast cultures in a 6 month period. Bioinformatics analysis of this
dataset is currently ongoing to identify the genes and gene
networks involved in regulating the yeast ionome.
We thank Brett Lahner for developing the original spreadsheet
for analyzing the raw ICP-MS data. We are grateful to Tony
Hazbun for supplying most of the stock yeast for this work and
his helpful discussion concerning growing of the yeast, and we
thank Brad Kennedy of the Purdue University Discovery Park
Cyber Center for help developing the YeastPIIMS information
management system. The study was supported by the National
Institutes of Health (4 R33 DK070290-02).
1 D. E. Salt, I. Baxter and B. Lahner, Annu. Rev. Plant Biol., 2008, 59,
2 C. E. Outtern and T. V. O’Halloran, Science, 2001, 292, 2488.
3 R. J. P. Williams, Coord. Chem. Rev., 2001, 216, 583.
4 B. Lahner, J. Gong, M. Mahmoudian, E. L. Smith, K. B. Abid,
E. E. Rogers, M. L. Guerinot, J. F. Harper, J. M. Ward,
L. McIntyre, J. I. Schroeder and D. E. Salt, Nat. Biotechnol., 2003,
21, 1215.
5 D. E. Salt, Plant Physiol., 2004, 136, 2451.
6 D. J. Eide, S. Clark, T. M. Nair, M. Gehl, M. Gribskov,
M. L. Guerinot and J. F. Harper, Genome Biol., 2005, 6, R77.
7 C. B. Brachmann, A. Davies, G. J. Cost, E. Caputo, J. Li, P. Hieter
and J. D. Boeke, Yeast, 1998, 143, 115.
8 E. A. Einzeler, D. D. Shoemaker, A. Astromoff, H. Liang,
K. Anderson, B. Andre, R. Bangham, R. Benito, J. D. Boeke,
H. Bussey, A. M. Chu, C. Connelly, K. Davis, F. Dietrich,
S. W. Dow, M. El Bakkoury, F. Foury, S. H. Friend, E. Gentalen,
G. Giaever, J. H. Hegemann, T. Jones, M. Laub, H. Liao,
N. Liebundguth, D. J. Lockhart, A. Lucau-Danila, M. Lussier,
N. M’Rabet, P. Menard, M. Mittmann, C. Pai, C. Rebischung,
J. L. Revuelta, L. Riles, C. J. Roberts, P. Ross-MacDonald,
B. Scherens, M. Snyder, S. Sookhai-Mahadeo, R. K. Storms,
S. Ve
´ronneau, M. Voet, G. Volckaert, T. R. Ward, R. Wysocki,
G. S. Yen, K. Yu, K. Zimmermann, P. Philippsen, M. Johnston
and R. W. Davis, Science, 1999, 285, 901.
9 P. K. Herman, Curr. Opin. Microbiol., 2002, 5, 602.
10 I. Baxter, M. Ouzzani, S. Orcun, B. Kennedy, S. S. Jandhyala and
D. E. Salt, Plant Physiol., 2007, 143, 600.
11 W. A. Duetz, L. Ru
¨edi, R. Hermann, K. O’Connor, J. Bu
¨chs and
B. Witholt, Appl. Environ. Microbiol., 2000, 66, 2641.
12 D. A. Skoog and J. J. Leary, Principles of Instrumental Analysis,
Saunders College Publishing, Philadelphia, USA, 4th edn, 1992.
Table 1 Percent relative standard deviation (%RSD) for each element calculated across all yeast strains analyzed (n¼6888) at different confidence
Confidence level Na Mg P S K Ca Mn Fe Co Ni Cu Zn Mo Cd
50% 6.0 4.3 3.8 5.4 5.1 8.5 4.5 8.2 3.9 4.6 4.4 4.2 7.3 3.8
75% 8.9 6.1 5.4 7.9 7.2 13.9 6.1 11.8 5.2 6.6 6.2 5.9 10.7 5.1
90% 13.2 8.4 7.6 12.2 9.5 22.4 8.2 16.2 6.6 9.3 8.1 7.7 14.8 6.7
95% 17.6 10.8 10.8 17.4 11.3 32.1 9.7 19.3 7.8 11.7 9.7 9.2 17.6 7.8
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... Inductively coupled plasma mass spectrometry (ICP-MS) is a technology used in systems biology and clinical research to profile the concentration of elements within samples and cells of living organisms (Amais et al., 2020;Barkla et al., 2016;Baxter, 2010;Konz et al., 2017;Meyer et al., 2018). This technology has been coupled with screening experiments using genetic modifications to study genome-wide genetic associations that are revealed by phenotypical cellular responses at the level of element abundances, in diverse model organisms, including yeast (Danku et al., 2009;Eide et al., 2005;Yu et al., 2012), and plants Chao et al., 2011;Salt et al., 2008). ...
... ICP-MS data were normalised by optical density (OD). Experimental details of the dataset are described in (Danku et al., 2009). Most of the cell lines (4207) were measured in 4 replicates, 684 lines in 8 replicates, 48 lines in 12 replicates, and 2 lines in 16 replicates giving a total of 26,976 samples screened in 305 different plates. ...
... It is of interest to compare Fig. 2D with a previously reported diploid S.Cerevisia ionome quantification (Cyert & Philpott, 2013;Eide et al., 2005) to gain insights into the genetic background and the external cellular environment and how they play a role in the internal elemental balance of the cell. Despite the level of potentially toxic elements such as cadmium and sodium being artificially increased in the yeast growth media for the data set under study (Danku et al., 2009) most of the elements agree in order of magnitude with the level reported in (Cyert & Philpott, 2013). ...
Full-text available
Introduction Inductively coupled plasma mass spectrometry (ICP-MS) experiments generate complex multi-dimensional data sets that require specialist data analysis tools. Objective Here we describe tools to facilitate analysis of the ionome composed of high-throughput elemental profiling data. Methods IonFlow is a Galaxy tool written in R for ionomics data analysis and is freely accessible at . It is designed as a pipeline that can process raw data to enable exploration and interpretation using multivariate statistical techniques and network-based algorithms, including principal components analysis, hierarchical clustering, relevance network extraction and analysis, and gene set enrichment analysis. Results and Conclusion The pipeline is described and tested on two benchmark data sets of the haploid S. Cerevisiae ionome and of the human HeLa cell ionome.
... The most reliable and accurate data are those for potassium, calcium, manganese, copper, zinc, molybdenum and cadmium. Exact quantitative determination of elements was conducted according to the method of Salt and co-workers (Danku et al., 2009;Salt et al., 2008) with the calculation based on Lahner et al. (2003) -and after removing the elements contained in the artificial sea water used in the short-term experiments. The choice of elements was determined both by what are the routine elements analyzed in relatively closely related higher plants and by the analytical capabilities available (Salt et al., 2008). ...
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The Little Neptune grass Cymodocea nodosa is a key seagrass species in the Mediterranean Sea, forming extensive and patchy meadows in shallow coastal and transitional ecosystems. In such habitats, high temperatures and salinities, separately and in combination, can be significant stressors in the context of climate change, particularly during heatwave events, and seawater desalination plant effluents. Despite well-documented negative, macroscopic effects, the underlying cellular and molecular processes of the combined effects of increasing temperature and salinities have remained largely elusive in C. nodosa – which are addressed by the present study. High salinity and high temperature, alone and in combination, affected ion equilibrium in the plant cells. Non-synonymous mutations marked the transcriptomic response to salinity and temperature stress at loci related to osmotic stress. Cell structure, especially the nucleus, chloroplasts, mitochondria and organization of the MT cytoskeleton, was also altered. Both temperature and salinity stress negatively affected photosynthetic activity as evidenced by ΔF/Fm’, following an antagonistic interaction type. Overall, this study showed that all biological levels investigated were strongly affected by temperature and salinity stress, however, with the latter having more severe effects. The results have implications for the operation of desalination plants and for assessing the impacts of marine heat waves.
... In particular, we focus on testing these measures in two fundamental tasks of omics data analysis, namely the retrieval of known biological information, and the detection of potential undiscovered functional associations between genes. To do that, we consider here three experimental benchmark data sets: Information and details concerning these data sets, including quality control analyses, can be found in [7,33,34]. To allow for an unbiased comparison of the different measures performance across the different data sets, all data are processed using the same pipeline starting from the raw measured concentration values. ...
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Mass spectrometry technologies are widely used in the fields of ionomics and metabolomics to simultaneously profile the intracellular concentrations of, e.g., amino acids or elements in genome-wide mutant libraries. These molecular or sub-molecular features are generally non-Gaussian and their covariance reveals patterns of correlations that reflect the system nature of the cell biochemistry and biology. Here, we introduce two similarity measures, the Mahalanobis cosine and the hybrid Mahalanobis cosine, that enforce information from the empirical covariance matrix of omics data from high-throughput screening and that can be used to quantify similarities between the profiled features of different mutants. We evaluate the performance of these similarity measures in the task of inferring and integrating genetic networks from short-profile ionomics/metabolomics data through an analysis of experimental data sets related to the ionome and the metabolome of the model organism S. cerevisiae. The study of the resulting ionome–metabolome Saccharomyces cerevisiae multilayer genetic network, which encodes multiple omic-specific levels of correlations between genes, shows that the proposed measures can provide an alternative description of relations between biological processes when compared to the commonly used Pearson’s correlation coefficient and have the potential to guide the construction of novel hypotheses on the function of uncharacterised genes.
... Processing the quantity of data received (almost the entire periodic table) from a large number of cells necessitates a multivariate data analysis to derive information on the state of the cells and the cell type. The capability of combining ionomic profiling and multivariate approach has been verified by several studies on a variety of cells, but has not yet been tested or applied to datasets obtained from ICP-ToF-MS analyses [28][29][30][31]. The use of this combination makes it possible to reveal the ecotoxicological effects of various individual metals as well as a combination (combined toxicity) of metals to algae. ...
Diatoms play a key role in assessing the ecotoxicology of metals and are already part of several national and international guidelines and regulations. Data on metal uptake and its correlation with a natural metal composition of the diatoms are mostly lacking on a cellular basis - mainly due to the lack of a suitable method on both the preparation and detection side. Therefore, within this work a fully automated approach based on the on-line coupling of a high performance liquid chromatograph (HPLC) and an inductively coupled plasma-time of flight-mass spectrometer (ICP-ToF-MS) was applied to analyze single cells of the alga Cyclotella meneghiniana multi-elementally in order to provide a deeper insight into the metal composition and its response to environmental stress. Multi-elemental analysis in single diatoms also enables assessment of combined toxicity of a set of metals. A set of four fingerprint elements, characteristically for diatoms (Mg, P, Si, Fe), were identified and hence the investigation of environmental stress onto the cells was enabled by performing incubation experiments with environmentally relevant toxic elements. It could be shown at moderate environmental stress caused by increasing the metal concentration in the medium (zinc) that the fingerprint element concentrations remained stable and thus the suitability of the selected elements for algae tracing was demonstrated. With regard to further ecotoxicological assessments, a multivariate approach was successfully applied allowing for cell classification upon different incubation concentration levels. This multivariate approach also facilitated an effective identification of three different diatom species (Cyclotella meneghiniana, Thalassiosira weissflogii and Thalassiosira pseudonana).
... Ionomics is the study of the ionome and involves the simultaneous measurement of the elemental composition of an organism as well as the changes in this composition in response to environmental, physiological, or genetic modifications [9]. It requires the use of rapid, sensitive, and precise multi-element analytical techniques such as inductively coupled plasma mass spectrometry (ICP-MS) [10][11][12][13][14][15]. Ionomics has the ability to capture information about the functional state of an organism under different conditions. ...
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The purpose of this study was to provide a new insight on the response of canines to stress exposure; the ionomic profiles of canine hair (2.8 ± 0.3 years, 15.17 ± 2.1 kg) (n = 10) was determined before and after lipopolysaccharide (LPS) injections. LPS was intramuscularly injected to induce inflammatory stress responses which were confirmed by observing increases in the level of serum cortisol, aldosterone, and inflammatory cytokines such as IL-6, IL-1β, and TNF-α. The hair contents of 17 elements were obtained by applying analytical procedures using the inductively coupled plasma mass spectrometry (ICP-MS). The following elements: sodium(Na) and potassium(K) among macro-elements, iron(Fe) and manganese(Mn) among micro-elements, and aluminum(Al), nickel(Ni), and lead(Pb) for toxic elements, showed significant increased levels with the immunological stress. The degree of increase in toxic elements was remarkable with the stress exposure. A forty-five-fold increase seen in Al accumulation with the stress exposure was noteworthy. Although mercury(Hg) and cadmium(Cd) showed decreased levels with the stress exposure, the degree was negligible compared to the level of increase. Correlation pattern between the elements was changed with the immunological stress. Toxic elements became more correlated with macro- or micro-elements than with toxic elements themselves after the stress exposure. Principal component analysis (PCA) showed that LPS challenge shifted the overall hair mineral profiles to a consistent direction changing Al and K up, even in animals with different hair mineral profiles before LPS treatment. In conclusion, the multivariate data processing and study of element distribution patterns provided new information about the ionomic response of the canine hairs to immunological stress, i.e., the ionomic profiles of canine hairs is strongly affected by the stress induced by LPS injections.
The ocean, which is called the “mother of origin of life,” is also the source of structurally unique natural products that are mainly accumulated in living organisms. Several of these compounds show pharmacological activities and are helpful for the invention and discovery of bioactive compounds. Marine biotechnology is the science in which marine organisms are used in full or partially to make or modify products, to improve plants or animals, or to develop microorganisms for specific uses. With the help of different molecular and biotechnological techniques, humans have been able to elucidate many biological methods applicable to both aquatic and terrestrial organisms. Marine biotechnology is an innovative field of research in science and technology concerning the support of living organisms with marine products and tools. To understand the omics of the living species, it is a novel way to produce genetically modified food, drugs, and energy to overcome global demand. The exploitation of biotechnology for drug discovery, including enzymes, antibiotics, and biopolymers, and chemical compounds from marine sources is deliberated in this Chapter. In addition, well-known and broadly used analytical techniques are derived from marine molecules or enzymes, including green fluorescence protein gene tagging methods and heat-resistant polymerases used in the polymerase chain reaction. Advances in bacterial identification, metabolic profiling, and physical handling of cells are being revolutionized by techniques such as mass spectrometric analysis of bacterial proteins. Advances in instrumentation and a combination of these physical advances with progress in proteomics and bioinformatics are accelerating our ability to harness biology for commercial gain. The objective of this review is to highlight some of the recent developments and findings in the area of marine biotechnology with special reference to the biomedical potential of marine natural organisms.
Circadian clocks are fundamental to the biology of most eukaryotes, coordinating behaviour and physiology to resonate with the environmental cycle of day and night through complex networks of clock-controlled genes. A fundamental knowledge gap exists, however, between circadian gene expression cycles and the biochemical mechanisms that ultimately facilitate circadian regulation of cell biology. Here we report circadian rhythms in the intracellular concentration of magnesium ions, [Mg(2+)]i, which act as a cell-autonomous timekeeping component to determine key clock properties both in a human cell line and in a unicellular alga that diverged from each other more than 1 billion years ago. Given the essential role of Mg(2+) as a cofactor for ATP, a functional consequence of [Mg(2+)]i oscillations is dynamic regulation of cellular energy expenditure over the daily cycle. Mechanistically, we find that these rhythms provide bilateral feedback linking rhythmic metabolism to clock-controlled gene expression. The global regulation of nucleotide triphosphate turnover by intracellular Mg(2+) availability has potential to impact upon many of the cell's more than 600 MgATP-dependent enzymes and every cellular system where MgNTP hydrolysis becomes rate limiting. Indeed, we find that circadian control of translation by mTOR is regulated through [Mg(2+)]i oscillations. It will now be important to identify which additional biological processes are subject to this form of regulation in tissues of multicellular organisms such as plants and humans, in the context of health and disease.
Possible changes in the ionome of Nicotiana langsdorffii wild-type and transgenic specimens following heat stress have been explored, considering both variations in the total concentration of 28 major and trace elements and their root/aerial part partitioning. In order to reveal the statistically significant effects, the natural variability was evaluated by the analysis of 10 individual specimens for each genotype. It was found that heat stress affects the plant ionome in a complex way, depending on the element and the genetic modification. A significant decrease in the total concentration was observed for several elements (e.g. Fe and Zn), whereas fewer elements (e.g. Ca and Mg) showed the opposite trend. The heat stress also affected the elemental distribution within the plants, determining accumulation in roots (e.g. Mo, P and Zn in transgenic plants) or in the aerial part (e.g. K) and indicating an altered efficiency of ion transport or uptake processes, respectively. Finally, analytical data for the heat stress were merged with those previously obtained for chemical and water stresses and analysed by multivariate analysis, making it possible to display the different ionomic signatures of each stress and suggest suitable indicators for plant biomonitoring studies.
In the post-genomic era, biological studies are characterized by the rapid development and wide application of a series of "omics" technologies, including genomics, proteomics, metabolomics, transcriptomics, lipidomics, cytomics, metallomics, ionomics, interactomics, and phenomics. These "omics" are often based on global analyses of biological samples using high through-put analytical approaches and bioinformatics and may provide new insights into biological phenomena. In this paper, the development and advances in these omics made in the past decades are reviewed, especially genomics, transcriptomics, proteomics and metabolomics; the applications of omics technologies in pharmaceutical research are then summarized in the fields of drug target discovery, toxicity evaluation, personalized medicine, and traditional Chinese medicine; and finally, the limitations of omics are discussed, along with the future challenges associated with the multi-omics data processing, dynamics omics analysis, and analytical approaches, as well as amenable solutions and future prospects. Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.
Open-vessel acid digestion is a low cost and easily automated sample decomposition method commonly used in laboratories involved in routine plant analysis. However, large amounts of reagents are required, the decomposition efficiency is limited by the boiling point of the digestion mixture and sample contamination and losses of volatile elements can frequently occur. In addition, the venting of large amounts of acid fume is undesirable due to its impact on the analyst and environment. For this reason, a simple, rugged and low cost closed-vessel conductively heated digestion system was developed and evaluated. The system accommodates 38 reaction vessels and enables the digestion of 200 mg of plant samples for subsequent elemental determination. The digestion procedure was optimized with the help of video images. The accuracy was confirmed by analyzing five certified reference materials digested by the proposed system. The digestion efficiency was evaluated by determining the residual carbon content and residual acidity. When plant samples were digested using the proposed system, results for Al, B, Ca, Cu, Fe, K, Mg, Mn, P, S and Zn by inductively coupled plasma optical emission spectroscopy were in agreement with those obtained after closed-vessel microwave-assisted digestion.
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Miniaturized growth systems for heterogeneous culture collections are not only attractive in reducing demands for incubation space and medium but also in making the parallel handling of large numbers of strains more practicable. We report here on the optimization of oxygen transfer rates in deep-well microtiter plates and the development of a replication system allowing the simultaneous and reproducible sampling of 96 frozen glycerol stock cultures while the remaining culture volume remains frozen. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and from rates of oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite. Maximum oxygen transfer rates (38 mmol liter−1 h−1, corresponding to a mass transfer coefficient of 188 h−1) were measured during orbital shaking at 300 rpm at a shaking diameter of 5 cm and a culture volume of 0.5 ml. A shaking diameter of 2.5 cm resulted in threefold-lower values. These high oxygen transfer rates allowed P. putida to reach a cell density of approximately 9 g (dry weight) liter−1 during growth on a glucose mineral medium at culture volumes of up to 1 ml. The growth-and-replication system was evaluated for a culture collection consisting of aerobic strains, mainly from the generaPseudomonas, Rhodococcus, andAlcaligenes, using mineral media and rich media. Cross-contamination and excessive evaporation during vigorous aeration were adequately prevented by the use of a sandwich cover of spongy silicone and cotton wool on top of the microtiter plates.
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Intracellular zinc is thought to be available in a cytosolic pool of free or loosely bound Zn(II) ions in the micromolar to picomolar range. To test this, we determined the mechanism of zinc sensors that control metal uptake or export in Escherichia coli and calibrated their response against the thermodynamically defined free zinc concentration. Whereas the cellular zinc quota is millimolar, free Zn(II) concentrations that trigger transcription of zinc uptake or efflux machinery are femtomolar, or six orders of magnitude less than one atom per cell. This is not consistent with a cytosolic pool of free Zn(II) and suggests an extraordinary intracellular zinc-binding capacity. Thus, cells exert tight control over cytosolic metal concentrations, even for relatively low-toxicity metals such as zinc.
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Understanding the functional connections between genes, proteins, metabolites and mineral ions is one of biology's greatest challenges in the postgenomic era. We describe here the use of mineral nutrient and trace element profiling as a tool to determine the biological significance of connections between a plant's genome and its elemental profile. Using inductively coupled plasma spectroscopy, we quantified 18 elements, including essential macro- and micronutrients and various nonessential elements, in shoots of 6,000 mutagenized M2 Arabidopsis thaliana plants. We isolated 51 mutants with altered elemental profiles. One mutant contains a deletion in FRD3, a gene known to control iron-deficiency responses in A. thaliana. Based on the frequency of elemental profile mutations, we estimate 2-4% of the A. thaliana genome is involved in regulating the plant's nutrient and trace element content. These results demonstrate the utility of elemental profiling as a useful functional genomics tool.
A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes. © 1998 John Wiley & Sons, Ltd.
The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.
The selection of the chemical elements by a particular cell from the environment involves a series of steps, the complexity of which depends upon the organism. There are usually two membranes to be crossed (bacteria) but there may be as many as ten (higher animals which distribute elements from intake fluid, through cells of organs to circulating fluids through a further set of cells to fixed locations in particular parts of space). The individual steps can be thermodynamically controlled or kinetically managed. In the second case energy can be used. The elements may remain in relatively fast exchange in their final condition or in non-exchanging chemical combination. The variety of paths which individual elements follow in any organism adds to the specific character of the organism. Clearly the paths have evolved to create an element distribution which we shall call the metallome, to parallel the nomenclature of protein distribution, the proteome.
A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.
Eukaryotic cell proliferation is controlled by specific growth factors and the availability of essential nutrients. If either of these signals is lacking, cells may enter into a specialized nondividing resting state, known as stationary phase or G(0). The entry into such resting states is typically accompanied by a dramatic decrease in the overall growth rate and an increased resistance to a variety of environmental stresses. Since most cells spend most of their life in these quiescent states, it is important that we develop a full understanding of the biology of the stationary phase/G(0) cell. This knowledge would provide important insights into the control of two of the most fundamental aspects of eukaryotic cell biology: cell proliferation and long-term cell survival. This review will discuss some recent advances in our understanding of the stationary phase of growth in the budding yeast, Saccharomyces cerevisiae.