proteomic data obtained from pooled samples do not allow to
look for any potential correlation between protein profiles and
individual features (length, weight, color, sex, etc.); this type of
information is lost after pooling.
3. Confounding factors. Any experiment must be designed in
order to control for any potentially important confounding
factor; otherwise, results and derived conclusions could be
misleading. Sex, length, weight, age, sampling collection, and
processing dates are just a few examples of potential confounding factors [1, 21]. Along this line, it is very useful to include
randomization in the experimental design to control for any
undesirable (unknown, systematic) source of variation
[22]. Despite this might be perceived as a very basic, obvious
point, there are still a relevant number of published studies
where important confounding factors have been completely
ignored, producing results that cannot be often replicated by
further independent studies.
4. Whole organism versus tissue–organ level analysis. Unless there
are clear restrictions caused by the (tiny) size of the organism
under study, it is advisable to focus proteomic analysis on
specific tissues–organs in order to decrease the proteome complexity and any potential bias due to tissue–organ heterogeneities across different individuals (for example, see refs. 21, 23).
Likewise, results should be easier to interpret, and more meaningful conclusions would be derived if an appropriate tissue–
organ is selected. For instance, the use of liver or gametes in
toxicological and fertilization studies, respectively. Under some
circumstances, working at subcellular or single-cell level could
be more informative [24–26].
3.2 Total Protein
Extraction
and Quantification
1. Add an appropriate volume (500–1000 μL) of protein solubilization lysis buffer (for instance, 7 M urea:2 M thiourea:2–4%
CHAPS solution) to a piece of tissue, organ, or cells that were
previously snap frozen in liquid nitrogen and further preserved
at À80
C (see Note 2).
2. Use an ultrasonic probe (sonicator) to accelerate tissue disaggregation, cellular lysis, and protein solubilization. Usually,
6–12 cycles of ultrasonic pulses on ice, 5 s each (pulse on)
followed by a 30 s break (pulse off) to avoid overheating and
chemical modifications, setting ultrasonic amplitude at
15–20%, are enough (see Note 3).
3. Centrifuge (21,000 Â g, 30 min, 4
C) to separate cellular
debris from solubilized proteins and collect the supernatant
(see Note 4). Total protein concentration is measured using
any modified version of Lowry, BCA, or Bradford method
which is compatible with chemical components of protein
solubilization buffers.
84
Angel P. Diz and Paula Sa ´ nchez-Marı ´n
look for any potential correlation between protein profiles and
individual features (length, weight, color, sex, etc.); this type of
information is lost after pooling.
3. Confounding factors. Any experiment must be designed in
order to control for any potentially important confounding
factor; otherwise, results and derived conclusions could be
misleading. Sex, length, weight, age, sampling collection, and
processing dates are just a few examples of potential confounding factors [1, 21]. Along this line, it is very useful to include
randomization in the experimental design to control for any
undesirable (unknown, systematic) source of variation
[22]. Despite this might be perceived as a very basic, obvious
point, there are still a relevant number of published studies
where important confounding factors have been completely
ignored, producing results that cannot be often replicated by
further independent studies.
4. Whole organism versus tissue–organ level analysis. Unless there
are clear restrictions caused by the (tiny) size of the organism
under study, it is advisable to focus proteomic analysis on
specific tissues–organs in order to decrease the proteome complexity and any potential bias due to tissue–organ heterogeneities across different individuals (for example, see refs. 21, 23).
Likewise, results should be easier to interpret, and more meaningful conclusions would be derived if an appropriate tissue–
organ is selected. For instance, the use of liver or gametes in
toxicological and fertilization studies, respectively. Under some
circumstances, working at subcellular or single-cell level could
be more informative [24–26].
3.2 Total Protein
Extraction
and Quantification
1. Add an appropriate volume (500–1000 μL) of protein solubilization lysis buffer (for instance, 7 M urea:2 M thiourea:2–4%
CHAPS solution) to a piece of tissue, organ, or cells that were
previously snap frozen in liquid nitrogen and further preserved
at À80
C (see Note 2).
2. Use an ultrasonic probe (sonicator) to accelerate tissue disaggregation, cellular lysis, and protein solubilization. Usually,
6–12 cycles of ultrasonic pulses on ice, 5 s each (pulse on)
followed by a 30 s break (pulse off) to avoid overheating and
chemical modifications, setting ultrasonic amplitude at
15–20%, are enough (see Note 3).
3. Centrifuge (21,000 Â g, 30 min, 4
C) to separate cellular
debris from solubilized proteins and collect the supernatant
(see Note 4). Total protein concentration is measured using
any modified version of Lowry, BCA, or Bradford method
which is compatible with chemical components of protein
solubilization buffers.
84
Angel P. Diz and Paula Sa ´ nchez-Marı ´n