13. Thermoblock with shaking capacity.
14. Speed-vacuum concentrator device.
15. Protein low bind microtubes.
2.6 Peptides
Resuspension and LC–
MS/MS Analysis
1. 0.1% Formic acid.
2. Ultrasonic bath.
3. High-resolution LC–MS/MS instrument.
3 Methods
3.1 Experimental
Design: Preliminary
Considerations
The first important step in every research project is to check carefully that the experiments have been correctly designed in order to
test confidently the working hypotheses. There are several key
aspects worth to be thoughtfully considered; some of them are
highlighted below:
1. Biological and technical replication. Identify clearly the number and type of replicates to be analyzed. Remind that statistical
power, that is, the probability of detecting an effect when there
is a true effect, depends on within-group variation (biological
and technical) of the dependent variable, the effect size, and
sample size [17, 18]. For instance, under the expectation that
analyzed proteins could present generalized patterns of high
biological variation and expected low-mid effect sizes for factor/s under study, an appropriate (usually high) number of
biological replicates would be needed. There are several online
tools to calculate a priori estimations of statistical power (or the
necessary sample size for a prefixed statistical power) if previous
information is known beforehand (preliminary pilot studies can
be carried out on this purpose). The inclusion of technical
replicates allows getting an estimation about the technical
error and to assess whether there is significant biological (signal) over technical (noise) variation in our data set, which is
very useful information in order to validate our experimental
procedure and further statistical inference [19].
2. Pooling or not pooling samples. When there exist limited
economic resources, high biological variation, and
low-moderate expected effect sizes for a substantial number
of analyzed proteins, following a pooling sample strategy to
make biological replicates could be an interesting option to be
considered [20]. Pooled samples are made by mixing equal
amounts of protein extracted from a number of individual
samples. Generally speaking, it is better to include a high rather
than a low number of individual samples within pools in order
to minimize any potential (bias) effect from outlier individuals
in pooled samples. However, it should be borne in mind that
Shotgun Proteomics in Non-model Organisms
83
14. Speed-vacuum concentrator device.
15. Protein low bind microtubes.
2.6 Peptides
Resuspension and LC–
MS/MS Analysis
1. 0.1% Formic acid.
2. Ultrasonic bath.
3. High-resolution LC–MS/MS instrument.
3 Methods
3.1 Experimental
Design: Preliminary
Considerations
The first important step in every research project is to check carefully that the experiments have been correctly designed in order to
test confidently the working hypotheses. There are several key
aspects worth to be thoughtfully considered; some of them are
highlighted below:
1. Biological and technical replication. Identify clearly the number and type of replicates to be analyzed. Remind that statistical
power, that is, the probability of detecting an effect when there
is a true effect, depends on within-group variation (biological
and technical) of the dependent variable, the effect size, and
sample size [17, 18]. For instance, under the expectation that
analyzed proteins could present generalized patterns of high
biological variation and expected low-mid effect sizes for factor/s under study, an appropriate (usually high) number of
biological replicates would be needed. There are several online
tools to calculate a priori estimations of statistical power (or the
necessary sample size for a prefixed statistical power) if previous
information is known beforehand (preliminary pilot studies can
be carried out on this purpose). The inclusion of technical
replicates allows getting an estimation about the technical
error and to assess whether there is significant biological (signal) over technical (noise) variation in our data set, which is
very useful information in order to validate our experimental
procedure and further statistical inference [19].
2. Pooling or not pooling samples. When there exist limited
economic resources, high biological variation, and
low-moderate expected effect sizes for a substantial number
of analyzed proteins, following a pooling sample strategy to
make biological replicates could be an interesting option to be
considered [20]. Pooled samples are made by mixing equal
amounts of protein extracted from a number of individual
samples. Generally speaking, it is better to include a high rather
than a low number of individual samples within pools in order
to minimize any potential (bias) effect from outlier individuals
in pooled samples. However, it should be borne in mind that
Shotgun Proteomics in Non-model Organisms
83