inconvenient of staining methods based on fluorescent dyes
(with high sensitivity such as Sypro Ruby) is that they are
usually much more expensive and need to use specific equipment to record images of gels with fluorescently labeled
proteins.
8. Scalpels should be cleaned with methanol or ethanol and precautions should be taken to avoid cross contamination among
samples during sample fractionation procedure (cutting gel
fractions within each sample lane). There are also other important sources of potential contamination (the most common is
keratin) that make necessary to work with material and
equipment thoughtfully cleaned with ethanol and under
laminar-flow cabinet. Contaminant load needs to be kept to a
minimum; otherwise, interference with mass spectrometry analyses could be important, decreasing the number of protein
identifications. It is important to wear gloves (avoid latex), a lab
coat, and hair cover. Avoid wearing clothes made of wool.
9. Make special care to ensure that there are no air bubbles at the
bottom of the microtube since these will interfere with sample
loading into the LC–MS/MS.
10. This protocol can be used with TMT6plex™, TMT10plex™,
or TMT11plex™. Volumes and quantities used in some steps
differ from those recommended for TMTpro™ 16 plex.
11. Thermo Fisher Scientific protocol state a safe working range
between 25 and 100 μg of protein.
12. If protein content has been solubilized in lysis buffer containing urea, then it is necessary to bring protein solution to <1 M
urea to avoid saturation in acetone solution and problems
during protein digestion (alternatively, one could try to fix
this inconvenience by increasing the acetone volume).
13. Protein pellet might not completely dissolve at this stage (short
ultrasonic pulses can be applied using an ultrasonic bath);
however, it will completely dissolve after protein reduction
and digestion steps at 55
C and 37
C, respectively.
14. Thermo Fisher Scientific TMT10plex (or TMT6plex) protocol
states that it is necessary to dissolve each vial with 0.8 mg of
each TMT label reagent with 41 μL of anhydrous acetonitrile.
However, we suggest doubling this volume of acetonitrile,
which allows to run two independent instead of one TMT
experiment (halving the costs of each experiment while still
working in the safe confidence range indicated by the own
company). This is true if 50 instead of 100 μg of total protein
is labeled for each experiment/sample, as far as this procedure
would allow to still work within the safe working range of
protein amount (25–100 μg) to be labeled indicated by the
original protocol. If the samples from the two experiments are
Shotgun Proteomics in Non-model Organisms
97
(with high sensitivity such as Sypro Ruby) is that they are
usually much more expensive and need to use specific equipment to record images of gels with fluorescently labeled
proteins.
8. Scalpels should be cleaned with methanol or ethanol and precautions should be taken to avoid cross contamination among
samples during sample fractionation procedure (cutting gel
fractions within each sample lane). There are also other important sources of potential contamination (the most common is
keratin) that make necessary to work with material and
equipment thoughtfully cleaned with ethanol and under
laminar-flow cabinet. Contaminant load needs to be kept to a
minimum; otherwise, interference with mass spectrometry analyses could be important, decreasing the number of protein
identifications. It is important to wear gloves (avoid latex), a lab
coat, and hair cover. Avoid wearing clothes made of wool.
9. Make special care to ensure that there are no air bubbles at the
bottom of the microtube since these will interfere with sample
loading into the LC–MS/MS.
10. This protocol can be used with TMT6plex™, TMT10plex™,
or TMT11plex™. Volumes and quantities used in some steps
differ from those recommended for TMTpro™ 16 plex.
11. Thermo Fisher Scientific protocol state a safe working range
between 25 and 100 μg of protein.
12. If protein content has been solubilized in lysis buffer containing urea, then it is necessary to bring protein solution to <1 M
urea to avoid saturation in acetone solution and problems
during protein digestion (alternatively, one could try to fix
this inconvenience by increasing the acetone volume).
13. Protein pellet might not completely dissolve at this stage (short
ultrasonic pulses can be applied using an ultrasonic bath);
however, it will completely dissolve after protein reduction
and digestion steps at 55
C and 37
C, respectively.
14. Thermo Fisher Scientific TMT10plex (or TMT6plex) protocol
states that it is necessary to dissolve each vial with 0.8 mg of
each TMT label reagent with 41 μL of anhydrous acetonitrile.
However, we suggest doubling this volume of acetonitrile,
which allows to run two independent instead of one TMT
experiment (halving the costs of each experiment while still
working in the safe confidence range indicated by the own
company). This is true if 50 instead of 100 μg of total protein
is labeled for each experiment/sample, as far as this procedure
would allow to still work within the safe working range of
protein amount (25–100 μg) to be labeled indicated by the
original protocol. If the samples from the two experiments are
Shotgun Proteomics in Non-model Organisms
97