5. Add enough volume (~150 μL) of HPLC-grade acetonitrile to
dehydrate gel pieces for 10 min. Spin and remove carefully the
acetonitrile solution.
6. Add enough volume (~100 μL) of 25 mM ammonium bicarbonate to rehydrate gel pieces for 10 min. Use occasional
vortexing. Then add the same volume (~100 μL) of HPLCgrade acetonitrile over the ammonium bicarbonate solution,
mixing everything well for 15 min using occasional vortexing.
Spin and remove all liquid. Dry gel plugs under speed-vacuum
or let microtubes open to air dry under a laminar flow hood.
7. Add an equivalent volume to that of gel plugs (~20–40 μL) of
sequencing-grade modified trypsin (12.5 ng/μL). Keep on ice
(4
C) for 45 min to allow rehydration of gel plugs while
keeping the enzyme inactive.
8. Remove excess trypsin solution, add 100 μL of 12.5 mM
ammonium bicarbonate, and incubate at 37
C overnight.
Stop trypsin digestion by adding 20–30 μL of 0.5% formic
acid solution (final pH should be around 2–3 in order to
inactivate the enzyme).
9. Transfer the solution with tryptic peptides to a new protein low
bind microtube (microtube II). In order to maximize the
peptide recovering, add 75 μL of 50% acetonitrile/0.1% formic
acid solution over gel plugs in microtube I for 10 min (use
vortex), spin and transfer the new solution with extracted peptides over that deposited in microtube II. This step can be
repeated 1–2 times more, taking care of leaving gel plugs on
microtube I (discarded).
10. Dry peptides (microtube II) under speed-vacuum. Avoid overdrying! At this point, dried peptides can be preserved longterm (months) in the freezer (À80
C).
11. Dried peptides (peptide amount for each fraction will depend
on the initial protein amount loaded and the number of fractions) are resuspended in 13 μL of 0.1% formic acid aided by
sonication (in an ultrasonic bath) for 5 min and centrifuged at
high speed for 5 min before being loaded in a mass spectrometry device (see Note 9).
12. For mass spectrometry analysis, we work with an
LTQ-Orbitrap Elite mass spectrometer (ESI-MS/MS) coupled to a Proxeon EASY-nLC 1000 UHPLC system (Thermo
Fisher Scientific, San Jose, CA, USA) (any other modern highresolution mass spectrometer is valid) under the following
conditions. Peptide separation is performed on a reversedphase (RP) column (EASY-Spray column, 50 cm  75 μm
ID, PepMap C18, 2 μm particles, 100 A ˚ pore size, Thermo
Fisher Scientific) with pre-column (Acclaim PepMap
100, 75 μm  2 cm, Thermo Fisher Scientific) using a
Shotgun Proteomics in Non-model Organisms
87
dehydrate gel pieces for 10 min. Spin and remove carefully the
acetonitrile solution.
6. Add enough volume (~100 μL) of 25 mM ammonium bicarbonate to rehydrate gel pieces for 10 min. Use occasional
vortexing. Then add the same volume (~100 μL) of HPLCgrade acetonitrile over the ammonium bicarbonate solution,
mixing everything well for 15 min using occasional vortexing.
Spin and remove all liquid. Dry gel plugs under speed-vacuum
or let microtubes open to air dry under a laminar flow hood.
7. Add an equivalent volume to that of gel plugs (~20–40 μL) of
sequencing-grade modified trypsin (12.5 ng/μL). Keep on ice
(4
C) for 45 min to allow rehydration of gel plugs while
keeping the enzyme inactive.
8. Remove excess trypsin solution, add 100 μL of 12.5 mM
ammonium bicarbonate, and incubate at 37
C overnight.
Stop trypsin digestion by adding 20–30 μL of 0.5% formic
acid solution (final pH should be around 2–3 in order to
inactivate the enzyme).
9. Transfer the solution with tryptic peptides to a new protein low
bind microtube (microtube II). In order to maximize the
peptide recovering, add 75 μL of 50% acetonitrile/0.1% formic
acid solution over gel plugs in microtube I for 10 min (use
vortex), spin and transfer the new solution with extracted peptides over that deposited in microtube II. This step can be
repeated 1–2 times more, taking care of leaving gel plugs on
microtube I (discarded).
10. Dry peptides (microtube II) under speed-vacuum. Avoid overdrying! At this point, dried peptides can be preserved longterm (months) in the freezer (À80
C).
11. Dried peptides (peptide amount for each fraction will depend
on the initial protein amount loaded and the number of fractions) are resuspended in 13 μL of 0.1% formic acid aided by
sonication (in an ultrasonic bath) for 5 min and centrifuged at
high speed for 5 min before being loaded in a mass spectrometry device (see Note 9).
12. For mass spectrometry analysis, we work with an
LTQ-Orbitrap Elite mass spectrometer (ESI-MS/MS) coupled to a Proxeon EASY-nLC 1000 UHPLC system (Thermo
Fisher Scientific, San Jose, CA, USA) (any other modern highresolution mass spectrometer is valid) under the following
conditions. Peptide separation is performed on a reversedphase (RP) column (EASY-Spray column, 50 cm  75 μm
ID, PepMap C18, 2 μm particles, 100 A ˚ pore size, Thermo
Fisher Scientific) with pre-column (Acclaim PepMap
100, 75 μm  2 cm, Thermo Fisher Scientific) using a
Shotgun Proteomics in Non-model Organisms
87