240 min linear gradient from 5% to 35% acetonitrile at a flow
rate of 300 nL/min. All other MS parameters were set as
described by Romero et al. [27].
3.6 Quantitative
Proteomic Analysis:
Isobaric-Labeling
Using TMT Multiplex
Proceed for multiplex relative quantitation by mass spectrometry
with the protocol described in TMT10plex™ Mass Tag Labeling
Kits and Reagents (Thermo Fisher Scientific; Pub.
No. MAN0016969) with minor modifications described below
(see Note 10).
1. Precipitate 50 μg of extracted protein (see Note 11) diluted in
HPLC-grade water or 100 mM TEAB (see Note 12) without
exceeding a total final volume of 100 μL in 6 volumes of
pre-chilled (À20
C) acetone. Mix well using vortex and
allow protein precipitation for at least 2 h at À20
C (overnight
if convenient). Use protein low bind microtubes.
2. Centrifuge 8000 Â g, 4
C, for 10 min. Remove acetone by
decanting carefully to avoid removing the white protein pellet
and keeping the microtube drying about 5–10 min (do not
overdry in order to avoid that dissolving protein pellet turns
into an even more difficult task!). Resuspend protein pellet in
100 μL of 100 mM TEAB (see Note 13).
3. Reduce and alkylate the protein content in two consecutive
steps. First, add 5 μL of 200 mM TCEP (reducing agent) and
incubate at 55
C for 1 h. Use a heater thermoblock with
shaking capacity (low speed, 300 rpm, is enough). Second,
add 5 μL of 375 mM iodoacetamide (IAM) in 100 mM
TEAB and incubate at room temperature avoiding light
exposure.
4. After resuspending sequencing-grade modified trypsin (20 μg
in 200 μL of 100 mM TEAB), add 10 μL to each (previously
acetone-precipitated, resuspended, reduced, and alkylated)
protein sample. Keep trypsin to protein ratio about 1:50
(even up to 1:100 should work fine). Allow protein digestion
overnight, 37
C (do not leave more than 12–16 h in order to
limit trypsin autolysis).
5. Equilibrate TMT label reagents to room temperature, then add
82 μL (see Note 14) of anhydrous acetonitrile to each 0.8 mg
TMT label vial (6, 10, or 11 vials, depending on the multiplexing capacity), and apply occasional vortexing for 5 min to
fully dissolve the reagent.
6. Add 41 μL of each TMT label to each of the ten reduced and
alkylated protein digested samples, incubate for 1 h, and
quench the labeling reaction by adding 8 μL of 5% hydroxylamine and incubating for another 15 min at room temperature.
88
Angel P. Diz and Paula Sa ´ nchez-Marı ´n
rate of 300 nL/min. All other MS parameters were set as
described by Romero et al. [27].
3.6 Quantitative
Proteomic Analysis:
Isobaric-Labeling
Using TMT Multiplex
Proceed for multiplex relative quantitation by mass spectrometry
with the protocol described in TMT10plex™ Mass Tag Labeling
Kits and Reagents (Thermo Fisher Scientific; Pub.
No. MAN0016969) with minor modifications described below
(see Note 10).
1. Precipitate 50 μg of extracted protein (see Note 11) diluted in
HPLC-grade water or 100 mM TEAB (see Note 12) without
exceeding a total final volume of 100 μL in 6 volumes of
pre-chilled (À20
C) acetone. Mix well using vortex and
allow protein precipitation for at least 2 h at À20
C (overnight
if convenient). Use protein low bind microtubes.
2. Centrifuge 8000 Â g, 4
C, for 10 min. Remove acetone by
decanting carefully to avoid removing the white protein pellet
and keeping the microtube drying about 5–10 min (do not
overdry in order to avoid that dissolving protein pellet turns
into an even more difficult task!). Resuspend protein pellet in
100 μL of 100 mM TEAB (see Note 13).
3. Reduce and alkylate the protein content in two consecutive
steps. First, add 5 μL of 200 mM TCEP (reducing agent) and
incubate at 55
C for 1 h. Use a heater thermoblock with
shaking capacity (low speed, 300 rpm, is enough). Second,
add 5 μL of 375 mM iodoacetamide (IAM) in 100 mM
TEAB and incubate at room temperature avoiding light
exposure.
4. After resuspending sequencing-grade modified trypsin (20 μg
in 200 μL of 100 mM TEAB), add 10 μL to each (previously
acetone-precipitated, resuspended, reduced, and alkylated)
protein sample. Keep trypsin to protein ratio about 1:50
(even up to 1:100 should work fine). Allow protein digestion
overnight, 37
C (do not leave more than 12–16 h in order to
limit trypsin autolysis).
5. Equilibrate TMT label reagents to room temperature, then add
82 μL (see Note 14) of anhydrous acetonitrile to each 0.8 mg
TMT label vial (6, 10, or 11 vials, depending on the multiplexing capacity), and apply occasional vortexing for 5 min to
fully dissolve the reagent.
6. Add 41 μL of each TMT label to each of the ten reduced and
alkylated protein digested samples, incubate for 1 h, and
quench the labeling reaction by adding 8 μL of 5% hydroxylamine and incubating for another 15 min at room temperature.
88
Angel P. Diz and Paula Sa ´ nchez-Marı ´n