11. To isolate the ROIs cut, remove the PBS, wash the dish gently
with filtered PBS, aspirate the PBS, and carefully lift the ring
from the microdissection chamber with tweezers. All of the
desired ROIs should be left on the sticky membrane of the
microdissection chamber, while the rest of the cells will be
removed along with the ring. It is imperative to go back to
the LCM and look at the isolated ROIs left on the microdissection chamber to ensure that the expected ROIs are present
and that no whole cells or other cellular debris are present. If a
cell is found, it must be obliterated with the laser.
12. Once the ROIs have been examined and validated, the cellular
protrusions can be lifted from the microdissection chamber
with a micropipette by taking a small volume of lysis buffer
(i.e., 5 μL) and pipetting up and down all over the microdissection chamber where the cuts are located (see Note 14).
Avoid making bubbles. The cuts should be transferred to a
1.5-mL centrifuge tube, and the process can be repeated to
ensure that all cuts have been lifted by observing the dish under
the microscope. The final volume should be small (i.e., 15 μL
total for all of your samples).
13. For the whole cell lysate sample, cut the membrane from the
ring that was lifted away from the dissection chamber in step
11 above with a scalpel and put it in a 1.5-mL centrifuge tube
with lysis buffer (i.e., 40 μL). Make sure to push the membrane
at the bottom of the tube and that it is immersed in lysis buffer
(see Note 15).
3.6 Protein
Extraction
The optimization of the protein extraction protocol was performed
to maximize the de-cross-linking of the fixative agents while
keeping the lysate as concentrated as possible. In addition to the
experimental samples, the following controls should also be considered: (1) a positive whole cell lysate control to compare with the
proteome of the protrusion sample and (2) a negative control to
determine the background proteins that are released to the media
and can be found on the membrane, such as exosomes. In this case,
the same number of cuts that were isolated for cellular protrusions
should be isolated in empty areas surrounding cells. Finally, when
setting up conditions, silver staining (e.g., ProteoSilver™ Silver
Stain Kit) might be used to determine the quality of the proteins
present in the isolated samples even in those with low amounts of
proteins, such as the cellular protrusion samples (see Note 16).
1. Take the isolated LCM samples from steps 12 and 13 in
Subheading 3.5 and thaw them if they were stored at À80
C
(see Note 17).
2. Sonicate for 5 min and do a quick spin to bring all
samples down.
Proteome of Cellular Protrusions Identified by LCM/MS
37
with filtered PBS, aspirate the PBS, and carefully lift the ring
from the microdissection chamber with tweezers. All of the
desired ROIs should be left on the sticky membrane of the
microdissection chamber, while the rest of the cells will be
removed along with the ring. It is imperative to go back to
the LCM and look at the isolated ROIs left on the microdissection chamber to ensure that the expected ROIs are present
and that no whole cells or other cellular debris are present. If a
cell is found, it must be obliterated with the laser.
12. Once the ROIs have been examined and validated, the cellular
protrusions can be lifted from the microdissection chamber
with a micropipette by taking a small volume of lysis buffer
(i.e., 5 μL) and pipetting up and down all over the microdissection chamber where the cuts are located (see Note 14).
Avoid making bubbles. The cuts should be transferred to a
1.5-mL centrifuge tube, and the process can be repeated to
ensure that all cuts have been lifted by observing the dish under
the microscope. The final volume should be small (i.e., 15 μL
total for all of your samples).
13. For the whole cell lysate sample, cut the membrane from the
ring that was lifted away from the dissection chamber in step
11 above with a scalpel and put it in a 1.5-mL centrifuge tube
with lysis buffer (i.e., 40 μL). Make sure to push the membrane
at the bottom of the tube and that it is immersed in lysis buffer
(see Note 15).
3.6 Protein
Extraction
The optimization of the protein extraction protocol was performed
to maximize the de-cross-linking of the fixative agents while
keeping the lysate as concentrated as possible. In addition to the
experimental samples, the following controls should also be considered: (1) a positive whole cell lysate control to compare with the
proteome of the protrusion sample and (2) a negative control to
determine the background proteins that are released to the media
and can be found on the membrane, such as exosomes. In this case,
the same number of cuts that were isolated for cellular protrusions
should be isolated in empty areas surrounding cells. Finally, when
setting up conditions, silver staining (e.g., ProteoSilver™ Silver
Stain Kit) might be used to determine the quality of the proteins
present in the isolated samples even in those with low amounts of
proteins, such as the cellular protrusion samples (see Note 16).
1. Take the isolated LCM samples from steps 12 and 13 in
Subheading 3.5 and thaw them if they were stored at À80
C
(see Note 17).
2. Sonicate for 5 min and do a quick spin to bring all
samples down.
Proteome of Cellular Protrusions Identified by LCM/MS
37