microproteomic analyses. Fixative solutions with glutaraldehyde are
ideal for maintaining cellular protrusions, including fragile
protrusions such as tunneling nanotubes [23]. Unfortunately, glutaraldehyde fixations, even in low concentrations, create thorough
cross-linking that seriously hampers the quality sample for MS
analysis [24]. Here, we use dimethyl-3-3
0 -dithiobispropiomimidate
(DTBP), which can be de-cross-linked by using a reducing agent
and a temperature-step process [25, 26]. In fact, the currently
optimized fixation with DTBP not only preserves cellular protrusions but also allows for the identification of high-quality and highquantity proteins, which reduces the number of cellular protrusions
needed for MS analysis [27]. The current protocol allows one to
specifically select any type of cellular protrusion based on morphological and/or fluorescence markers, thus greatly improving its
selectivity compared to previous methods [6, 7, 19–22]. Importantly, we were able to isolate TNTs, a very delicate and specialized
cell-to-cell communication conduit, which have been related to
several diseases, such as cancer, human immunodeficiency virus,
or prion infection [28–31].
2 Materials
2.1 Cell Culture
1. CAD (cath-a-differentiated) cell line.
2. 25-cm
2 cell culture flasks.
3. 5-, 10-, and 25-mL individually wrapped pipettes.
4. 1-mL aspirating pipettes.
5. 15- and 50-mL conical tubes.
6. An optical biological inverted microscope.
7. A biological safety cabinet, e.g., 1300 Series Class II, Type A2
Biological Safety Cabinet.
8. CO 2 incubator and an aspiration pump.
9. CAD cell culture medium: Add 50 mL of 10% fetal bovine
serum (FBS) to 450 mL of OPTI-MEM. Filter-sterilize and
store at 4
C for no longer than 4 weeks.
10. 0.4% trypan blue solution.
11. Metalized hemocytometer.
2.2 Preparation
for LCM
As stated above, there are different types of LCM. In our studies,
we used a laser microdissection system controlled by the Molecular
Machines & Industries (MMI) Cell Tools Software.
1. MMI live cell chamber with membrane and petri dish. This
includes a membrane ring for the initial cultivation of cells and
a UV-permeable microdissection chamber to isolate the cellular protrusions of interest.
Proteome of Cellular Protrusions Identified by LCM/MS
27
ideal for maintaining cellular protrusions, including fragile
protrusions such as tunneling nanotubes [23]. Unfortunately, glutaraldehyde fixations, even in low concentrations, create thorough
cross-linking that seriously hampers the quality sample for MS
analysis [24]. Here, we use dimethyl-3-3
0 -dithiobispropiomimidate
(DTBP), which can be de-cross-linked by using a reducing agent
and a temperature-step process [25, 26]. In fact, the currently
optimized fixation with DTBP not only preserves cellular protrusions but also allows for the identification of high-quality and highquantity proteins, which reduces the number of cellular protrusions
needed for MS analysis [27]. The current protocol allows one to
specifically select any type of cellular protrusion based on morphological and/or fluorescence markers, thus greatly improving its
selectivity compared to previous methods [6, 7, 19–22]. Importantly, we were able to isolate TNTs, a very delicate and specialized
cell-to-cell communication conduit, which have been related to
several diseases, such as cancer, human immunodeficiency virus,
or prion infection [28–31].
2 Materials
2.1 Cell Culture
1. CAD (cath-a-differentiated) cell line.
2. 25-cm
2 cell culture flasks.
3. 5-, 10-, and 25-mL individually wrapped pipettes.
4. 1-mL aspirating pipettes.
5. 15- and 50-mL conical tubes.
6. An optical biological inverted microscope.
7. A biological safety cabinet, e.g., 1300 Series Class II, Type A2
Biological Safety Cabinet.
8. CO 2 incubator and an aspiration pump.
9. CAD cell culture medium: Add 50 mL of 10% fetal bovine
serum (FBS) to 450 mL of OPTI-MEM. Filter-sterilize and
store at 4
C for no longer than 4 weeks.
10. 0.4% trypan blue solution.
11. Metalized hemocytometer.
2.2 Preparation
for LCM
As stated above, there are different types of LCM. In our studies,
we used a laser microdissection system controlled by the Molecular
Machines & Industries (MMI) Cell Tools Software.
1. MMI live cell chamber with membrane and petri dish. This
includes a membrane ring for the initial cultivation of cells and
a UV-permeable microdissection chamber to isolate the cellular protrusions of interest.
Proteome of Cellular Protrusions Identified by LCM/MS
27