2.6 Mass
Spectrometry Sample
Preparation
1. 5Â Laemmli buffer: 25 mM Tris–HCl (pH 6.8), 25% glycerol,
10% SDS, 0.02% bromophenol blue, 150 mM DTT, and
molecular water (see Note 4).
2. An 8% acrylamide Bis-Tris gel for mass spectrometry: Prepare a
1.25 M Bis-Tris stock and adjust pH to 6.5–6.8. For the
resolving gel, calculate the necessary volume of the stock solution to get a final concentration of 350 mM Bis-Tris and add
the corresponding volume of 30% acrylamide/bis-acrylamide
(37.5:1) to get a final concentration of 8%. Add H 2 O to make
up a final volume of 5 mL. Finally, add 10% ammonium persulfate (APS) and TEMED (10 μL) to polymerize it. For the
stacking gel, calculate the necessary volume of the stock solution to get a final concentration of 350 mM Bis-Tris and add
the corresponding volume of 30% acrylamide to get a final
concentration of 4%. Add H 2 O to make up a final volume of
1.75 mL. Finally, add 10% APS and TEMED (10 μL) to
polymerize it.
3. 20Â MOPS buffer: 1 M 3-(N-morpholino) propane sulfonic
acid (MOPS), 1 M Tris Base, 20 mM EDTA, 2% SDS, and
molecular water (see Note 5).
4. Running buffer: 20% MOPS buffer, 0.5% sodium bisulfite, and
molecular water (see Note 1).
5. Fixing solution for gels: methanol, acetic acid, and molecular
water (40:40:8, respectively) (see Note 1).
6. Precision Plus Protein™ Dual Color Standards (Bio-Rad,
#161-0374), loading tips.
7. Powerpac™ Basic Power Supply and Mini-PROTEAN
® Tetra
cell system.
8. Compact digital rocker.
9. Speed vacuum.
10. iST Sample Preparation Kit from PreOmics.
2.7 Mass
Spectrometers
1. Thermo Scientific Orbitrap Fusion mass spectrometer or timsTOF Pro mass spectrometer from Bruker.
3 Methods
3.1 Cell Culture
The method described here is to get an enrichment of cellular
protrusions and, subsequently, to extract the proteins and analyze
them by MS. Cell density is a critical step when isolating cellular
protrusions. It can vary not only with the types of cellular protrusions that need to be isolated but also with the cell types used for
the experiments. For example, if you are interested in isolating
filopodia, we recommend plating low cell density in order to ensure
Proteome of Cellular Protrusions Identified by LCM/MS
29
Spectrometry Sample
Preparation
1. 5Â Laemmli buffer: 25 mM Tris–HCl (pH 6.8), 25% glycerol,
10% SDS, 0.02% bromophenol blue, 150 mM DTT, and
molecular water (see Note 4).
2. An 8% acrylamide Bis-Tris gel for mass spectrometry: Prepare a
1.25 M Bis-Tris stock and adjust pH to 6.5–6.8. For the
resolving gel, calculate the necessary volume of the stock solution to get a final concentration of 350 mM Bis-Tris and add
the corresponding volume of 30% acrylamide/bis-acrylamide
(37.5:1) to get a final concentration of 8%. Add H 2 O to make
up a final volume of 5 mL. Finally, add 10% ammonium persulfate (APS) and TEMED (10 μL) to polymerize it. For the
stacking gel, calculate the necessary volume of the stock solution to get a final concentration of 350 mM Bis-Tris and add
the corresponding volume of 30% acrylamide to get a final
concentration of 4%. Add H 2 O to make up a final volume of
1.75 mL. Finally, add 10% APS and TEMED (10 μL) to
polymerize it.
3. 20Â MOPS buffer: 1 M 3-(N-morpholino) propane sulfonic
acid (MOPS), 1 M Tris Base, 20 mM EDTA, 2% SDS, and
molecular water (see Note 5).
4. Running buffer: 20% MOPS buffer, 0.5% sodium bisulfite, and
molecular water (see Note 1).
5. Fixing solution for gels: methanol, acetic acid, and molecular
water (40:40:8, respectively) (see Note 1).
6. Precision Plus Protein™ Dual Color Standards (Bio-Rad,
#161-0374), loading tips.
7. Powerpac™ Basic Power Supply and Mini-PROTEAN
® Tetra
cell system.
8. Compact digital rocker.
9. Speed vacuum.
10. iST Sample Preparation Kit from PreOmics.
2.7 Mass
Spectrometers
1. Thermo Scientific Orbitrap Fusion mass spectrometer or timsTOF Pro mass spectrometer from Bruker.
3 Methods
3.1 Cell Culture
The method described here is to get an enrichment of cellular
protrusions and, subsequently, to extract the proteins and analyze
them by MS. Cell density is a critical step when isolating cellular
protrusions. It can vary not only with the types of cellular protrusions that need to be isolated but also with the cell types used for
the experiments. For example, if you are interested in isolating
filopodia, we recommend plating low cell density in order to ensure
Proteome of Cellular Protrusions Identified by LCM/MS
29