18. DTBP-cross-linked proteins can be cleaved by reducing the
disulfide bond of the spacer arm with 100–150 mM DTT at
37
C for 30 min (see reagent setup), thus freeing the isolated
proteins from the cross-linked product.
19. RCDC is compatible with detergents (such as SDS) and reducing agents (such as DTT). Bradford and BCA are compatible
with reducing agents and detergents, respectively. Make a standard curve with BSA and use as blank the protein extraction
supernatant of a membrane from a prepared MMI live chamber
dish but never seeded with cells. Fibronectin is a protein, and
for that reason, it is likely that fibronectin coating increases
protein results.
20. RCDC can be less reliable for very small samples of isolated
cellular protrusions, and it takes away a large amount of the
isolated samples. In order to use all of the LCM-isolated cellular protrusions for MS analysis, we recommend determining
what the total number of cuts required is and avoid using the
RCDC. The number of cuts required will vary depending on
the cellular protrusion types and should be predetermined by
each investigator. When using the orbitrap LC-MS/MS, we
ran 2 and 5 μg of total protein or 3,000 to 6,000 cuts, depending on the types of cellular protrusions isolated. When using
the timsTOF Pro mass spectrometer, the number of cuts was
reduced due to the greater sensitivity of this mass spectrometer
compared to the Orbitrap.
21. To avoid keratin contamination, all these steps and the
required solutions should be done in a biological hood and
with molecular grade water.
22. This “limited gel” allows for the separation of the proteins by
size in preparation for MS, and each lane can be run as one
sample by MS.
23. It is easier to cut the samples if they are loaded between stained
ladders.
24. Because of the minute sample size, only very sensitive mass
spectrometers should be used. We recommend using either the
Thermo Scientific Orbitrap Fusion mass spectrometer or the
timsTOF Pro mass spectrometer from Bruker.
25. The iST sample preparation takes about 2½ h and includes all
required steps, such as lysis to denature, reduce, and alkylate
proteins, digestion using a LysC and Trypsin mix, and peptide
purification, which includes removal of hydrophilic
contaminants.
Proteome of Cellular Protrusions Identified by LCM/MS
43
disulfide bond of the spacer arm with 100–150 mM DTT at
37
C for 30 min (see reagent setup), thus freeing the isolated
proteins from the cross-linked product.
19. RCDC is compatible with detergents (such as SDS) and reducing agents (such as DTT). Bradford and BCA are compatible
with reducing agents and detergents, respectively. Make a standard curve with BSA and use as blank the protein extraction
supernatant of a membrane from a prepared MMI live chamber
dish but never seeded with cells. Fibronectin is a protein, and
for that reason, it is likely that fibronectin coating increases
protein results.
20. RCDC can be less reliable for very small samples of isolated
cellular protrusions, and it takes away a large amount of the
isolated samples. In order to use all of the LCM-isolated cellular protrusions for MS analysis, we recommend determining
what the total number of cuts required is and avoid using the
RCDC. The number of cuts required will vary depending on
the cellular protrusion types and should be predetermined by
each investigator. When using the orbitrap LC-MS/MS, we
ran 2 and 5 μg of total protein or 3,000 to 6,000 cuts, depending on the types of cellular protrusions isolated. When using
the timsTOF Pro mass spectrometer, the number of cuts was
reduced due to the greater sensitivity of this mass spectrometer
compared to the Orbitrap.
21. To avoid keratin contamination, all these steps and the
required solutions should be done in a biological hood and
with molecular grade water.
22. This “limited gel” allows for the separation of the proteins by
size in preparation for MS, and each lane can be run as one
sample by MS.
23. It is easier to cut the samples if they are loaded between stained
ladders.
24. Because of the minute sample size, only very sensitive mass
spectrometers should be used. We recommend using either the
Thermo Scientific Orbitrap Fusion mass spectrometer or the
timsTOF Pro mass spectrometer from Bruker.
25. The iST sample preparation takes about 2½ h and includes all
required steps, such as lysis to denature, reduce, and alkylate
proteins, digestion using a LysC and Trypsin mix, and peptide
purification, which includes removal of hydrophilic
contaminants.
Proteome of Cellular Protrusions Identified by LCM/MS
43