13. Remove the residual solvent and perform alkylation using
10 mM propionamide in 50 mM ammonium bicarbonate for
30 min at room temperature.
14. Rinse the gel pieces with 50% acetonitrile and 50 mM ammonium bicarbonate and place in a speed vacuum for 5 min.
15. Digest with trypsin/LysC overnight at 37
C.
16. Peptide extraction: Spin down the tubes and collect the solvent, which contains the peptides. Add 60% acetonitrile, 39.9%
water, and 0.1% formic acid and incubate for 10–15 min. The
peptide pools can be dried in a speed vacuum.
17. Reconstitute the digested peptide pools and inject onto a
100 μm I.D. C18 reversed-phase analytical column (2.4 μM
Reprosil-Pur), 25–50 cm in length. The UPLC was a Waters M
class, operated at 300 nL/min using a linear gradient from 4%
mobile phase B to 35% B. Mobile phase A consisted of 0.2%
formic acid, 5% DMSO, and water; mobile phase B was 0.2%
formic acid, 5% DMSO, and acetonitrile.
18. For data collected using the Orbitrap Fusion mass spectrometer, it was set to acquire data in a data-dependent fashion
selecting and fragmenting by collision-induced dissociation
the most intense precursor ions optimized to maximize duty
cycle. An exclusion window of 60 s was used to improve
proteomic depth, and multiple charge states of the same ion
were not sampled.
19. For data collected using the timsTOF Pro mass spectrometer in
PASEF mode, the mobility range used was 0.7 1/K 0 to 1.5
1/K 0 and the ramp time was 100 ms. The effective duty cycle
was 120 Hz, where the dynamic exclusion setting was set to
0.4 min.
3.8 Sample
Preparation for Mass
Spectrometry Using
iST Sample
Preparation Kit from
PreOmics
As an alternative to using limited gels, the samples can be directly
prepared using the iST Sample Preparation Kit from PreOmics.
This allows for a fast and complete sample preparation that minimizes possible background contamination. The kit can be used
with samples as small as 1 μg of protein. In our case, we were able
to use it for small cellular protrusions, like filopodia. The kit
includes lysis, digest, and peptide purification protocols (see
Note 25).
1. Follow the manufacturer’s instructions.
2. The peptide pool is obtained at the end, ready to be run
for MS.
3.9 Data Analysis
1. MS data analysis will vary depending on the mass spectrometer
and software used. In our case, the MS data were analyzed
using Preview and Byonic v2.6.49 as well as custom tools for
Proteome of Cellular Protrusions Identified by LCM/MS
39
10 mM propionamide in 50 mM ammonium bicarbonate for
30 min at room temperature.
14. Rinse the gel pieces with 50% acetonitrile and 50 mM ammonium bicarbonate and place in a speed vacuum for 5 min.
15. Digest with trypsin/LysC overnight at 37
C.
16. Peptide extraction: Spin down the tubes and collect the solvent, which contains the peptides. Add 60% acetonitrile, 39.9%
water, and 0.1% formic acid and incubate for 10–15 min. The
peptide pools can be dried in a speed vacuum.
17. Reconstitute the digested peptide pools and inject onto a
100 μm I.D. C18 reversed-phase analytical column (2.4 μM
Reprosil-Pur), 25–50 cm in length. The UPLC was a Waters M
class, operated at 300 nL/min using a linear gradient from 4%
mobile phase B to 35% B. Mobile phase A consisted of 0.2%
formic acid, 5% DMSO, and water; mobile phase B was 0.2%
formic acid, 5% DMSO, and acetonitrile.
18. For data collected using the Orbitrap Fusion mass spectrometer, it was set to acquire data in a data-dependent fashion
selecting and fragmenting by collision-induced dissociation
the most intense precursor ions optimized to maximize duty
cycle. An exclusion window of 60 s was used to improve
proteomic depth, and multiple charge states of the same ion
were not sampled.
19. For data collected using the timsTOF Pro mass spectrometer in
PASEF mode, the mobility range used was 0.7 1/K 0 to 1.5
1/K 0 and the ramp time was 100 ms. The effective duty cycle
was 120 Hz, where the dynamic exclusion setting was set to
0.4 min.
3.8 Sample
Preparation for Mass
Spectrometry Using
iST Sample
Preparation Kit from
PreOmics
As an alternative to using limited gels, the samples can be directly
prepared using the iST Sample Preparation Kit from PreOmics.
This allows for a fast and complete sample preparation that minimizes possible background contamination. The kit can be used
with samples as small as 1 μg of protein. In our case, we were able
to use it for small cellular protrusions, like filopodia. The kit
includes lysis, digest, and peptide purification protocols (see
Note 25).
1. Follow the manufacturer’s instructions.
2. The peptide pool is obtained at the end, ready to be run
for MS.
3.9 Data Analysis
1. MS data analysis will vary depending on the mass spectrometer
and software used. In our case, the MS data were analyzed
using Preview and Byonic v2.6.49 as well as custom tools for
Proteome of Cellular Protrusions Identified by LCM/MS
39