(e) Even if gel pieces still appear slightly blue after the final
incubation, do not perform destaining more than four
times total.
27. If the protein amount is unknown, 3–5 μL of trypsin solution
can be used for single protein bands.
28. Trypsin solution is usually completely absorbed by gel pieces in
15 min or less.
29. Allow 10–15 min for swelling of the gel pieces between additions of ammonium bicarbonate. Large gel spots/bands (for
example, when a whole gel lane is processed as a single sample)
may take as much as 100 μL to fully rehydrate.
30. Sample should not dry to completion in the vacuum concentrator as this may result in low peptide recovery for LC-MS due
to poor solubility of some precipitated peptides in traditional
LC-MS loading buffers; rather, samples should be removed in
$5 μL of solution. It is reasonable to assume that the residual
solution is acidified water and compatible with traditional
LC-MS initial buffer conditions.
31. MS loading solvent may be added to samples to bring all
samples up to an equal volume. If the concentration is
unknown, 20 μL per sample is recommended.
32. For sample with no salts or other contaminants, such as those
dialyzed in ammonium bicarbonate or other volatile buffer,
30 μg of protein can be dried down in a vacuum concentrator
instead of performing the acetone precipitation. After sample is
dried, protocol can be continued at step 6.
33. Incubation up to 18 h is acceptable.
34. Before LC-MS/MS analysis, sample needs to be desalted using
a C18 spin column or C18 desalting tips with binding capacity
of at least 30 μg. Follow manufacturer’s protocol.
Acknowledgement
This work was supported by ATCC contract 2010-0516-0005
(a subcontract of NIH-NIAID Contract HHSN272201000027c)
and ATCC contract 2016-0550-0002 (a subcontract of
NIH-NIAID contract HHSN272201600013c.
References
1. Jean Beltran PM, Federspiel JD, Sheng X, Cristea IM (2017) Proteomics and integrative omic
approaches for understanding host-pathogen
interactions and infectious diseases. Mol Syst
Biol 13(3):922. https://doi.org/10.15252/
msb.20167062
2. Albeldas C, Ganief N, Calder B, Nakedi KC,
Garnett S, Nel AJM, Blackburn JM, Soares NC
(2018) Global proteome and phosphoproteome dynamics indicate novel mechanisms of
vitamin C induced dormancy in mycobacterium smegmatis. J Proteome 180:1–10.
546
Carolina Mehaffy et al.
incubation, do not perform destaining more than four
times total.
27. If the protein amount is unknown, 3–5 μL of trypsin solution
can be used for single protein bands.
28. Trypsin solution is usually completely absorbed by gel pieces in
15 min or less.
29. Allow 10–15 min for swelling of the gel pieces between additions of ammonium bicarbonate. Large gel spots/bands (for
example, when a whole gel lane is processed as a single sample)
may take as much as 100 μL to fully rehydrate.
30. Sample should not dry to completion in the vacuum concentrator as this may result in low peptide recovery for LC-MS due
to poor solubility of some precipitated peptides in traditional
LC-MS loading buffers; rather, samples should be removed in
$5 μL of solution. It is reasonable to assume that the residual
solution is acidified water and compatible with traditional
LC-MS initial buffer conditions.
31. MS loading solvent may be added to samples to bring all
samples up to an equal volume. If the concentration is
unknown, 20 μL per sample is recommended.
32. For sample with no salts or other contaminants, such as those
dialyzed in ammonium bicarbonate or other volatile buffer,
30 μg of protein can be dried down in a vacuum concentrator
instead of performing the acetone precipitation. After sample is
dried, protocol can be continued at step 6.
33. Incubation up to 18 h is acceptable.
34. Before LC-MS/MS analysis, sample needs to be desalted using
a C18 spin column or C18 desalting tips with binding capacity
of at least 30 μg. Follow manufacturer’s protocol.
Acknowledgement
This work was supported by ATCC contract 2010-0516-0005
(a subcontract of NIH-NIAID Contract HHSN272201000027c)
and ATCC contract 2016-0550-0002 (a subcontract of
NIH-NIAID contract HHSN272201600013c.
References
1. Jean Beltran PM, Federspiel JD, Sheng X, Cristea IM (2017) Proteomics and integrative omic
approaches for understanding host-pathogen
interactions and infectious diseases. Mol Syst
Biol 13(3):922. https://doi.org/10.15252/
msb.20167062
2. Albeldas C, Ganief N, Calder B, Nakedi KC,
Garnett S, Nel AJM, Blackburn JM, Soares NC
(2018) Global proteome and phosphoproteome dynamics indicate novel mechanisms of
vitamin C induced dormancy in mycobacterium smegmatis. J Proteome 180:1–10.
546
Carolina Mehaffy et al.