6. The gel should be stained with MS compatible SDS-PAGE dye
such as Coomassie or SimplyBlue SafeStain.
7. The gel can either be run to completely resolve individual
bands or alternatively, as a means of removing potential contaminants (i.e., detergents and buffer components) prior to
digestion of a complex sample such as whole cell lysate, culture
filtrate proteins, or subcellular fractions. For the latter method,
the gel is run for roughly 5 min at 200 V (depending on
electrophoresis system) or until sample has migrated roughly
1 cm into the gel. The entire lane is then cut and processed as a
single sample. This protocol is also suitable for digestion of
individual protein spots from 2D-GE.
8. Dissolve 25 μg of trypsin in 250 μL of 0.2 M Ammonium
bicarbonate. Reconstituted trypsin should be stored at 4
C
and can be used for up to 2 weeks.
9. This procedure can be used with proteases other than trypsin;
however, the reconstitution buffer may differ for other
proteases.
10. MS loading solvent is used to resuspend the final digest prior to
injection in the LC-MS/MS. This solvent is dependent on the
solvent system used for analysis. For most commonly used
LC-MS/MS analysis in positive mode, a common loading
solution is 3–5% v/v acetonitrile in 0.1% v/v formic acid.
11. The protocol outlined here is ideal for 30 μg of protein. Other
amounts of protein require optimization and volumes adjusted
accordingly.
12. DTT should be prepared fresh, immediately before addition to
the sample.
13. Alternatively, alkylation of cysteines can be carried out using
iodoacetic acid. Alkylation with Iodoacetamide results in carbamidomethylation of cysteines (+57 Delta mass). Alkylation
with Iodoacetic acid results in carboxymethylation of cysteines
(+58 Delta mass). Alkylating reagents are light sensitive. Alkylating solutions should be prepared immediately before use and
should always be protected from light.
14. ProteaseMAX is a surfactant that increases the efficiency of
trypsin’s enzymatic activity. This protocol can be modified if
ProteaseMAX is not available by increasing trypsin incubation
time with the sample to 18 h.
15. Intact proteins can also be analyzed by direct infusion in a
LC-MS/MS analyzer. Please refer to the subsequent sections
in this chapter if this is needed for your instrument.
16. If any of the samples contain ethylene glycol or other somewhat viscous substances that do not dry quickly, allow plate to
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Carolina Mehaffy et al.
such as Coomassie or SimplyBlue SafeStain.
7. The gel can either be run to completely resolve individual
bands or alternatively, as a means of removing potential contaminants (i.e., detergents and buffer components) prior to
digestion of a complex sample such as whole cell lysate, culture
filtrate proteins, or subcellular fractions. For the latter method,
the gel is run for roughly 5 min at 200 V (depending on
electrophoresis system) or until sample has migrated roughly
1 cm into the gel. The entire lane is then cut and processed as a
single sample. This protocol is also suitable for digestion of
individual protein spots from 2D-GE.
8. Dissolve 25 μg of trypsin in 250 μL of 0.2 M Ammonium
bicarbonate. Reconstituted trypsin should be stored at 4
C
and can be used for up to 2 weeks.
9. This procedure can be used with proteases other than trypsin;
however, the reconstitution buffer may differ for other
proteases.
10. MS loading solvent is used to resuspend the final digest prior to
injection in the LC-MS/MS. This solvent is dependent on the
solvent system used for analysis. For most commonly used
LC-MS/MS analysis in positive mode, a common loading
solution is 3–5% v/v acetonitrile in 0.1% v/v formic acid.
11. The protocol outlined here is ideal for 30 μg of protein. Other
amounts of protein require optimization and volumes adjusted
accordingly.
12. DTT should be prepared fresh, immediately before addition to
the sample.
13. Alternatively, alkylation of cysteines can be carried out using
iodoacetic acid. Alkylation with Iodoacetamide results in carbamidomethylation of cysteines (+57 Delta mass). Alkylation
with Iodoacetic acid results in carboxymethylation of cysteines
(+58 Delta mass). Alkylating reagents are light sensitive. Alkylating solutions should be prepared immediately before use and
should always be protected from light.
14. ProteaseMAX is a surfactant that increases the efficiency of
trypsin’s enzymatic activity. This protocol can be modified if
ProteaseMAX is not available by increasing trypsin incubation
time with the sample to 18 h.
15. Intact proteins can also be analyzed by direct infusion in a
LC-MS/MS analyzer. Please refer to the subsequent sections
in this chapter if this is needed for your instrument.
16. If any of the samples contain ethylene glycol or other somewhat viscous substances that do not dry quickly, allow plate to
544
Carolina Mehaffy et al.