Biology Reference
In-Depth Information
sequencing. MS-grade trypsin is chemically modifi ed to minimize
autolysis and the resulting MS “background” peaks. It is important
for effi cient digestion that proteins are denatured and fully avail-
able to the enzyme. This is ensured by S-S bond reduction and
cysteine alkylation before, and often also after, electrophoresis ((
1
)
and see Chapter 10). Different techniques have been reported to
improve the effi ciency of the digestion process, including sonica-
tion (
3
), microwave irradiation (
4
), high pressure (
5
), chemical
additives, and gel shredding (
6
). After enzymatic digestion, peptides
are extracted out of the gel into the solution.
Extraction protocols vary depending on the MS system and
the method of analysis. Solid-phase extraction using C18 ZipTips
is often employed to clean samples before plating them on the
target plate for MALDI MS. Only low concentrations of organic
solvents are allowed if an extract is to be directly loaded onto
reverse-phase chromatographic material such as in C18 ZipTips,
peptide microtraps, and LC columns. Although multiple extraction
steps increase the amount of peptides extracted from a gel plug,
signifi cant sample loss may result from the subsequent sample
transfer to a different tube and from solvent evaporation in vacuum
evaporators. Trifl uoroacetic acid (TFA) is commonly used to
acidify peptide extracts for better ionization effi ciency during
MALDI-MS analysis but should not be used for electrospray ion-
ization (ESI) MS analysis.
Mass spectral analysis can be performed with a variety of mass spec-
trometers with varying ionization mechanisms and mass analyzers
(
2
). Several different ionization sources are utilized in mass spec-
trometry, with ESI and MALDI being the most popular types.
MALDI and ESI ionization modes create different ionization con-
ditions that affect the peptide's charge state, ionization effi ciency,
and observed mass spectrum. In general, MALDI produces singly
charged peptide ions under vacuum, while ESI produces multiply
charged ions at atmospheric pressure (AP). However, AP-MALDI
is a relatively new MALDI ionization procedure that is increasingly
employed with many types of mass analyzers. Since peptides
respond differently to these ionization conditions, MALDI and
ESI should be considered as complementary techniques.
Mass analyzers also differ in design and utility, often exploiting
fragmentation of peptide ions in MS/MS applications. Fragmen-
tation is typically collisionally induced after ionization and is often
employed because it generally provides the most sensitive approach
to peptide sequencing and offers one of the most reliable methods
for protein identifi cations. Several mass analyzers dominate the
fi eld: linear ion traps, triple stage quadrupole, and TOF mass ana-
lyzers. Linear ion traps and various modern hybrid trap instruments
produce high-quality MS/MS fragmentation spectra due to high
ion storage capacity and fast scan times. Triple stage quadrupole
mass spectrometers are ideally suited for targeted proteomics
1.5. Mass Spectral
Analysis
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