Biology Reference
In-Depth Information
consequent transfer of singly charged analyte ions
into the gas phase. Because MALDI is more
tolerant to background contaminants that
suppress ionization in ESI, such as detergents,
extensive sample cleanup is not mandatory.
Proteins or peptides can be separated of
Mass Spectrometry Instrumentation
The past decade has seen substantial improve-
ments in tandem MS instrumentation, ef
ci-
ency of ion transfer by ion optics, and data
interpretation algorithms. Within a decade,
protein identi
ine into
multiple fractions and spotted onto a MALDI
matrix plate prior to MS analysis. Recently,
MALDI-TOF (time-of-
cation increased from a few dozen
proteins to the routine identi
cation of more than
10,000 proteins in mammalian cells.
53,54
MALDI-MS is widely used in combination
with the time-of-
ight mass spectrometry)
emerged as a technique for imaging mass spec-
trometry (IMS) intended for the analysis of small
molecules and intact proteins in cells and human
tissues.
51
IMS requires pretreatment of thin tissue
slices with the MALDI matrix followed by scan-
ning of the tissue by laser beam, thereby
providing a two- and even three-dimensional
spatial distribution of intensities of protein,
peptide, and small-molecule ions.
52
IMS holds
a promise to replace immunohistochemical stain-
ing (IHC) of tissues and facilitate high-throughput
approaches to veri
ight (TOF) instruments, as
both ionization mode and mass measurement
occur in a pulsed fashion. TOF analyzers derive
the mass of an analyte by measuring the
flight
time of each ion in a vacuum tube. Because
TOF instruments have been one of the earlier
instruments capable of high mass accuracy,
they were very frequently used for top-down
proteomics
and studies
of protein post-
translational modi
cations.
As opposed to TOF instruments with pulsed
mode of ion separation, ion-trapping (IT) instru-
ments accumulate ions prior to their mass
measurement. Since ions are given suf
cation of tissue biomarkers.
52
Unlike MALDI, ESI involves an online intro-
duction of samples into the mass spectrometer
in a solvated state and is currently the most
widely used technique for the proteomic
biomarker discovery. Application of voltage,
typically 2,000 to 5,000 V to the sample emitter
tip, leads to formation of highly charged drop-
lets that eventually evaporate, allowing ions to
enter the mass spectrometer.
49
Presence of
high-abundance peptides, organic molecules,
solvent additives, and detergents can signifi-
cient
time to
fill the trap, IT instruments have reason-
ably high sensitivity. In addition, IT instruments
employ ESI, have fast scanning speeds, and offer
the ability to performmultiple levels of fragmen-
tation of the same analyte, but at the expense of
poor mass accuracy (100 to 200 ppm) and resolu-
tion (2,000
full width at half maximum
[FWHM]).
Quadrupole instruments use the principle of
-
cantly reduce ionization ef
ciency of low-
abundance peptides, an effect referred to as
ionization suppression. A variety of protein
and peptide depletion or fractionation
approaches is frequently used to reduce compe-
tition of low-abundance analytes for charge,
diminish ion suppression, and thus increase
the number of peptide and protein identi
filtering peptide ions in the oscillating electric
fields, transmitting only ions within the narrow
and prede
ned m/z range. Advantages of quad-
rupoles include fast scan times and high sensi-
tivity; resolution of quadrupoles, however,
remains relatively low (1,000 FWHM). Triple
quadrupole mass spectrometers employ consec-
utive
ca-
tions.
18
To alleviate the effect of contaminants,
differential proteomic
filtering of precursor peptide ions and frag-
mentation and
ling
should employ identical sample preparation
protocols, LC-MS instrumentation, and bio-
informatics algorithms.
biomarker pro
filtering of
fragments,
thus
increasing selectivity of analysis.
The introduction of hybrid instruments that
combined different modes of
ion selection,
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