Biology Reference
In-Depth Information
fragmentation, and separation has revolution-
ized the
experimentally derived mass spectra to compile
spectral libraries. This process is often referred
to as peptide-spectrum matching; it offers
faster data analysis and fewer false-positive
identi
field of proteomics. For example, the
combination of an ion-trap with an Orbitrap,
an analyzer that traps ions in an orbit and uses
Fourier transform algorithm to derive m/z,
allows for a two-stage identi
cations. 62 Probability of the correct
peptide matching at the MS and MS/MS levels
is based on deviation of experimental parent
and fragment m/z from theoretical m/z and is
assessed using various scoring algorithms, such
as Sequest, Mascot, Tandem, SpectumMill, Phe-
nyx, OMSSA, and others. 61,63 e 66 As a result,
peptide sequences are derived with certain
statistical probabilities and false discovery rates.
The use of high-resolution-accuracy instruments
reduces the number of peptides that fall within
the theoretical m/z range in database, thereby
reducing the number of false-positive peptide-
spectrum matches. 57 Not all spectra match the
theoretical database, as some spectra originate
from peptides with PTMs that are not de
cation of peptides,
solely in the ion-trap, or in the Orbitrap, 55,56 or
concurrently in both ion-trap and Orbitrap.
Such a setup provides high-mass accuracy (1 to
5 ppm) in MS and MS/MS modes, resolution
up to 240,000, and relatively fast scan speeds.
LTQ-FTICR, an instrument based on Fourier
transform ion cyclotron resonance, offers capa-
bilities of an Orbitrap with resolutions up to
750,000. 57 The newest hybrid TOF analyzers
also provide high sensitivity, high mass accuracy
(2 to 5 ppm) and resolution (10,000 to 40,000) in
MS1 and MS/MS modes, and fast scan time. 58
Such instruments are equipped with two quad-
rupoles in front of the TOF analyzer and enable
analysis of either whole proteins or tryptic
peptides in complex samples. High mass-
accuracy and resolution allow for
ned
in the search algorithm, from peptides with
SNPs, miscleaved peptides, solvent ions,
contaminant small molecules, lipids, or even
airborne molecules of building materials. 67
An approach to circumvent
filtering out
exact ion masses, thereby reducing background
noise and eliminating co-eluting contaminants.
the issue of
nonspeci
c or naturally occurring cleavage
products is to perform de novo sequencing in
which peptide sequences are derived directly
from MS1 precursor and MS/MS fragment
ions, without matching to the theoretical data-
base. This challenging task, however, requires
clean MS/MS spectra and no interference from
fragment ions originating from co-eluting
peptides and contaminants. The advantage of
this approach is the identi
Deconvolution and Database Search
of Tandem Mass Spectra
Regardless of proteomic platform and choice
of the MS instrument, the general method of
protein or peptide sequencing remains the
same. In all cases, measurement of m/z of
precursor ion is followed by its fragmentation
by collision-induced dissociation (CID), electron-
capture dissociation (ECD) 59 or electron-transfer
dissociation (ETD). 60 The resulting raw spectrum
cation of PTMs and
unexpected proteolytic peptide fragments.
files contain an m/z ratio of precursor ions and
its MS/MS fragments. In the bottom-up proteo-
mic approach, peptides are identi
POST-TRANSLATIONAL
MODIFICATIONS AS DISEASE
BIOMARKERS
ed via match-
ing of experimental MS/MS spectra to
theoretical spectra derived from an in silico
digest of a database containing all known
protein sequences. 61 Another search approach
uses the vast number of publicly available
Possible disease-speci
c post-translational
modi
cations include phosphorylation, glycosyl-
ation, methylation, acetylation, ubiquitination,
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