Biology Reference
In-Depth Information
isotope-labeled ( 13 C and 15 N) amino acids into
the cell culture media and consecutive incorpo-
ration of these amino acids into protein sequence
upon its translation in the cell. 83 In SILAC exper-
iments, treated and control cells are cultured in
the media with heavy ( 13 C and 15 N) or light
( 12 C and 14 N) isotope-labeled lysine and argi-
nine, respectively. Upon
counting the number of times that all peptides
corresponding to a protein were sequenced.
The more abundant the protein, the higher
number of tryptic peptides is available for
sequencing, resulting in more MS/MS events,
referred to as spectral counts. Spectral counting
is applied to relative and absolute protein quan-
ti
cation between different MS runs. Absolute
protein quanti
five or more cell divi-
sions, an equimolar mixture of both cell lysates
is subjected to the sample preparation protocol.
Heavy peptides in such a mixture have physical
and chemical properties identical to those of
light peptides but show an MS1 mass shift. Ratio
of heavy-to-light peptide intensities corresponds
to relative protein abundances between treated
and control cells. SILAC experiments have excel-
lent precision as any run-to-run variation in
LC-MS does not affect the peptide ratio;
however, performing SILAC on complex
samples using slow scanning instruments and
dynamic exclusion settings results in missed
protein identi
cation requires normalization of
spectral counts by correcting for protein length
(normalized spectral
abundance
factor, or
NSAF) 79
or the possible number of
tryptic
peptides (exponentially modi
ed protein abun-
dance index, emPAI). 80 This method has
a dynamic range of about two to three orders
of magnitude but suffers from low precision,
accuracy, and reproducibility, especially for
low abundance proteins that are identi
ed by
few spectral counts. 81
XIC-based quanti
cation methods rely on
measuring the three-dimensional space of
peptide ion intensity, m/z, and chromato-
graphic elution time. Because XIC quanti
cations due to the doubled
sample complexity. Only actively dividing cells,
such as established cancer cell lines, are
amenable to SILAC. Some primary and slow
dividing cells can hardly be cultured for
ca-
tion is more accurate and suitable for
measuring relative abundances of medium-
abundance proteins, even a single MS/MS
spectral count event will have a corresponding
MS1 chromatographic peak that can be inte-
grated. 81 MS/MS fragmentation is still per-
formed to determine identity of each peak but
is not used for quanti
ve
divisions and, as a result, cannot be fully labeled.
Labeling of proteins of whole organisms, such as
bacteria, yeast, fruit
flies, and even mice, is also
possible by feeding them a diet containing
heavy-labeled amino acids. 84 e 89 A heavy SILAC
protein mixture can also be used as a reference
standard when spiked into nonlabeled normal
and disease biological
cation. Another variant
of XIC quanti
cation, intensity-based absolute
quanti
cation (iBAQ), involves dividing the
sum of XIC peptide intensities by the number
of
fluids.90 90 On the negative
side, SILAC experiments are relatively expensive
and have a quite narrow differential quanti
theoretically observable peptides. 82
XIC
quanti
cation requires reproducible chroma-
tography to enable alignment of peptide peaks
and achieves a dynamic range of four orders of
magnitude.
ca-
tion range of approximately twentyfold. 91
Another approach to incorporate heavy
isotopes into peptides involves exchange of two
16 O atoms for two 18 O atoms on C-terminal
peptides during enzymatic digestion of proteins
in deuterated water (H 18 O). 92 As a result, an
MS1 shift of 4 Da between 16 O- and 18 O-labeled
peptides is observed. The major caveat of this
methodology, however,
Metabolic and Enzymatic Labeling
A common metabolic labeling strategy,
SILAC (stable isotope labeling with amino acids
in cell culture),
involves addition of heavy
is a nonhomogenous
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