Biology Reference
In-Depth Information
proteins in blood may indicate an ongoing path-
ological process in a distant organ.
69,70
Further-
more, disturbed glycosylation patterns may be
tissue-speci
quanti
cation of proteins in large numbers of
biological samples. In some biological processes,
a small change in protein levels may be crucial
and lead to substantial changes in cell signaling
outcome or cellular phenotype.
74
Quantitative
proteomic methods that are accurate and repro-
ducible
c even in the case if the protein itself
is expressed in multiple tissues. A differential
phosphorylation pattern has been noted in
several neurodegenerative diseases.
71
Disease-speci
enough to reveal
relatively small
c PTMs are often missed in the
bottom-up proteomics studies because peptides
with PTMs are often poorly ionized by ESI or
missed in the consequent bioinformatics analysis
that does not search for all possible PTMs.
Further advances in bottom-up proteomics will
eventually lead to the more detailed investiga-
tion of PTMs in disease. To enable ef
changes in protein levels (
20%) are essential.
Multiple strategies available for protein quantifi-
-
cation (
Figure 3
) can be categorized as either
label-free methods or methods involving protein
and peptide labeling with chemical tags or heavy
isotopes of carbon (C
13
) or nitrogen (N
15
). The
major advantage of label-assisted over label-
free methods is the ability of former methods
to derive differential protein ratios within
a single MS analysis, as well as higher quanti-
tative accuracy and precision.
75
Label-free
approaches generally have a wide dynamic
range of quanti
cient
PTM analysis, multiple approaches to enrich
PTM peptides, such as lectin
72
or titanium
oxide
73
chromatography, can be used. Analysis
of highly branched and heterogeneous oligosac-
charide chains would require ef
cient
de novo
sequencing methods. High-resolution mass
spectrometry has a lot of potential to enable
robust top-down analysis of PTM variations in
pathological states.
five orders of
magnitude and allow for quantitative compar-
ison of large numbers of samples.
cation of four or
Label-Free Quanti
cation
cation, such as spectral
counting and extracted ion chromatograms
(XIC), offered low sample preparation costs
and was greatly improved lately with the use
of high-resolution instruments, reproducible
chromatography, and powerful data analysis
software.
76
e
78
Label-free quanti
PROTEIN QUANTIFICATION
BY MASS SPECTROMETRY
ows allow for
cataloging proteomes of biological samples but
cannot provide
Protein identi
cation work
accurate
and reproducible
Spectral
counting
relies
on
=
FIGURE 3
Quantitative mass spectrometry approaches. (A) Metabolic labeling, or SILAC (stable isotope labeling by amino
acids in cell culture). Control and treated cells are grown in the media with light- or heavy-isotope labeled lysine and arginine
to allow for
five or six cell divisions, then lysed, mixed in equimolar amounts based on total protein, digested by trypsin, and
analyzed by LC-MS/MS. Heavy-isotope labeled peptides show an MS1 mass shift of 6 to 10 Da. (B) Chemical labeling by
ICAT (isotope-coded af
nity tags). Cysteine residues are labeled with light or heavy tags, proteins are mixed and trypsin
digested, peptides are puri
nity methods, and analyzed by LC-MS/MS. As a result, peptides with heavy isotope-
labeled tags show a mass shift in the MS1 spectrum. (C) Chemical labeling by iTRAQ (isobaric tags for relative and absolute
quanti
ed by af
cation) or TMT (tandem mass tags). Equimolar amounts of total protein extracts are digested by trypsin, peptides are
labeled with isobaric amine-reactive tags, mixed and analyzed by LC-MS/MS. Following peptide fragmentation, reporter ions
show a mass shift in the MS/MS spectrum. (D) Label-free approaches including XIC (extracted ion chromatogram) and
spectral counting. Following protein digestion, each sample type is analyzed separately by high-resolution mass spectrom-
etry. XIC measures integrated MS1 intensity of a precursor ion; spectral counting measures the number of times the precursor
ion was fragmented by MS/MS.
Search WWH ::
Custom Search