Biology Reference
In-Depth Information
cations (PTMs).
1,2
Speciation of genes pro-
vides the proteome with the molecular machinery
necessary to drive diverse structural, metabolic,
and cellular signaling events. Classical molecular
biologyandbiochemical tools (e.g., gel electropho-
resis, Western blots, or enzyme-linked immuno-
sorbent assay [ELISA]) have limited speci
modi
Over the last decade, similar top-down exam-
ples have highlighted how a bird
s-eye view
5
of intact proteins enables investigations into
a variety of biological systems and processes,
namely characterizing the temporal dynamics of
PTMs, deciphering cross-talk between PTMs
that regulate protein interactions, characterizing
discrepancies in genome annotation, character-
izing allele ratios at heterozygous loci, and unrav-
eling protein and PTM-based diagnostic markers
of disease.
3,6,7
The top-down philosophy is
implemented through different MS work
'
city
for resolving protein variation that occurs in
combination (i.e., proteotyping).
3
For example,
recent work by Zabrouskov et al. characterized
cardiac troponin I (cTnI) with 1D gel electropho-
resis as a single, tightly focused band at approxi-
mately 26 kDa (
Figure 1
,inset).
4
When analyzing
cTnI with modern high-resolution mass spectro-
metry (MS), numerous cTnI variants are shown
to exist with masses ranging from 23.4
e
24.1
kDa (
Figure 1
). Fragmentation of the individual
cTnI species with tandem mass spectrometry
(MS/MS orMS
n
) serves to elucidate themolecular
identity and spatial distribution of the modi
ows
including: (1) the characterization of protein
heterogeneity via 2D gel electrophoresis,
8
(2)
pro
uids with surface-enhanced laser
desorption/ionization/time-of-
ling bio
ight (SELDI-
TOF),
9
(3) MS imaging and MS biotyping of
tissues, cells, and microorganisms with matrix-
assisted laser desorption ionization (MALDI)-
TOF,
10,11
(4) MS immunoassays,
12
e
14
and (5)
use of ultra-high-resolution MS and MS/MS to
ca-
tions that occur in parallel.
FIGURE 1
Top-down analysis of cardiac troponin I (cTnI).
4
High-resolution mass spectrometry enables the character-
ization of ~30 cTnI species in a single mass spectrum, contrasting 1D gel electrophoresis (inset), which shows cTnI running as
a single band. Mass spectrometry not only has improved analysis speci
city but also provides accurate molecular mass
information for each cTnI species. Peaks labeled with roman numerals represent truncated cTnI species. Open circles
¼
oxidation.
þ
P&
þ
2P
¼
mono and diphosphorylation.
(Figure reprinted with permission from reference #4.)
Search WWH ::
Custom Search