Biology Reference
In-Depth Information
these, polystyrene-divinylbenzene copolymer
columns are gaining use due to improved
mechanical and chemical stability of polymer
columns compared to silica-based columns.
75
Similarly, super
including bacteria, human plasma, tissues, and
leukocytes.
3,24,40,85
Hydrophilic interaction liquid chromatog-
raphy (HILIC) exploits interactions between
proteins and a polar stationary phase in the pres-
ence of a less polar mixture of organic solvent
and water. When the polar stationary phase is
supplied by IEC columns, the hydrophilic inter-
actions are complemented by ionic interactions
and provide high selectivity and resolution,
particularly for modi
cially porous (SP) RPLC resin,
in which a nonporous silica core is coated with
a thin layer of porous silica, has been shown to
have mass transfer ef
ciencies similar to those
of nonporous silica while maintaining high
loading capacities at back pressures consistent
with standard HPLC pumping systems (i.e.,
no need for UPLC).
84
Our recent work used SP
resins online with small-bore (75
ed peptides and proteins.
For both top-down and middle-down studies,
HILIC on an IEC column has been shown to
improve the dynamic range of histone-codes
by subfractionating intact histone proteins into
PTM isomers.
86,87
For example, Tian et al. created
an online 2D WCX-HILIC/RPLC system for
high-throughput investigations into the major
histone family members isolated from human
m
m) capillary
columns and showed that low attomole levels
of intact proteins were routinely achieved
with typical elution periods of approximately
6 s for proteins analyzed from tissue and cell
lysates.
41
fibroblasts.88
88
Similarly, Young et al. showed
that capillary WCX-HILIC with a saltless pH
gradient supported high-throughput analysis
of hypermodi
Orthogonal and Multidimensional
Separations
Reducing the complexity of protein samples
using orthogonal separation strategies that
separate proteins based upon physicochemical
properties (e.g., polarity, isoelectric point, and
molecular mass) can enable improved dynamic
range and greater numbers of identi
ed histone-codes, characteriz-
ing
200 H3.2 and 70 H4 species from 1
m
gof
>
material in 2 h.
89
Electrophoretic approaches have also re-
ceived considerable attention for intact protein
analysis owing to their high separation effi-
cations
in proteomics research. Similar to bottom-up
approaches, diverse HPLC-based approaches
for separation of proteins have been utilized
for top-down multidimensional platforms as
reviewed by Capriotti et al.
77
Ion exchange
chromatography (IEC)
d
that is, weak anion
exchange (WAX); weak cation exchange
(WCX); strong anion exchange (SAX); strong
cation exchange
-
ciencies.
90
e
92
Separations based upon isoelec-
tric point (pI), such as chromatofocusing (CF)
and isoelectric focusing (IEF), have become
commonplace for top-down. For example, Pat-
rie et al. performed CF on an IEC column with
a descending linear pH gradient to separate
M. acetivorans
proteins prior to top-down anal-
ysis.
39
CF has also been used to characterize
membrane proteins
81,93
and has been exploited
for clinical proteomics in the characterization
of diverse cancers.
94
Solution-phase IEF is also
an effective means for separating intact proteins
and provides information on the pI of proteins
for improved con
(SCX);
and immobilized
metal ion af
nity chromatography (IMAC)
d
separates proteins based upon charge
e
charge
interactions between a protein and a charged
resin, using a stepwise or linear gradient in
counterion concentrations (supplied by salts or
changes in pH) to elute proteins. Intact protein
fractionation by IEC has been used to separate
proteins
ca-
tion. A variety of commercial IEF systems
are available and are typically intended for
of
dence in protein identi
from diverse
biological
sources
ine
first-dimension prefractionation of large
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