Biology Reference
In-Depth Information
sensitivity of intact protein analysis.
74
As a result,
signi
complex mixtures according to protein hydro-
phobicity. Technological advancements com-
monly exploited in LC/MS for bottom-up
applications are poised to have a profound
impact on intact protein separations for top-
down. Improved chromatographic resolution
for biomolecules is typically observed with
smaller resin particle sizes (
cant efforts in sample preparation and
chromatographic separations have been made
in the last decade to extend the dynamic range
and detection limits of top-down analysis.
Sample Preparation
ESI is largely considered intolerant to the
presence of background components in
samples. Protein samples are often prepared
with biological buffers (e.g., Tris, PBS), salts
(e.g., sodium, potassium, and phosphate), cha-
otropic agents (e.g., urea), and many detergents
(e.g., sodium dodecyl sulfate [SDS]). Excess salts
and detergents lead to chemical noise that
competes with protein analyte for charge in
the ESI solution and the creation of isobaric
background species that can obscure protein
signal. Similarly, contaminants such as plasti-
cizers and silicone additives in low-grade
solvents, sample tubes, and laboratory equip-
ment may adversely affect analysis.
35
It is
common practice to use MS-compatible volatile
salts and buffers such as ammonium acetate or
ammonium bicarbonate and remove detergents
by protein precipitation and salts by reversed-
phase liquid chromatography (RPLC) or desalt-
ing columns. Detergent removal is critically
important for charged detergents (e.g., SDS)
where protein adduction is prevalent even at
low detergent concentrations, necessitating
extreme measures such as cold acetone or fulch
precipitation to remove the detergent.
22,75
A
variety of MS-compatible detergents such as
acid labile surfactants (ALS) have been devel-
oped for top-down analysis.
76
m), although
this comes at the expense of high backpressures,
which necessitate lower solvent
5
m
ow rates and
extended analysis times.
78
However, the imple-
mentation of ultra-high-performance liquid
chromatography (UHPLC or UPLC) for protein
separations at high backpressure (400
e
1,600
bar) has been shown to permit high
ow rates
and fast elution times (~
1
/
3
thetimefortradi-
tional LC/MS).
78
Elevated column tempera-
tures (e.g., 50
e
65
C) lower mobile-phase
viscosity and system backpressure while
enhancing adsorption/desorption kinetics,
resulting in reduced band broadening associ-
ated with protein diffusion into and out of pores
in the stationary phase. Similarly, large pore
sizes (1,000 Å) may increase resolution for
some resin chemistries and pore architectures.
75
Also, the mobile phase composition (e.g., pH
and eluotropic strength of solvents) dramati-
cally affects protein conformation, solubility,
ionization ef
ciency, and charging for ESI.
79
For example, acetonitrile (ACN) is a more polar,
aprotic solvent that better denatures proteins
and the less polar isopropanol (IPA) is a stronger
eluent that better solubilizes proteins and
improves elution ef
ciencies for hydrophobic
proteins.
80
For membrane proteins a 1:1 (v/v)
ACN:IPA mobile phase is commonly employed
for improved protein recovery.
81
Various reversed-phase resins with varying
column sizes and stationary phases have been
utilized for online LC/MS of intact proteins.
Reports on the use of monolithic,
82
polymeric,
75
porous silica,
40
and nonporous silica
83
as
RPLC phases in top-down LC/MS demon-
strate varying
High-Performance Liquid
Chromatography
High-performance liquid chromatography
(HPLC) plays a pivotal role in both bottom-up
and top-down analyses.
77
In particular,
RPLC improves dynamic range by separating
figures-of-merit for chromato-
graphic resolution, speed, and sensitivity. Of
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