On-Line Two-Dimensional Separation of Intact Proteins
CAN
105
Karl Burgess and Andrew Pitt,
Functional Genomics Facility, University of Glasgow, UK
Introduction
While 2-D-electrophoresis is a powerful tool for protein separation,
it is difficult to automate, and has limited utility for the analysis of
many classes of proteins. Multidimensional liquid chromatography
of peptides, exemplified by shotgun peptide analysis, is highly
automated, but requires the proteolytic digestion of the sample,
which greatly increases sample complexity and therefore the
required resolution of subsequent separations. Furthermore, much
of the information on differential post-translational modification and
isoform expression is lost. The use of Dionex ProPac® and Dionex
PepSwiftTM polystyrene divinylbenzne (PS-DVB) columns allow
improved resolution, as well as rapid and automated separation of
intact proteins from complex samples.
Off-line two-dimensional (2-D) separation of proteins is an effective
method for simplifying complex samples; however, when using an
intermediate fraction collection step it is difficult to guarantee the
reinjection of the entire collected fraction, and there may be sample
loss due to the binding of protein to the plastic in the microplates.
Finally, frequent reconfiguration of the system to alternatively run
ion-exchange and reversed-phase columns may be challenging for
some scientists.
On-line 2-D separation allows more precise control over the
gradient, and reduces sample loss. In addition, the process is
significantly more automated.
Dionex UltiMate 3000 Proteomics MDLC, consisting of:
LPG-3400M micropump
LPG-3100 pump
WPS-3000 autosampler
2 x FLM-3100 column compartment
UVD-3000 detector
Chromeleon® 6.80 Chromatography Data System
Probot Microfraction Collector/Spotter
ProPac SAX-10, 1.0 mm i.d. × 25 cm
PepSwift 500 µm i.d. × 5 cm, PS-DVB monolith column
Bruker HCTultraTM Discovery System
Passion. Power. Productivity.
Acetonitrile (CH3CN), Fluka HPLC Grade
Water, Milli-Q® Ultrapure Water Purification System
Formic Acid (FA), MS Grade, Fluka
Hydrochloric Acid, Analar, Fluka
Tris, Analar. Sigma
NaCl, Analar, Sigma
Preparation of Mobile Phase and
Standards
1 L of ion-exchange mobile phase A was prepared by adding 1.21g
Tris to 1 L Milli-Q water for a 10 mM concentration. Mobile phase pH
was reduced to 8.0 by careful addition of 5.0 M HCl.
1 L of ion-exchange mobile phase B was prepared by adding 1.21g
Tris to 1 L Milli-Q water for a 10 mM concentration. Mobile phase
pH was reduced to 8.0 by addition of 5.0 M HCl. 58.44g NaCl was
added to produce a 1 M salt concentration.
1 L of reversed-phase mobile phase A was prepared by adding 20 mL
HPLC grade CH3CN and 1 mL MS grade FA to 979 mL of MilliQ water.
1 L of reversed-phase mobile phase B was prepared by adding
199.2 mL Milli-Q water and 0.8 mL MS grade FA to 800 mL of HPLC
grade CH3CN.
Preparation of Samples
Equipment
®
Reagents and Standards
Leishmania donovani promastigotes were grown in medium 199
supplemented with 10% fetal calf serum at 25 °C and harvested
during the mid-log phase of growth. Cell pellets were washed twice
with PBS and lysed in DiGE lysis buffer by three cycles of sonication
for 3 s and cooling. 200 µg of each lysate was acetone precipitated
and resuspended in ion-exchange buffer A prior to injection onto the
system.
This is a Customer submitted application note published as is. No ISO data available for included figures
Figure 1: Schematic of online intact protein separation system plumbing. An initial loading step is performed using the isocratic
pump. This flow path is equipped with an in-line microfilter to trap any particulates to preclude contamination of the ProPac column
downstream. During the injection step, proteins are trapped either on the ProPac strong anion-exchange column, or failing that, on
a 500 µm ProSwift reversed-phase column downstream of it. Post-injection, a very shallow gradient is applied to the ion-exchange
column, trapping eluted proteins on alternating ProSwift columns while the other column has a standard, 20 min reversed-phase
gradient applied to it.
Chromatographic Conditions
The LC system was configured according to the workflow shown
in Figure 1. Sample washed off the ProPac column by increasing
salt concentration is trapped by a ProSwift column. While this is
occurring, the other ProSwift column is being eluted by a short
reversed phase gradient, and fractions are collected. Reversed
phase columns alternate between trapping and elution during the
extended ion exchange gradient.
Column:
Mobile Phase:
Gradient:
Flow Rate: Right Pump: Inj. Volume:
Detection:
Column Temp.
2
Ion-Exchange:
ProPac PS-DVB pellicular SAX, 1 mm × 25 cm
Reversed-Phase:
PepSwift PS-DVD monolith, 500 µm × 5 cm
IE A: 10 mM Tris-Cl pH 8.0
IE B: 10 mM Tris-Cl pH 8.0 1M NaCl
RP A: 2% acetonitrile, 0.1% FA in H2O
RP B: 80% acetonitrile, 0.08% FA in H2O
See Table 1
Left Pump: 25 µL/min
20 µL/min
180 µL
UV Absorbance at 214 and 280 nm
Upper oven: 37 °C, Lower oven: 60 °C
On-Line Two Dimensional Separation of Intact Proteins
Table 1: Gradient Table per Fraction
Time (min)
CH3CN (%)
NaCl (%)
0.0
2
n
1.0
2
21.0
56
22
72
26
72
27
2
36
2
n+2.85 (for 16
fraction separations)
ESI-MS/MS Conditions
Ion Mode: Positive, Enhanced
Mass Range: 500-3000 m/z
Max Fill Time: 250 ms
MS/MS Scans: 5 per MS (Ultrascan).
This application note has been kindly provided by a Dionex customer, and in the
opinion of Dionex represents an innovative application of Dionex products. This
application has not been tested in a Dionex applications lab and therefore its
performance is not guaranteed.
This is a Customer submitted application note published as is. No ISO data available for included figures.
Figure 2: UV (214 nm absorbance) pseudogel map of wild type (left) and pentamidine resistant Leishmania donovani analysed
using the DeCyder DIA software (GE Healthcare). Spot detection software was set to detect 1000 spots. 925 spots were detected,
with 102 decreasing and 181 increasing in intensity in pentamidine-resistant Leishmania.
Results and Discussion
Conclusions
200 µg of pentamidine-resistant Leishmania donovani lysate were
separated using the methodology described above. A 16-fraction
separation was performed, with 24 fractions collected in the
reversed phase dimension for a total of 384 fractions. Collected
fractions were subjected to tryptic digestion and analysed by
LC-MS/MS. A total of 499 unique significantly scoring proteins were
identified using the MASCOTR software (Matrix Science). The UV
absorbance data was extracted from the Chromeleon software, and
plotted to produce pseudogel style maps (Figure 2).
On-line 2-D separation of intact proteins provides an additional step
of automation for researchers performing a large number of intact
protein separations. Reduction in sample loss due to reinjection
is also a benefit. Finally, this system allows the possibility of
performing entirely automated top-down analysis of complex
samples.
This is a Customer submitted application note published as is.
No ISO data available for included figures.
PepSwift is a trademark and ProPac, UltiMate, and Chromeleon are registered trademarks of
Dionex Corporation.
Mascot is a registered trademark of Matric Science Ltd.
Milli-Q is a registered trademark of Millipore Corporation.
Passion. Power. Productivity.
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LPN 2254 PDF 04/09
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