online hydrophobic interaction chromatography- asms 2015 ... · online hydrophobic interaction...
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Bifan Chena, Ying Pengb, Santosh G. Valejab, Lichen Xiua, Andrew J. Alpertb,c, and Ying Gea,b,d
a Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, b Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin,c PolyLC Inc., Columbia, Maryland, d Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin
A series of new hydrophobic interaction chromatography
(HIC) materials was developed and evaluated.
For the first time, HIC and mass spectrometry (MS) have
been coupled successfully using ammonium acetate
(NH4OAc) as the salt in the mobile phase.
The separation of standard proteins is demonstrated using
online HIC-MS.
In top-down proteomics, protein separations are usually performed
using reverse phase chromatography (RPC). RPC conditions tend to
denature proteins, exposing numerous hydrophobic residues in the
protein core. This can lead to unusually strong retention, aggregation,
precipitation, poor recovery and wide peaks. An alternative mode is
hydrophobic interaction chromatography (HIC). HIC interacts with
hydrophobic residues on the surface of a protein, maintaining the
tertiary structure. A decreasing concentration gradient of a salt high in
the Hofmeister series is used. Proteins elute in order of increasing
surface hydrophobicity1. While ammonium sulfate is most commonly
used in HIC, it is not compatible with mass spectrometry (MS).
Adequate retention of proteins with the MS-compatible salt ammonium
acetate (NH4OAc) would require concentrations in the range 3-4 M2,
which is impractical for online MS.
This study explores the hypothesis that a HIC material of sufficiently
hydrophobic character could retain proteins with low enough
concentrations of NH4OAc to make online HIC-MS possible. However,
an excessively hydrophobic material could promote denaturation on
contact3,4. A new series of HIC materials was synthesized in a search
for materials that strike a balance between these two trends.
Columns: PolyPROPYL A, PolyBUTYL A, PolyPENTYL A, PolyHEXYL A,
PolyHEPTYL A, PolyOCTYL A, PolyNONYL A, and PolyDECYL A with the
side chains indicated were synthesized as described1 using 3-µm, 1500-Å
silica (PolyLC Inc. [Columbia, MD]). Initial studies were performed with
100×4.6-mm columns and HIC-MS was performed with 100×0.2-mm i.d.
capillaries.
Reagents: NH4OAc and acetonitrile (ACN) were HPLC-grade. Protein
standards including cytochrome C (CytC; equine heart), ribonuclease A
(RNAse; bovine pancreas), aprotinin (Apr; bovine lung),lysozyme (LYS;
chicken egg white), and chymotrypsinogen (CHYGEN; bovine pancreas),
were purchased from Sigma-Aldrich (St. Louis, MO).
HPLC: This was performed with an Essence system from Scientific
Systems Inc. (State College, PA). Flow rate: 1.0 mL/min. Detection:
Absorbance at 280 nm.
LC-MS: Bruker Nano-Advance HPLC system was used with a
PolyHEPTYL A or a PolyHEPTYL A capillary column. Mobile phase A
contained 1 M NH4OAc and mobile phase B contained 20mM NH4OAc in
50:50 water/ ACN; pH was not adjusted. A 15 min gradient (from 100%
MPA to 100% MPB) followed by 100% MPB for 5 min was used at a flow
rate of 3.0 or 2.4 µL/min. Samples eluted from HIC columns were
electrosprayed into a MaXis Plus Q-TOF mass spectrometer (Bruker,
Germany).
Base peak chromatogram of 4-protein mixture HIC-MS run on
PolyHEXYL A at 2.4 µL/min for 15 min (top), 30 min (middle),
and 50 min (bottom) gradient.
Online Hydrophobic Interaction Chromatography-
Mass Spectrometry for Top-Down Proteomics
ASMS 2015
Poster THP 396
With the existing materials PolyPROPYL A and PolyBUTYL A, adequate retention of
proteins requires impractically high concentrations of NH4OAc.
The new, more hydrophobic HIC materials do retain proteins adequately with practical
concentrations of NH4OAc but require the addition of some organic solvent for elution in a
reasonable time. Retention time is also less tied to ligand length: a HIC-RPC hybrid mode.
Effect of salt concentration
Demonstration of online HIC-MS feasibility
using a single protein
y = 137804x - 156837R² = 0.9987
0.E+00
2.E+06
4.E+06
6.E+06
0 10 20 30 40
Pea
k A
rea
Concentration (µM)
Working Curve
Demonstration of online HIC-MS feasibility
using a 4-protein mixture
Online HIC-MS for protein quantification
Different gradient time on PolyHEXYL ABase peak chromatogram and mass spectrum of 2 µL of 0.06 mg/mL Lys at 3
µL/min; 15-min gradient.
Overlaid HIC-MS chromatograms and working curve of serial diluted Lys on PolyHEPTYL
A at 3 µL/min for 15-min gradient, demonstrating decent quantitation capability in HIC-MS.
Isotopic resolution of each protein on a chromatographic scale
Base peak chromatogram of 4-protein mixture HIC-MS run on
PolyHEXYL A (top) and in PolyHEPTYL A (bottom) at 2.4 µL/min;
15-min gradient.
0 2 4 (min)
RNAse CHYGEN
2.5M
1.0M
0.75M
0.5M
0.25M
PolyPROPYL A PolyBUTYL A
(min)0 2 4 6 8 10 12 14
0.5M
0.75M
1.0M
2.5M
20mM
RNAse CHYGEN
3.3M
2.5M
1.0M
0.75M
0.5M
3.3M
2.5M
1.0M
0.75M
0.5M
1
2
3
inte
nsityx1
04
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 (min)
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5
v
HIC-MS : PolyHEXYL A vs. PolyHEPTYL A
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 (min)
PolyHEXYL A
PolyHEPTYL A
Contact with more hydrophobic material denatures
protein CytC to some extent.
High initial salt concentration doesn’t promote retention
much, but at least 0.75 M is required to minimize
denaturation.
RNAse
Apr
LYS CHYGEN
4-protein mixture
PolyHEPTYL A at 3 µL/min for 15 min
Methods
Introduction
Overview Results
Conclusions
References
Acknowledgements
The authors would like to thank the Bruker engineers for
technical assistance and all members of the Ge group for helpful
discussions.
Characterization of a series of new HIC columns
Increasing salting-out effect
Increasing salting-in effect
Anions: PO43-, SO4
2-, CH3COO-, Cl-, Br-, NO3-, ClO4
-, I-, SCN-
Cations: NH4+, Rb+, K+, Na+, Li+, Mg2+, Ca2+, Ba2+
Hofmeister series
(min)0 2 4 6 8
RNAse
CHYGENPolyHEPTYL A
PolyOCTYL A
Partial elution between 10-15’
10’ A B; 5’ B
The new HIC combination works as an intermediate
between HIC and RPC. It requires some organic solvent
and denatures certain proteins to some degree. The
function of the salt seems to be to preserve protein
structure rather than promote retention5.
The less hydrophobic materials PolyPENTYL A,
PolyHEXYL A and PolyHEPTYL A offer the promise of
elution of proteins in high yield and reasonably well-
shaped peaks with retention of at least some tertiary
structure.
We have successfully demonstrated the feasibility of
online HIC-MS for top-down proteomics.
1) A.J. Alpert, J. Chromatogr. 359 (1986) 85.2) D.L. Gooding, M.N. Schmuck, M.P. Nowlan, and K.M.
Gooding, J. Chromatogr. 359 (1986) 331.3) E. Haimer, A. Tscheliessnig, R. Hahn, and A. Jungbauer, J.
Chromatogr. A, 1139 (2007) 84.4) C.T. Mant and R.S. Hodges, in High-Performance Liquid
Chromatography of Peptides and Proteins, CRC Press 1991, pp. 437-450.
5) J.M. McNay, J.P. O’Connell, and E.J. Fernandez, Biotechnol. Bioeng. 76 (2001) 233.
15’ A B
A: NH4OAc concentration as noted
B: 20 mM NH4OAc
In this HIC-RPC hybrid mode, going to low concentration
of NH4OAc without any organic solvent results in no
elution even if organic solvent is subsequently supplied.
(min)0 5 10 15 20
10’ A B; 5’ B C; 5’ C
Lysozyme (LYS)
PolyPENTYL A
10’ B C; 5’ C
10’ A C; 5’ C
A: 2.5 M NH4OAc
B: 20 mM NH4OAc
C: 20 mM NH4OAc w/ 50% ACN
Hysteresis in elution
LYS
Expt’l: 14296.4762
+Na
4 6 8 10 12 (min)0.0
0.4
0.8
1.2
Inte
nsity
X 1
05
LYS
RNAse
Apr
LYS
CHYGEN
Overlaid HIC-MS chromatograms of
individual proteins
HIC-MS chromatogram of the
proteins when mixed
Minimal
adducts
LYS
CHYGEN
4-protein mixture
RNAseApr
LYS CHYGEN
RNAse
Apr
Conventional HIC columns
mV
olts
PolyHEXYL A
PolyHEPTYL A
PolyPENTYL A
CytC (- ACN)
(min)0 4 8 12 16
CytC (+ ACN)
PolyPENTYL A
PolyHEXYL A
PolyHEPTYL A
A: 1 M NH4OAc
B: 20 mM NH4OAc w/ 37.5% ACN
10’ A B; 5’ B
Effect of addition of organic solvent
A: 1M NH4OAc
B: 20 mM NH4OAc
Protein denaturation is more severe on the more hydrophobic
materials, but longer chain materials provide better separation.
PolyHEPTYL A and PolyHEXYL A were determined to be the
best compromise for this HIC-MS investigation.
PolyOCTYL A
PolyPENTYL A
Column comparison
PolyDECYL A LYS
(min)0 2 4 6 8 10 12
PolyHEXYL A
PolyHEPTYL A
PolyNONYL A
PolyDECYL A
Conventional: PolyBUTYL A
RNAse CHYGEN
Denatured form
10’ A B; 5’ B
PolyNONYL A
PolyOCTYL A
PolyHEPTYL A
PolyHEXYL A
PolyPENTYL A
10’ A B; 5’ B
LYS
RNAse
Expt’l: 13674.9416
Apr
Expt’l: 6507.8210
Expt’l: 14296.4762
Expt’l: 25647.1535
CHYGEN
CHYGEN
LYS
LYS
RNAse
Apr
15 min
30 min
50 min
(min)
(min)
1789.0679
2044.5067
2566.5208
1000 1800 2600 m/z
‒ 35.0 µm
‒ 17.5 µm
‒ 8.75 µm
‒ 4.38 µm
‒ 2.19 µm
4-protein mixture
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