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