Clemson University
TigerPrints
All Theses
12-2015
CAPILLARY-CHANNELED POLYMER
FIBERS AS STATIONARY PHASES FOR
PROTEIN CHROMATOGRAPHY
Marissa Pierson
Clemson University, [email protected]
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Pierson, Marissa, "CAPILLARY-CHANNELED POLYMER FIBERS AS STATIONARY PHASES FOR PROTEIN
CHROMATOGRAPHY" (2015). All Theses. Paper 2280.
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Theses
CAPILLARY-CHANNELED POLYMER FIBERS AS STATIONARY PHASES
FOR PROTEIN CHROMATOGRAPHY
A Thesis
Presented to
the Graduate School of
Clemson University
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
Chemistry
by
Marissa Pierson
December 2015
Accepted by:
Dr. R. Kenneth Marcus, Committee Chair
Dr. Jeffrey Anker
Dr. Phillip Brown
ABSTRACT
Capillary-channeled polymer (C-CP) fibers have been studied in this
laboratory as stationary phases for protein separations in high-performance liquid
chromatography (HPLC). C-CP fibers are uniquely shaped so as to include eight
continuous capillary-channels which interdigitate once packed into a column. The
packed column resembles a monolithic structure of unobstructed flow through
capillary channels which reduces backpressure and increases linear velocity,
reducing separation time. Fibers are effectively nonporous with respect to
macromolecules, resulting in fast mass transfer and high sample recovery. C-CP
fibers made from polypropylene (PP) yield a fairly homogenous hydrophobic
surface suitable for reversed phase (RP) chromatography. In a microbore C-CP
column, separations can be done very quickly, in less than ten minutes, even at
low flow rates, saving time as well as money in column cost, solvent cost and
waste generation.
Electrospray ionization mass spectrometry (ESI-MS) analysis provides
more analytical information than multi-wavelength detectors. However, the low
flow rates needed (<0.5 mL min-1) for optimal spectral clarity generally imply low
linear velocities and slow separations if liquid chromatography (LC) in tandem
with mass spectrometry (MS) is desired. To this end, decreased C-CP column
diameters (0.5 mm i.d.) were employed to increase linear velocity, and therefore
speed separation time, without the need to increase flow rates. Ribonuclease A,
cytochrome c, myoglobin and lysozyme were loaded in phosphate buffered
ii
saline and urine. Matrix was removed using a water loading phase before
applying a gradient of ACN to elute proteins according to hydrophobicity into ESIMS.
The truly interesting and research relevant proteins are often found in
small concentrations in serum. Analysis of biofluids for potential biomarkers (i.e.
proteins) has been of great interest in medicinal research; however, most
biomarkers are typically found in minute concentrations and masked by more
abundant proteins, prominently, serum albumin. A large portion of albumin
hinders the collection of the proteins of interest. Modifying nylon C-CP fibers with
cibacron blue dye, an albumin-specific ligand has the potential to selectively
remove albumin from serum samples.
iii
DEDICATION
I would like to dedicate this thesis to my family, my husband and my two
best friends. To my family, thank you for being my audience from age 3 to 25.
Thank you, my dear siblings, for fueling my sibling rivalry and for listening to my
scientific ramblings even though I was probably boring you to tears. Thank you to
my parents for the sacrifices you have made so that I might live to learn for a
lifetime without limits. Thank you to my G-Ma, for being my example that women
are strong and smart and capable, especially under pressure.
To Jane, for supporting my ambitions and always picking up where we left
off like no time has passed at all. Kirstin, this place would have crushed me if you
were not here with me. If Clemson gave me one thing, it was you.
To Chuck, there is not space enough to thank you here. You have given
me everything. And while this thesis is not the gift you deserve, it is what I give to
you, for now.
iv
ACKNOWLEDGMENTS
I would like to acknowledge the many mentors in chemistry that have
encouraged me to pursue higher levels of scientific understanding. Notably, I
extend many thanks to my very first mentor in chemistry Dr. Dominic Frollini of
Upper St. Clair High School. His willingness to answer my many questions, even
when I was not the most capable student, opened my eyes to the avenues of
chemistry beyond general curriculum and inspired my undergraduate major
decision. I would also like to extend my gratitude to the dedicated chemistry
professors at Indiana University of Pennsylvania. I learned invaluable techniques
and concepts. I truly appreciate the time and genuine support given to not just
me, but all chemistry students.
I would also like to acknowledge my fellow graduate students at Clemson
University. Through all of the stress and frustration, I am eternally thankful for my
friends who always lent an ear or a shoulder or a glass of wine.
I would also like to acknowledge my advisor, Dr. Marcus, without whom I
would not be earning this Master’s degree.
v
TABLE OF CONTENTS
Page
TITLE PAGE ....................................................................................................... i
ABSTRACT ........................................................................................................ ii
DEDICATION .................................................................................................... iv
ACKNOWLEDGMENTS .................................................................................... v
LIST OF FIGURES ......................................................................................... viii
CHAPTER
I.
INTRODUCTION ............................................................................... 1
II.
POLYPROPYLENE CAPILLARY-CHANNELED POLYMER (C-CP)
FIBERS FOR REVERSED PHASE HIGH PERFORMANCE
LIQUID CHROMATOGRAPHY MASS SPECTROMETRY
SEPARATION AND ANALYSIS OF FOUR PROTEINS
FROM BIOLOGICAL MATRICES ................................................. 6
Introduction................................................................................... 6
Experimental ................................................................................ 9
Results and Discussion .............................................................. 11
Conclusion.................................................................................. 18
III.
CIBACRON BLUE TRIAZING DYE LIGAND MODIFIED
CAPILLARY-CHANNELED POLYMER (C-CP) FIBERS
FOR THE REMOVAL OF SERUM ALBUMIN ............................ 21
Introduction................................................................................. 22
Experimental .............................................................................. 23
Results and Discussion .............................................................. 24
Conclusion.................................................................................. 32
IV.
CONCLUSIONS AND FUTURE WORK .......................................... 35
REFERENCES ................................................................................................ 38
vi
LIST OF TABLES
Table
2.1
Page
Resolution calculated from peak 1; ribonuclease A, and peak 2;
cytochrome c, in Figure 2.2. ....................................................... 13
vii
LIST OF FIGURES
Figure
Page
2.1
Linear velocity comparison on 0.5 mm and 1.0 mm ID columns. Two
columns of equal length and packed to an interstitial fraction of
~0.63 were compared. Uracil injections denoted system retention
times at varying flow rates so linear velocities could be
calculated ................................................................................... 12
2.2
Gradient elution of 100 ppm each: ribonuclease a, cytochrome c and
transferrin from a 30cm x .5 mm ID column packed with
polypropylene C-CP fibers. Flow rate was varied ....................... 14
2.3
Resolutions calculated between neighboring peak pairs. Gradients
were varied linearly by change in ACN+0.01%TFA and are
represented by %B/min, which is a gradient rate of 25 %B/min
would take 2 minutes to reach 50% B. ...................................... 15
2.4
Chromatographic traces of the a) UV at 280nm and b) TIC of the four
protein suite solution made in synthetic urine matrix. Flow rate of
0.25 mL min-1 gradient conditions: 1 min hold in 100% MilliQ water
+ 0.01% TFA, 0-10% ACN + 0.01% TFA in 30 seconds, 10%-50%
ACN in 5 min .............................................................................. 16
2.5
Mass spectra shown are representative of each of the four peaks, peak
1 represented by a), peak 2 by b) and so on. nH+ charge states are
labeled. X-axis denotes m/z ....................................................... 16
3.1
550 Nylon-6 C-CP fibers are pulled through FEP microbore tubing.
Columns are cut into 2.5cm lengths and press fit onto 1mL
micropipette tips. In order, bare fibers, fibers exposed to dye
solution during centrifugation, fibers dyed in dye bath ................ 25
3.2
5 wash solvents; DMSO, PBST, NH4OH, EtOH and H2O, were loaded
in 800uL aliquots. Each was reloaded in order to pass a total of 3
times. Eluent absorbance was read at 280nm on the Tecan plate
reader.1mL HCl, 1mg CB/mL of nylon-HCl, and 1 inch of dyed
nylon in 1mL of HCl, from left to right ......................................... 26
3.3
Quantification of CB on nylon 6 fibers. Vials contain 5mg nylon in 1mL
HCl, 1mg CB/mL of nylon-HCl, and 1 inch of dyed nylon in 1mL of
HCl, from left to right................................................................... 27
viii
List of Figures (Continued)
Figure
Page
3.4
Mass of BSA loaded on cibacron blue dyed and native nylon 1 inch
tips. 100 ppm BSA loaded in PBS, washed with PBS+.1% tween
20. Difference suggests mass remaining on fiber ....................... 28
3.5
BSA tagged with Rhodamine Red Fluorescent marker was passed
over native (b) and cibacron blue dyed (d) tips. (a) and (c) had no
BSA exposure. No change in overall counts observed between (c)
and (d) ........................................................................................ 29
3.6
PBS solution of BSA and CB was incubated for 30 min and then
separated using a PD10 SEC column (5000 Da exclusion limit).
Image (a) shows the second fraction containing CB. Image (b)
shows an aliquot of the first 3.5 mL void fraction following ultracentrifugation (1000 Da exclusion limit). Upper blue solution would
be excluded BSA-CB complex ................................................... 30
3.7
Fluorescent microscope images of native PP-4 tips and those treated
with cibacron blue LTL. Second row images show those tips with
rhodamine red modified BSA loaded on the surface. Bottom row
images show the tips after the r-BSA has experienced a wash in
PBS+.01% Tween-20 ................................................................. 32
ix
CHAPTER ONE
INTRODUCTION
Understanding the proteome has become an area of ever increasing
interest in the scientific and medical communities. Since the term proteomics was
first coined in the early 1990’s scientists have been working to uncover the many
truths that proteins hold [1]. It is generally known that proteins are foundational in
living beings; they make up tissues, exist in, on and around cells, and perform
functions based on their shape. Proteins are integral in every function in the
body, from contracting a muscle to fighting a disease. It is for this reason that
illuminating the many intricacies of protein mechanisms has become so
important. Knowing the roles proteins play allows for the manipulation and
exploitation of the proteome to a medical advantage. One of the fastest growing
areas of the pharmaceutical industry is that of protein therapeutics, especially
and most commonly disease treatment with antibodies [2, 3]. On the other hand,
with a more fully understood proteome, protein diagnostics are possible, using
either the presence of a protein, the absence of a protein or the abnormal level of
a protein [4, 5]. What is more, monitoring the specific protein levels can also help
guide treatments. All of these benefits of understanding the proteome rely on a
depth of understanding. In order to understand and use proteins to our
advantage we need ways to study protein interactions as well as detection
methods. To look at individual proteins demands the ability to separate.
The Marcus Lab has, over the past 12 years, explored the use of capillary
channeled polymer (C-CP) fibers as stationary phases for protein separations [6-
10]. Utilizing fibers generated in the Clemson University materials science
department, fibers are packed into microbore columns for use in high
performance liquid chromatography (HPLC) and as solid phase extraction (SPE)
spin-down tip formats [11, 12]. The fibers are made from several available
polymers; polypropylene (PP), nylon and polyester (PET), which can be used in
various modes of chromatography; reversed phase, ion exchange, hydrophobic
interaction chromatography and chemical modifications for other modes,
respectively [11, 13, 14]. Each fiber is extruded with a unique cross-sectional
shape with protrusions forming eight collinear channels which run down the
length of the fiber. As these fibers are mechanically drawn into microbore or
standard inner-diameter columns, the protrusions interlock with those of adjacent
fibers forming a series of much smaller channels.
The channels promote unobstructed fluid flow, which in HPLC reduces
back pressure and increases linear velocity; this is especially useful with viscous
samples. Another exploited property of the C-CP fibers is the nearly non-porous
surface. C-CP fibers have been specifically employed to separate proteins, as
the pore size is small enough that no protein can enter, thus virtually removing
the C-term broadening with respect to the van Deemter equation [14, 15]. The
downside to this feature is a severe reduction in surface area, and thus binding
capacity, when compared to porous stationary phases. Another potential
advantage of polymer stationary phases is the chemical stability over a broad
range of pH.
One clear benefit of the C-CP fibers has been for solid phase extraction
(SPE) format separations [12, 16-19]. SPE is a standard sample clean-up step
2
that serves to remove matrix salts prior to instrumental data collection; this is
especially the case in mass spectrometry (MS) [20, 21]. SPE works by adsorbing
analyte to a stationary phase while salts and matrix are washed off of the sample
and stationary phase, then a suitable eluent removes the analyte and can
proceed to further analysis. In order to analyze a sample in MS, analyte must first
be ionized. If the sample is in an ionic solution, salts are almost indefinitely more
ionizable and will be preferentially ionized; this can result in noisy spectra or the
loss of analyte signal all together. In previous publications, a small length of CCP fiber column was attached to a micropipette tip so as to form an SPE-tip
which could be used in a centrifuge to provide a driving force for fluid movement
[12, 22]. Protein samples in buffer and matrices are loaded into the tip, spun,
washed with water, spun, and finally eluted with 50/50 ACN/Water. Eluted
proteins can be spotted and analyzed using MALDI-MS. Similarly, full length
columns have been used as injection loops in electrospray ionization (ESI) mass
spectrometry to desalt inline [23]. The resulting spectra generally have an overall
increase in signal to noise ratio. SPE using C-CP fibers is fast, simple and
significantly less expensive than commercially available SPE cartridges, with a
single SPE-tip costing less than $0.25.
Capitalizing on the hydrophobic nature of the polypropylene fiber surface
allows not only SPE type separations, but reversed phase HPLC as well. It has
been demonstrated that three proteins can be separated using a gradient from
90:10 H2O:ACN, to 50:50 H2O:ACN over 2.5 minutes [24]. Optimum fiber
packing, in terms of interstitial fraction, was determined to be ~0.63 for reversed
phase chromatography on polypropylene. This fiber packing is translated into
3
rotation count for ease of understanding in the lab, and these rotation counts will
be specific to column inner diameter necessarily. A range of column inner
diameters and flow rates were also analyzed for resolution and plate height.
The old adage that “time is money” is a driving force in industry and
developmental research. Higher throughput, i.e. analyses per minute, increases
money made per minute. The low flow rates used to obtain low plate heights on
large i.d. columns do not promote high throughput. Meanwhile the high flow rates
are typically only suitable for absorbance spectral monitoring. Ideally, these fiber
columns could be coupled with electrospray ionization (ESI) mass spectrometry
(MS). ESI tends to ionize better at reduced flow rates, with preferred flow rates
between 200–600 μl min-1 [25]. This reduced flow rate also decreases solvent
consumption and waste generation. The benefit of using mass spectrometry for
detection is the added benefit of chemical and structural identification. Using
retention time as the only indicator of an analyte of interest leaves significant
room for error in identification. The challenge is to efficiently separate a sample in
a short period of time using a flow rate compatible with ESI.
The focus of this thesis will highlight a method of protein analysis using
microbore C-CP fiber columns coupled with ESI-MS. Multiple points of interest
will be combined to result in an ideal separation scheme. These points include;
reduced flow rates for use with ESI, reduced column i.d. for increased linear
velocity at low flow rates, and optimized gradient conditions for efficient protein
separation. Currently many lines of research in protein LC-MS use monolithic
stationary phases [26-29]. In general the comparisons of the C-CP fibers unique
to this research group to monolithic stationary phases include reduced back
4
pressure, as a result of through-pores, and chemical stability of the polymers.
The glaring difference tends to be the cost; monolithic columns tend to be difficult
to make with appreciable column to column variability, thus making commercial
columns very expensive, even for a very small column. C-CP columns,
conversely, are very easy to make with significantly inexpensive materials. Fibers
are generated in kilometers long spools such that the fibers from a single batch
are the same, decreasing the column variability.
Employing polypropylene C-CP fibers as reversed phase columns in line
with LC-MS and their potential as the first dimension in LC-LC-MS could impact
the fields of proteomics and urinomics as a method which reduces cost through
materials and increased throughput. Proteomics aims to discover and study
biomarkers as diagnostics and therapeutics for disease. Urinomics hopes to
apply proteomics and other “omics” to the study of urine as a non-invasive
method of biomarker collection. But the added challenges of protein separations
in high salt matrices demands a cost effective method of separation that includes
an inline analysis. Coupling C-CP fibers to LC-MS provides a possible answer to
these challenges through the removal of salts, reduced back pressure, higher
linear velocity, reversed phase separations all while in-line with an established
method of identification.
5
CHAPTER TWO
POLYPROPYLENE CAPILLARY-CHANNELED POLYMER (C-CP) FIBERS FOR
REVERSED PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
MASS SPECTROMETRY SEPARATION AND ANALYSIS OF
PROTEINS FROM BIOLOGICAL MATRICES
Introduction
One of the largest fields of biomedical research is the field of proteomics.
With the realization that proteins can serve as markers in disease diagnostics
and treatment, it is logical that research has aimed to decode the information
contained in protein compositions. With more complexity than the human
genome, the Human Proteome Project has attempted to map the vast protein
landscape [30]. The search for biomarkers and the thresholds of the marker
status; presence, absence, abundance, interaction as well as the structure of
proteins have been some of the goals of current research. In order to find and
use proteins as biomarkers, a sample must be drawn and prepared for analysis,
examples include urine, serum, saliva, tissue and sexual fluids [31]. While all of
these sample sources are of interest, urine has become increasingly popular as a
protein analyte source.
Urine’s popularity is due to the fact that collection is non-invasive, sample
size in most cases is large and the normal protein levels remain relatively stable
in a subject, so changes observed reveal information [32], [33]. Urine protein
levels have been used to profile pediatric cystinuria, diabetic nephropathy
progression among other diagnostics and health monitoring [34], [35], 7]. One of
6
the biggest challenges in the analysis of urine and other complex protein
samples is matrix effects. Proteins of interest are generally in low concentrations
and can be overshadowed by interfering compounds like salts and high
abundance serum proteins which provide little diagnostic value [36].
Because of the challenges in protein monitoring and analysis due to the
complexity of samples [37, 38], a separation step or multiple steps are standard
practice in proteomics [20, 39]. Current methods include solid phase extraction
(SPE), 2D gels, ligand binding assays, precipitation steps and filtration steps [4043]. Each of these methods adds time to analysis as the sample components are
prepared prior to a detection method such as mass spectrometry. SPE is often
used as a method of desalting to remove matrix and is usually a bottleneck point
in the throughput for sample analysis. After SPE preparation, sample is
separated using reversed phase chromatography or capillary electrophoresis
prior to coupling with ESI-MS for detection [20, 39]. This added step and the
inherent challenges of LC-MS have left room in the area of protein LC-MS for
novel research.
As a proposed method of filling this gap, novel capillary-channeled
polymer (C-CP) fiber stationary phases have been studied. The fibers unique 8
channeled shape allows for dense packing by interdigitation. But while packing is
dense and uniform, there is not an extreme increase in backpressure as there
are straight channels running the length of the column. The channels promote
fluid transport and are therefore especially well-suited for analysis of viscous
biological samples. Another advantage of the C-CP fiber columns is their virtually
non-porous surface [15]. The lack of pores reduces mass transport limitations for
7
large molecules, thus reducing the van Deemter c-term broadening. This allows
for increased linear velocities without significant peak broadening.
The current parallel to these unique fibers and their interlocking structure
is the field of monolithic stationary phases. Monoliths boast low backpressures as
the stationary phase is one piece which suffers little to no compression under
pressure, unlike packed bed columns. Monoliths can also be synthesized to allow
different modalities including ion exchange, reversed phase and affinity
chromatography. The monoliths are often generated in column; however the
formation of the structure can be non-uniform, irreproducible, incomplete and
expensive. Monolithic stationary phases have paired well with ESI-MS yielding
very fast baseline separation of a variety of compounds, including proteins [44].
Often the challenges in LC-MS come from physical limitations. Flow rates
that are ideal in chromatography are too voluminous to efficiently ionize and
desolvate in an ESI source. Ion pairing agents in chromatography, like TFA, are
ion suppressants in mass spectrometry [45]. Developing methods that are
reliable and maintain the sensitivity and speed can pose its own challenges [46].
Coupling liquid chromatography to mass spectrometry usually involves some
sample preparation method like filtration or SPE which quickly becomes the
analysis bottleneck because the time to prepare a sample is longer than the
instrument run time, and is usually not automated in line with detection [32]. In
some cases, differences in matrices can shift the retention time of an analyte
[47].
While the challenges in mass spectrometry can pose problems, the
advantages far outweigh the option of UV/Vis absorbance detection. UV/Vis does
8
not provide specific information about the structure or identity of an analyte,
merely its presence. A major goal of proteomics is to identify an analyte as well
as gain some understanding of the structure and function of the protein of
interest [48, 49].
Current trends in fast protein separations use monolithic columns and
reversed phase gradients. Nischang et. al. used a 15 cm poly(BuMA-co-EDMA)
monolithic capillary columns with a 0-50% ACN+0.1% formic acid gradient to
separate three proteins [50]. They were able to achieve baseline resolution in 6
minutes at a flow rate of 1.6 μL min-1. Similarly, Terborg et. al. added gold
nanoparticles to poly(glycidyl methacrylate-co-ethylene dimethacrylate) capillary
columns for separations of 3 proteins. The gradient conditions 5-70% ACN +
0.1% TFA in water over 7.5 min was unable to baseline resolve all proteins in 8
min at a flow rate of 2 μL min-1 [51]. Finally, Vaast et. al. using a poly(styrene-codivinylbenzene) monolithic capillary column separates a 5 protein suite in under 4
minutes with nearly baseline separation at a flow rate of 4 μL min-1 [52].
We describe here a method of protein separation using polypropylene CCP fibers for analysis using UV/Vis absorbance and ESI-MS detections.
Experimental
Polypropylene capillary channeled polymer fibers were melt-extruded by
the Clemson University College of Material Science and Engineering (Clemson,
SC, USA). Polypropylene, by in-house designation, PP4, was chosen based on
previous publications in this lab [11, 53]. With a uniform shape and maximum
cross-sectional area of 40 μm, it provides the highest separation efficiency. PP4
9
fibers were provided to the Marcus lab on a spool containing a continuous length
of fibers over 5 km. Fibers were bundled using a rotary counter to translate
optimum packing to a rotation count. After bundling, fibers are heat shrunk using
boiling water and then cleaned sequentially in water, acetonitrile and methanol.
Clean fibers are pulled by hand using a monofilament through a length of PEEK
tubing (Cole-Parmer, USA) of two different inner diameters; 0.8mm and 0.4mm.
Columns were packed to an interstitial fraction of ~0.63, determined by uracil
injection.
Stock protein solutions of 500 μg mL-1 each ribonuclease A, lysozyme,
myoglobin, transferrin and cytochrome C from Sigma Aldrich (St. Louis, MO,
USA) were made up in MilliQ water (conductivity 18 mΩ/cm) + 0.1% TFA and
kept frozen between uses. Mobile phase components of the same MilliQ water +
0.1% TFA and HPLC grade acetonitrile (ACN) +0.1% TFA were prepared in
house. For matrix studies, proteins were dissolved in 100 mM phosphate
buffered saline (PBS) with reagents from (Sigma), and synthetic urine matrix at
100 μg mL-1. Synthetic urine mimics the salt composition and pH of a human
sample.
Gradient conditions studied varied but all began at 100% MiliQ water +
0.1% TFA and ended at 50:50 H2O:ACN. Linear velocity and interstitial packing
studies were performed on a Dionex Ultimate 3000 HPLC system with a multiwavelength UV/Vis detector controlled by Chromeleon 6.7 software (Thermo).
The remaining experiments were performed on a Thermo Scientific Surveyor+
LC-ESI-MS system.
10
Results & Discussion
Previous work in this group has demonstrated the ability of C-CP fibers to
separate bio-molecules. In general, these separations have been done on 4.6
mm, 2.1 mm and 1.0 mm inner diameter (ID) C-CP fiber columns. One of the
driving factors of the fiber column success is the low back pressure exhibited
which allows for very high flow rates. As flow rates increase, so must linear
velocity, which in the case of virtually non-porous stationary media, increases the
rate of mass transfer, and ultimately the speed of separation [54]. It is with this
logic the following study was presupposed. By decreasing the inner diameter of
the column, linear velocity of a particular volume flow rate must increase. Figure
2.1 demonstrates this phenomenon. Uracil injections on columns of equal length
and interstitial fraction of approximately 0.63, were used to calculate linear
velocity at various flow rates. At a flow rate of 0.5 mL min-1 the microbore column,
1.0mm ID, shows a linear velocity of about 33 mm sec-1, while the column with
half the inner diameter exhibiting a linear velocity of over 77 mm sec-1. At flow
rates over the 0.5 mL min-1, the 0.5 mm ID column exhibits significant back
pressure. This is not considered a serious draw back because the intended
application of this system is to couple to electrospray ionization mass
spectrometry, which ionizes best in a range of flow rates between 0.25-0.5 mL
min-1.
11
Linear Velocity (mm sec-1)
90
80
70
60
50
40
0.4 mm ID
30
0.8 mm ID
20
10
0
0
0.2
0.4
0.6
0.8
1
1.2
Flow Rate (mL min-1)
Figure 2.1. Linear velocity comparison on 0.4 mm and 0.8 mm ID columns. Two
columns of equal length and packed to an interstitial fraction of ~0.63 were
compared. Uracil injections denoted system retention times at varying flow rates
so linear velocities could be calculated.
C-CP fiber columns have been studied for the use of reversed phase
separations of biomolecules for several years in the Marcus lab [7, 8, 11, 55]. In
order to couple the fiber columns to an HPLC-ESI-MS system, several
parameters needed to be optimized. A comparison of the separation of three
proteins was used to understand the effect of flow rate. Figure 2.2 shows the
separation of ribonuclease A, cytochrome c and human transferrin at 100 µg mL-1
each in PBS buffer, with a single gradient program of 100% MilliQ water + 0.1%
TFA to 100% ACN + 0.1% TFA in 10 minutes. The flow rate of the separation
was varied from 0.1-0.5 mL min-1. Overall, peak width decreases and resolution
increase with increasing flow rate. The resolution of the first and second peaks,
12
ribonuclease A and cytochrome c respectively, reach 2.04 +/- .03 at the
maximum flow rate, as seen in Table 2.1.
Table 2Error! No text of specified style in document..1 Resolution calculated from
peak 1; ribonuclease A, and peak 2; cytochrome c, in Figure 2.2.
Flow Rate (mL min-1) Linear Velocity (mm sec-1)
Resolution +/0.5
62.0438
2.04 0.03
0.4
50.8982
1.76 0.06
0.3
37.77778
1.63 0.02
0.2
24.8538
1.38 0.02
0.1
12.14286
1.15 0.03
The overall time of separation is also reduced by more than half for the 0.5mL
min-1 compared to the 0.1 mL min-1 example. While it is clear that optimally a
higher flow rate is preferred, this figure also serves to support the use of a flow
rate near 0.25 mL min-1 as peak shape and resolution remain. As this experiment
used the larger 0.8 mm ID column, the pressures extolled on a 0.5 mm ID
column could become problematic. To use 0.25 mL min-1 provides a compromise
between separation characteristics and pressure constraints as well as ESI
operational flow rates.
13
.1ml/min
Abs
.2ml/min
.3ml/min
.4ml/min
.5ml/min
2
4
6
8
10
12
14
Time (min)
Figure 2.2 Gradient elution (100:00:100, H2O+0.1%TFA:ACN+0.1%TFA) of
100 µg mL-1 each: ribonuclease A, cytochrome c and transferrin from a 30cm x
0.4mm ID column packed with polypropylene C-CP fibers. Flow rate was varied.
After chooosing the flow rate, gradients were varied at the 0.25 mL min -1
flow rate. Each gradient program had a one minute loading segment before the
initiation of the gradient. Gradients in this experiment were kept linear, that is, at
the initiation of the gradient, the rate was constant over the time period.
Gradients each ran from 100% MilliQ water + 0.01% TFA to 50% ACN + 0.01%
TFA. Figure2.3 shows the effects each gradient had on the pairs of peaks.
Resolution of each peak-pair was calculated using the resolution equation:
.
Four proteins seperated in each experiment; ribonuclease a, cytochrome c,
lysozyme and myoglobin. While there is a trend toward increased resolution with
decreasing gradient rate, there are some points where the trend is not followed.
14
1.6
1.4
Resolution
1.2
1
0.8
Peak1:Peak2
Peak2:Peak3
0.6
Peak3:Peak4
0.4
0.2
0
25
12.5
8.3
6.25
5
4.17
Gradient Rate (%B min-1)
Figure 2.3 Resolutions calculated between neighboring peak pairs. Gradients
were varied linearly by change in ACN+0.01%TFA and are represented by
%B/min, which is a gradient rate of 25 %B/min would take 2 minutes to reach
50% B.
These instances, such as gradient rates 6.25 and 5 %B min-1, in which the
resolution remains the same or decreases even at decreasing gradient rate can
be explained by longitudenal broadening. As it takes longer for proteins to elute
in the mobile phase, the proteins diffuse and broaden the peak shape, reducing
the resolution.
15
Figure 2.4 Chromatographic traces of the a) UV at 280nm and b) Total ion
chromatogram (TIC) of the four protein suite solution made in synthetic urine
matrix. Flow rate of 0.25 mL min-1 gradient conditions: 1 min hold in 100% MilliQ
water + 0.01% TFA, 0-10% ACN + 0.01% TFA in 30 seconds, 10%-50% ACN in
5 min.
Figure 2.5. Mass spectra shown are representative of each of the four
peaks in Figure 2.4, peak 1 represented by a), peak 2 by b) and so on. nH+
charge states are labeled. X-axis denotes m/z.
16
Figures 2.4 and 2.5 show the ability to separate a four protein mixture;
ribonnuclease A, cytochrome c, lysozyme, and myoglobin, at 100 µg mL -1 each,
from undiluted urine matrix for analysis by ESI-MS. Figure 2.4 shows the
chromatographic traces obtained. There is a shift in retention time and a
broadening of peak shape from the UV trace to the total ion chromatogram (TIC),
this is a result of a change in system tubing ID. Figure 2.5 are the peaks
analyzed by MS with the nH+ charge states labeled. Each peak’s spectrum is
determined by averaging the spectra of the center of the elution peak, while
subtracting the spectra of the neighboring baselines. The ability to deconvolve
individual proteins from a chromatograph increases the chances of identifying the
analyte. Typically a UV chomatogram gives little to no identification or structural
information. With the data collected the peaks can be correctly identified as a)
ribonuclease a, 13,700 Da, b) cytochrome c, 12,327 Da c) lysozyme, 14,300 Da
and d) myoglobin, 17,000 Da. It is also noteable that the charge states of the
eluted proteins are, in general, higher than those of proteins that are directly
injected into the system. Proteins tend to denature on the surface of the fibers
and remain this way upon elution. It is well understood that a protein will exhibit
higher charge states in ESI when denatured as more protonation sites are
available [56].
The ability to couple HPLC to ESI-MS has important advantages, and its
own set of challenges. Flow rate restrictions being one large factor, and
conditional constraints being another. In this case, flow rates were kept at 0.25
17
mL min-1 to maintain optimal ionization. Trifluoroacetic acid (TFA) is a very
common ion pairing agent used in reversed phase chromatography to control
mobile phase pH and peak shape. However, TFA is a known ion suppressant in
ESI-MS[45]. In order to reduce the ion suppressant effects of TFA, the
concentration has been reduced from a typical 0.1 v/v% to 0.01%, rather than
replacing with a different ion pairing agent like formic acid [44, 45]. In practice,
the loss in resolution from 0.1% TFA to 0.01% TFA is approximately 0.046.
Because of the minimal loss in resolution, 0.01% TFA was used in all ESI-MS
experiments.
Conclusion
This work has served to compile many past findings surrounding the use of CCP fibers as stationary phases for protein separations. Previously, this four
protein suite was separated over 25 min at 1.5 mL min-1 flow rate [55]. It is
notable that in the case of C-CP fiber columns, increased flow rates result in
increased resolution. This is attributed to the more favorable mass transfer rates
at higher flow rates allowing a protein that finds a favorable mobile phase
composition to be “swept” off the fiber quickly, resulting in better peak shape and
resolution. Decreasing column diameter has served to reduce the solvent
consumption in protein separations. While the times for resolved separations
may not be as short as those previously reported, solvent consumption is
decreased as flow rates are significantly reduced [57].
Further, the use of C-CP fibers as SPE desalting matrices has shown promise
in many practices. Tip format desalting prior to MALDI and ESI MS analysis has
18
shown significant reduction in noise and increased spectral clarity [12, 22, 58,
59]. Following these publications, the application of the C-CP fiber column as an
in-line desalting injection loop was demonstrated as a simple in-line clean-up
step to reduce noise in protein MS due to matrix effects [23]. Compiling these
methods of reversed phase chromatography using gradients, decreasing column
inner diameter and inline SPE desalting of matrices prior to ESI-MS has shown
the ability of the ever-cost efficient C-CP fiber column to provide a single step
method of protein sample preparation (SPE), separation (RP-HPLC) and analysis
(ESI-MS) in under 10 minutes. This is a comperable timescale to current
reversed phase protein separations on monolithic columns [27].
In the future, work would include employing these methods to quantify
proteins in matrices to further the application of C-CP fibers for proteomic
analysis. It would also be interesting to see the effect that temperature would
have on C-CP fiber column separations. A further reduction in back pressure
would be expected, but it would be interesting to see the effect on resolution,
peak shape and separation time, as the solvation strength should increase with
temperature. Other future studies should look into the transfer kinetics and
whether, in the case of C-CP fibers, the flow rate or the linear velocity is the
driving force of the separation efficiency. While the two parameters are clearly
dependent upon one another, it seems that even when the linear velocity is
comperable, if the flow rates are different, the separation time is is also different.
Literature searches surrounding this project have also spawned questions
into glycoproteomics and whether the C-CP fibers have the capacity to separate
19
glyoproteins to further diagnostics [60, 61]. Current trends in proteomics show a
desire to create single step bioreactor/analyzers by immobilizing trypsin on the
surface of a column to digest a protein and simultaneously separate the digest
products. Research in this group has shown promise in the field of surface
modifications via lipid-tethered ligands (LTL) [62, 63]. It would be conceivable to
immobilize a trypsin enzyme to the LTL to digest proteins on column.
20
CHAPTER THREE
CIBACRON BLUE TRIAZINE DYE LIGAND MODIFIED CAPILLARYCHANNELED POLYMER (C-CP) FIBERS FOR THE REMOVAL
OF SERUM ALBUMIN
Abstract
The affinity ligand quality of the triazine dye, cibacron blue, toward serum
albumin has been well studied [64-68]. By immobilizing the ligand to a stationary
phase, affinity separations can be performed in order to remove bulk albumin
from serum samples. This decrease in albumin concentration may serve as a
direct advantage for proteomics as large amounts of serum albumin hinder the
study of low abundance proteins of interest [69]. Capillary-channeled polymer (CCP) fiber columns have shown promise in protein separations as they
demonstrate low backpressures ideal for viscous protein samples, virtually nonporous surfaces with respect to proteins which reduce van Deemter C-term
effects, and high mass transfer rates to increase solute interactions at the
stationary phase surface. Combining these attributes with the specificity of
cibacron blue would generate a useful depletion column that could be used in
spin-down or column formats. Nylon’s terminal amine can react directly with the
chloride moiety of the triazine dye covalently. Complementarily, head-groupmodified PEG-lipids can be used on polypropylene (PP) fibers for the same end
goal [62, 63]. While this may be a useful study, results were far exceeded by
commercial and previous publications [64, 67-72].
21
Introduction
The ever growing field of proteomics is in need of evolving technologies in
order to handle the complexities and problems inherent in biological samples.
The study of proteins aims to solve medical problems and explain biological
processes, aiding in the progress of many fields including, pharmaceuticals, cell
biology and biotechnology. In general, the most abundant protein in any whole
serum sample will be albumin [73]. However, the truly interesting and research
worthy proteins are found in minute concentrations in serum. With the large
proportion of albumin in samples, collecting the proteins of interest can pose
serious problems.
The Marcus group has been developing novel stationary phases for the
separation of proteins via HPLC [7, 8, 10, 74]. With the help of the Materials
Science Department of Clemson University, several different versions of
capillary-channeled polymer (C-CP) fibers have been created [6]. The polymers
which make up the fiber backbone include nylon, polypropylene (PP), and
polyester (PET). These plastics are melt-extruded so as to provide channels
which run the length of the fibers, increasing the available surface area. When
pulled through an HPLC column, the fibers clasp together to form even smaller
channels that run straight through the column. The fibers are effectively nonporous, when used with macromolecules [15]. The closest current comparable
technology is monolithic solid phases [75]; the advantages C-CP fibers have over
the monoliths are low cost, low back-pressure, reproducibility and both pH and
22
physical stability. The polymeric structure of the fibers allows for reversed phase
(RP), hydrophobic interactions (HIC) as well as ion exchange (IEC).
With a focus on affinity chromatography, we have been able to
functionalize the fibers in order to achieve more specific separations [76].
Preliminary data and future publications include Cibacron Blue dying of nylon
fibers and PEG-lipid functionalization of PP fibers using hydrophobic interactions.
PEG-lipids have been shown to adsorb to the fibers through hydrophobic
interactions, leaving a functionalized head group to “hang” in the mobile phase to
interact with the analyte of interest. Cibacron Blue is known to have an affinity to
albumin [68, 77, 78]. Functionalizing the fibers with the dye should add the
affinity of the CB-albumin complex to the fibers’ already efficient fluid transport
and high surface area with negligible pore size for optimum mass transport. This
research is significant because the already defined separation capabilities of CCP fibers will be further specified via affinity interactions to provide simple yet
effective albumin depletion from samples. With fast and inexpensive sample
cleanup, those interested in proteins other than albumin can easily deplete the
abundant albumin from the bulk.
Experimental
Dying Fibers
Extruded nylon-6 fibers were obtained from the Materials Science
Department of Clemson University. Fibers were placed in a dye bath containing
~1mg mL-1 Cibacron Blue dye (Sigma Aldrich, St. Louis, MO, USA) kept at pH 8
with calcium carbonate. Bath was heated to 80° C with stirring, fibers soaked for
23
8hrs. Dyed fibers were washed successively with water, acetonitrile, acetone,
ethanol and methanol before being packed into columns.
Column Construction
550 cibacron blue dye-modified nylon-6 fibers were pulled collinearly
through fluorinated fluorinated ethylene propylene (FEP) microbore (0.8 mm
inner diameter) tubing from Cole Parmer, resulting in an interstitial fraction of
about 0.63. After a full wash using water, acetonitrile and ethanol, columns were
cut into 1 inch “tips” and press fit onto micropipette tips in order to facilitate use in
a centrifuge.
Experimental Setup
Column tips are loaded with protein solutions at 100 µg mL-1
concentrations and solution is driven through the column using centrifugal force
at 1900 rcf. Effluent is collected and analyzed using a Tecan microplate reader at
280 nm.
Dye Determination
In order to determine the mass of dye on the fibers a simple colorimetric
test was performed. 1 inch dyed nylon tips were dissolved in 1 mL concentrated
HCl. At extremely acidic pH, cibacron blue becomes pink, so dye quantification
was determined using a Thermo Genysis 10S UV/Vis spectrophotometer at
500nm.
Results & Discussion
Determination of dye on nylon
24
Nylon-6 fibers were spooled and washed with water, acetonitrile and
ethanol before dying. Fibers were left in a dye bath containing 1 mg mL-1
cibacron blue dye, MilliQ water and the pH was adjusted to ~9 with sodium
bicarbonate. The bath was maintained at 60°C and fibers were submerged for 68 hours. Following dye treatment, fibers were washed heavily again with water,
Figure 3.1: 550 Nylon-6 C-CP fibers are pulled through FEP microbore tubing.
Columns are cut into 2.5cm lengths and press fit onto 1mL micropipette tips. In
order, bare fibers, fibers exposed to dye solution during centrifugation, fibers dyed
in dye bath.
acetonitrile and ethanol before packing into FEP tubing. In order to determine the
best method of removing adsorbed dye, several solvents were passed through
tips three times in 800 µL aliquots. Figure 3.1 shows 3 different tips, the left tip
shows a bare nylon fiber tip, center is a tip exposed to dye via solution load and
centrifugation, the right tip is dye-bath-dyed nylon. The disparity in color between
the center and right tips may indicate that the dye has merely adsorbed to
25
the fiber during simple exposure and that the dye bath allows bonding and
maximum exposure of the fiber to the dye. Absorbance of eluent was read using
Figure 3.2: 5 wash solvents; DMSO, PBST, NH4OH, EtOH and H2O, were
loaded in 800uL aliquots. Each was reloaded in order to pass a total of 3 times.
Eluent absorbance was read at 280nm on the Tecan plate reader.
a Tecan plate reader at 280 nm. Each solvent was tested on 3 tips and 3
absorbance readings from each elution taken. Figure 3.2 confirms the use of
ethanol as a wash medium. Ammonia was added as a wash step as well.
Ammonia is suggested by textile dye manufactures as a way to remove cross
dye from laundered fabrics, so logic follows that it would be useful to remove
non-bonded dye here. A challenge that arises here is that, even though it is
apparent that ethanol is the best for removing non-bonded dye, it is not obvious
when all dye has been removed. It is worth mentioning here that the UV
absorbance intensity is much greater for the cibacron blue dye than for BSA. And
so any stray dye in subsequent steps may have an effect on BSA quantification
results.
26
Subsequent to removing non-bonded dye, total dye was determined. 2.5
cm tip portions, 5 mg nylon on average, were extracted from the FEP tubing and
Figure 3.3: Quantification of CB on nylon 6 fibers. Vials contain 5mg nylon in 1
mL HCl, 1 mg CB mL-1 of nylon-HCl, and 1 inch of dyed nylon in 1 mL of HCl,
from left to right.
the dyed nylon was dissolved in 1mL concentrated hydrochloric acid. At
extremely low pH, cibacron blue appears pink/red in color as seen in Figure 3.3.
Absorbance readings were taken using a Genysis 10S UV/Vis spectrometer at
500 nm. 5mg mL-1 Nylon in concentrated HCl was used as stock to
dissolve/dilute CB. Fibers extracted from dyed nylon tip, dissolved in 1 mL HCl
and read, shows 0.134 (+/- 0.0002) mg cibacron blue per dyed tip.
BSA Removal with CB-Nylon in tip format
Dyed and cleaned tips were compared to native nylon tips for the removal
of BSA from a PBS solution. Figure 3.4 shows the calculated mass of the load,
which is mass loaded minus mass eluted using UV/Vis calibration, the mass
27
Figure 3.4: Mass of BSA loaded on cibacron blue dyed and native nylon 1 inch
tips. 100 ppm BSA loaded in PBS, washed with PBS+.1% tween 20. Difference
suggests mass remaining on fiber.
washed, using a PBS + 0.1% tween 20 solution, and the calculated difference.
The difference between the load and the wash represents the mass of BSA
bound to the surface of the fibers. Native tips will bind BSA through hydrophobic
interactions while CB-dyed tips should bind BSA based on the ligand interaction
plus the hydrophobic interaction. In this case it seems that the dyed fibers
actually reduce the binding capacity. Previous papers published report the use of
tween20 as a method for the reduction in non-specific binding of proteins [79].
However, this data suggests that even tween20 will not completely remove
adsorbed BSA. It is also worth noting the overlap of the error bars which may
also suggest a statistically insignificant difference between the two sets of fibers.
28
In an attempt to further confirm or negate the presence of BSA on the fiber
surface, fluorescent imaging using fluorescently labeled proteins was used as a
qualitative control. Rhodamine-red labeled BSA (R-BSA) and yellow fluorescent
protein (YFP), were graciously provided by Dr. Kenneth Christensen. Figure 3.5
is a comparison of BSA loading between a native nylon tip and a dyed nylon tip.
Image (a) is a native nylon tip with no exposure to R-BSA while (b) is the same
tip after 100 µg mL-1 R-BSA in PBS has been passed through the tip 3 times.
Image (c) is a dyed nylon tip with no R-BSA exposure with image (d) is the same
tip exposed to the same R-BSA solution. There is an obvious increase in
29
fluorescence intensity between the native nylon tips before and after R-BSA
loading, i.e. a positive control for the R-BSA loading and imaging. There was a
background of fluorescence contributed from the dye itself as evidenced in (c).
There is no change in fluorescence
b
count between (c) and (d), suggesting
that no BSA is binding to the dyed
nylon, even via hydrophobic interaction.
There is a slight possibility that the
rhodamine dye attaches near or in the
di-nucleotide fold of BSA which is the
identified area via which cibacron blue
interacts.
Figure 3.6: PBS solution of BSA and
CB was incubated for 30 min and then
separated using a PD10 SEC column
Ligand Interaction
(5000 Da exclusion limit). Image (a)
shows the second fraction containing
Some steps were taken to
CB. Image (b) shows an aliquot of the
first 3.5 mL void fraction following
ensure that the interactions expected
ultra-centrifugation (1000 Da exclusion
were, in fact, foundational. First, that the limit). Upper blue solution would be
excluded BSA-CB complex.
dye-protein complex exists without a support phase was confirmed. A simple
incubation and separation was performed using buffer exchange and ultracentrifugation. A PBS solution of BSA and CB was incubated for 30 min with
stirring to allow the protein and dye to interact. An aliquot of the incubated
solution was loaded onto a PD10 buffer exchange column with an exclusion limit
of 5000 Da. The blue void fraction, 3.5 mL, was collected as it should contain
BSA (~65kDa) and any BSA+CB complex (~65kDa + 800 Da). Figure 3.6 (a)
30
shows the remaining fraction containing only the CB (~800 Da). To confirm the
strength of the interaction, ultra-centrifugation was employed as verification. A
fraction of the first fraction was loaded into an ultra-centrifugation filter and spun
down. Figure 3.6 (b) shows the final separation with the upper portion
maintaining its blue color from the dye. CB falls below the exclusion limit of the
filter (1000 Da) and would therefore pass through if not complexed with the much
larger BSA. Because the eluent in the ultra-filtration step remains uncolored,
while the filtrate is blue, the BSA and cibacron blue are concluded to interact
because without an interaction between the two, the cibacron blue would have
passed through the filter, coloring the eluent.
Cibacron Blue Head Group Modified PEG-Lipid
The final efforts in this exploration were following in the footsteps of other
methods studied in this lab to use poly ethylene glycol (PEG) lipids with a
modified head group. In this case, commercial PEG-lipids with a hydroxyl
modified head group were purchased from Nanocs. Triazine dyes have the ability
to react with both amine and hydroxyl reactive centers, though CB does so only
at elevated temperatures and pH. In this case the time and conditions needed to
generate this lipid-tethered ligand (LTL) would compromise the PEG-lipid. So a
complementary ligand, Reactive Blue 4 (RB4), was chosen as it has the same
ligand interaction with BSA but can react at a significantly lower temperature
(30°C vs. 60°C). After generation of LTLs, polypropylene (PP4) tips were
exposed to the LTL solution and then a PBS solution of 100 ppm R-BSA. Figure
3.7 shows controls of native PP4, and PP4 exposed to just the LTL (no
31
fluorophore). Each of those tips was exposed
to the R-BSA solution imaged and then
washed with PBS + 0.1% tween20 and
Native PP4
PP4 + LTL
PP4 + BSA
PP4 + LTL
+ BSA
PP4+ BSA +
PBS Wash
PP4+LTL +
BSA +Wash
imaged again. Side by side comparison
shows that both the native and LTL treated
tips capture R-BSA, while only LTL treated
PP4 retains any BSA after a PBS + 0.1%
tween20 wash. The retention is very low and
may be considered insignificant in
comparison to more robust stationary phases
with the same ligand. This may be due to
non-optimal lipid loading. There was also no
separation of PEG-lipid+CB from the reaction
solution which would also contain unreacted
dye and PEG-lipid. The unreacted PEG-lipid
would also adsorb to the fiber surface,
reducing protein capture.
Figure 3.7 Fluorescent
microscope images of native PP4 tips and those treated with
cibacron blue LTL. Second row
images show those tips with
rhodamine red modified BSA
loaded on the surface. Bottom
row images show the tips after
the r-BSA has experienced a
wash in PBS+0.01% Tween-20.
Conclusions
The results of the experiments performed in this study led to the
conclusion that at this point, albumin removal using the dye modified fiber system
fails to function in the expected manner. With the surety that the dye indeed acts
as a ligand for BSA, the addition of the dye to the fiber does not maintain this
interaction to the point of BSA depletion from a bulk solution as desired. The
postulated reasoning behind this is a surface ligand deficiency.
32
As a demonstration, 1 inch of nylon packed FEP tubing contains, on
average, 5 mg of fiber. While the exact monomer length is not known, for these
purposes assume 10,000 monomers per polymer length. Converting the mass of
the fiber divided by the molecular mass of nylon monomers, 226.32 g mol-1, the
moles of nylon in a tip amount to about 2.2 nanomole. The challenge arises
because each polymer has only 1 tertiary amine group which can bond to the
triazine dye, thus only 2.2 nanomoles of cibacron blue may bond. This is nearly
consistent with the 0.134 mg amount reported in Figure 3.3. There are two
orders of magnitude difference, which may be a result of the 10,000 monomer
assumption, or incomplete washing of the fiber. Then, based on the assertion
that biomolecules only interact at the surface, the number of “visible” ligands is
further reduced. Accounting for all of these and the fact that not all reactive
centers will react with the dye and not all dye molecules will interact with a BSA
means that the system may, in fact, capture BSA, but not at a detectable level
and not efficiently enough to be implemented.
Two future systems could possibly overcome these limitations. The first,
being a new nylon fiber using a shorter polymer length, which would increase the
number of end-groups per mass. The second would be to implement the lipid
tethered ligand format currently being studied in this lab. While attempts have
been made to use this format, a fundamental problem with the organic chemistry
may have interfered with results. The temperature and pH at which the reactive
dye needs to bond will degrade the PEG-lipid. It may be possible to create a
synthetic LTL with the dye which could subsequently increase ligand density and
perform as a functional system.
33
Should either of those systems perform efficiently, this opens a full line of
possible dye-protein ligand modes for selective capture of an array of proteins.
34
CHAPTER FOUR
CONCLUSIONS AND FUTURE WORK
The preceding thesis has contained two studies for the further application of
C-CP fibers to separate proteins. Through the fibers’ unique eight channeled
design, fluid flow is unobstructed and allows for the use of high linear velocities
without significant increases in backpressure. Because of this property, high
throughput analysis is the most notable advantage. Another key property for high
throughput protein analysis is the low porosity of the fibers. The innability of
macromolecules to enter the pores allows for increased mass ransfer rates and
high sample recovery. The high thorughput sample analysis and the low
materials cost to produce C-CP fibers make the fibers an exremely affordable
and efficient option for protein separations.
Increasing the speed of analysis is a common goal in any method. C-CP fiber
columns allow for increased flow rates because of their unobstructed fluid flow,
additionally, increased flow rates result in increased resolution. This is attributed
to the more favorable mass transfer rates at higher flow rates. While the times for
resolved separations may not be as short as those reported elsewhere, solvent
consumption decreases as flow rates are significantly reduced [57]. Further, the
use of C-CP fibers as SPE desalting matrices has shown promise in many
practices. Tip format desalting prior to MALDI and ESI MS analysis has shown
significant reduction in noise and increased spectral clarity [12, 22, 58, 59].
35
Following these publications, the application of the C-CP fiber column as an inline desalting injection loop was demonstrated as a simple in-line clean-up step
to reduce noise in protein MS due to matrix effects [23]. Compiling these
methods of reversed phase chromatography using gradients, decreasing column
inner diameter and inline SPE desalting of matrices prior to ESI-MS has shown
the ability of the ever-cost efficient C-CP fiber column to provide a single step
method of protein sample preparation (SPE), separation (RP-HPLC) and analysis
(ESI-MS) in under 10 minutes.
In the future, work would include employing these methods to quantify
proteins in matrices to further the application of C-CP fibers for proteomic
analysis. It would also be interesting to see the effect that temperature would
have on C-CP fiber column separations. A further reduction in back pressure
would be expected, but it would be interesting to see the effect on resolution,
peak shape and separation time, as the solvation strength should increase with
temperature. Other future studies should look into the transfer kinetics and
whether, in the case of C-CP fibers, the flow rate or the linear velocity is the
driving force of the separation efficiency. While the two parameters are clearly
dependent upon one another, it seems that even when the linear velocity is
comperable, if the flow rates are different, the separation time is is also different.
Literature searches surrounding this project have also spawned questions
into glycoproteomics and whether the C-CP fibers have the capacity to separate
glyoproteins to further diagnostics [60, 61]. Current trends in proteomics show a
desire to create single step bioreactor/analyzers by immobilizing trypsin on the
36
surface of a column to digest a protein and simultaneously separate the digest
products. Research in this group has shown promise in the field of surface
modifications via lipid-tethered ligands (LTL) [62, 63]. It would be conceivable to
immobilize a trypsin enzyme to the LTL to digest proteins on column.
Another application of the C-CP fibers is deployment as affinity ligand
substrates for affinity chromatography. The results of the experiments performed in
the cibacron blue study led to the conclusion that at this point, albumin removal using the
dye modified fiber system fails to function in the expected manner. With the surety that
the dye indeed acts as a ligand for BSA, the addition of the dye to nylon fibers does not
maintain this interaction to the point of BSA depletion from a bulk solution as desired.
The postulated reasoning behind this is a surface ligand deficiency, due to the fact that
the dye may only attach at the terminal amine.
Two future systems could possibly overcome these limitations. The first,
being a new nylon fiber using a shorter polymer length, which would increase the
number of end-groups per mass. The second would be to implement the lipid
tethered ligand format currently being studied in this lab. While attempts have
been made to use this format, a fundamental problem with the organic chemistry
may have interfered with results. The temperature and pH at which the reactive
dye needs to bond will degrade the PEG-lipid. It may be possible to create a
synthetic LTL with the dye which could subsequently increase ligand density and
perform as a functional system.
Should either of those systems perform efficiently, this opens a full line of
possible dye-protein ligand modes for selective capture of an array of proteins.
37
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31 (2008) 1923-1935.
38
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chromatography separations: Part II - applications, Journal of Separation
Science, 32 (2009) 695-705.
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Polymer (C-CP) Fiber Columns for Rapid Reversed Phase HPLC of Proteins,
Anal. Bioanal. Chem., 404 (2012) 721-729.
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solutions employing capillary-channeled polymer (C-CP) fibers as the stationary
phase, Analyst, 138 (2013) 1098-1106.
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characteristics on nylon 6 capillary-channeled polymer (C-CP) fiber stationary
phases by frontal analysis, Biotechnol Prog, 29 (2013) 1222-1229.
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68-75.
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1351 (2014) 82-89.
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Synthetic Urine Solution Prior to ESI-MS Analysis via a Capillary-Channeled
Polymer Fiber Microcolumn, Journal of the American Society For Mass
Spectrometry, 24 (2013) 975-978.
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fibers for the rapid extraction of proteins from urine matrices prior to detection
with MALDI-MS, Proteomics Clin Appl, 9 (2015) 522-530.
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aqueous systems employing capillary-channeled polymer (C-CP) fibers modified
with poly(acrylic acid) (PAA), Analytical Methods, 2 (2010) 461-469.
39
[19] A.J. Schadock-Hewitt, R.K. Marcus, Initial evaluation of protein A modified
capillary-channeled polymer fibers for the capture and recovery of
immunoglobulin G, J Sep Sci, 37 (2014) 495-504.
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fiber stationary phase extractions of proteins from MALDI-MS suppressing
media, Analytical Methods, 5 (2013) 3194-3200.
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synthetic urine solution prior to ESI-MS analysis via a capillary-channeled
polymer fiber microcolumn, J Am Soc Mass Spectrom, 24 (2013) 975-978.
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40
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43
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44
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45
[74] R.D. Stanelle, M. Mignanelli, P. Brown, R.K. Marcus, Capillary-channeled
polymer (C-CP) fibers as a stationary phase in microbore high-performance liquid
chromatography columns, Anal Bioanal Chem, 384 (2006) 250-258.
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Microextraction in packed sorbent (MEPS) and use of a bonded monolith as
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Analyte Extractions, Anal. Chem., submitted for publication.
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832 (2006) 216-223.
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stability of head group-functionalized PEG-lipid ligand tethers on polypropylene
capillary-channeled polymer fibers, Journal of Separation Science, Submitted for
Publication (2014).
46