Rutgers University Bioanalytical Chemistry Presentation

my author name is is govert somsen

you should use the review to provide information on 1-2 background slides and then have 5-6 slides on each paper with good explanation on results and discussion clearly

CE-MS for Proteomics and Intact
Protein Analysis
Valeriia O. Kuzyk, Govert W. Somsen,
and Rob Haselberg
Abstract
This chapter aims to explore various parameters involved in achieving high-end capillary
electrophoresis hyphenated to mass spectrometry (CE-MS) analysis of proteins, peptides,
and their posttranslational modifications. The
structure of the topics discussed in this book
chapter is conveniently mapped on the scheme
of the CE-MS system itself, starting from
sample preconcentration and injection techniques and finishing with mass analyzer considerations. After going through the technical
considerations, a variety of relevant applications for this analytical approach are presented,
including
posttranslational
modifications analysis, clinical biomarker discovery, and its growing use in the biotechnological industry.
Keywords
CE-MS, Proteomics · Intact protein analysis ·
Sample concentration techniques · CE
separation optimization · CE-MS interfacing ·
MS fragmentation · PTM analysis ·
V. O. Kuzyk · G. W. Somsen · R. Haselberg (*)
Division of Bioanalytical Chemistry, AIMMS:
Amsterdam Institute of Molecular and Life Sciences,
Vrije Universiteit Amsterdam, Amsterdam,
The Netherlands
e-mail: r.haselberg@vu.nl
Biopharmaceuticals analysis · Biomarker
discovery
Abbreviations
ADC
Antibody-drug conjugate
BGE
Background electrolyte
CE
Capillary electrophoresis
CE-MS Capillary electrophoresis-mass
spectrometry
CID
Collision-induced dissociation
CRP
C-reactive protein
CZE
Capillary zone electrophoresis
EKI
Electrokinetic injection
EKS
Electrokinetic supercharging
EOF
Electroosmotic flow
EPO
Erythropoietin
ESI
Electrospray ionization
ETD
Electron transfer dissociation
FASS Field-amplified sample stacking
FESI
Field-enhanced sample injection
FTICR Fourier transform ion cyclotron
resonance
HCD
Higher-energy collision dissociation
HDI
Hydrodynamic injection
IPA
Intact protein analysis
IT
Ion trap mass analyzer
ITP
Isotachophoresis
LB
Leading buffer
LC
Liquid chromatography
© Springer Nature Switzerland AG 2021
A. V. Colnaghi Simionato (ed.), Separation Techniques Applied to Omics Sciences, Advances in
Experimental Medicine and Biology 1336, https://doi.org/10.1007/978-3-030-77252-9_4
51
V. O. Kuzyk et al.
52
LC-MS Liquid chromatography-mass
spectrometry
LE
Leading electrolyte
LOD
Limit of detection
LOQ
Limit of quantitation
LTQ-FTICR Linear trap quadrupole-Fourier
transform ion cyclotron resonance
mAbs
Monoclonal antibodies
MALDI Matrix-assisted laser desorption/
ionization
MCE Microchip capillary
electrophoresis
MS
Mass spectrometry
MSI
Multisegment injection
PTM
Posttranslational modification
QTOF
Quadrupole time of flight
RPLC Reversed-phase liquid
chromatography
SPE
Solid-phase extraction
TE
Terminal electrolyte
t-ITP
Transient isotachophoresis
TOF
Time-of-flight mass analyzer
1
Introduction
Proteomics as a scientific area encompasses
large- and medium-scale studies of proteins
structure and function within a living system. The
proteome itself is a complex system with high
dynamic range of its components abundancy,
representation, and varieties. Whether researchers try to grasp the full diversity of this system
(untargeted approach) or aim for a search and
evaluation of certain components (targeted
approach), a specific toolbox is an absolute must.
The analytical techniques for proteome research
are quite distinct from the traditional protein
chemistry methods as they have to be sensitive
enough to detect low-abundance proteins and to
be sufficiently high-throughput for handling
large amounts of protein species [1].
A high-end analysis in proteomics field – no
matter whether peptides or whole proteins are
being measured – is based on two immanent factors. To begin with, a precise mass determination
with a sensitive detector is needed. Mass spectrometry (MS) is commonly acknowledged to be
the supreme candidate for this task, especially if
its configurations are able to fragment the analytes and gain more information about their primary structure.
However, for the complex samples that proteomics typically deals with, the value of accuracy and high resolution of mass spectrometers
gets nearly lost without proper separation of the
analytes prior to detection. A separation system
that allows analysis of one compound at a time
would be ideal. However, generally this is not
realistic, so highest peak capacity allied to the
best separation resolution should be pursued.
Currently, high-performance liquid chromatography (or more precisely, reversed-phase liquid
chromatography) is a widely recognized workhorse technology for proteomics applications in
both laboratory and clinical settings mainly due
to its efficiency, robustness, and reproducibility
[2–4].
Despite numerous advantages that LC-MS
hyphenation has to offer for proteomics, in some
analytical applications, it may be beneficial to
shift the balance towards capillary electrophoresis (CE) as separation step. Current developments
in MS-based proteomics are primarily aimed at
minimizing the analysis time while keeping the
quality of separation and high sensitivity. For
that, CE-MS is very suitable due to its short analysis times [5] without compromising the sensitivity [6]. Unlike most LC columns, CE capillaries
do not need regeneration prior to new injection;
therefore analytical throughput can be increased
by multiple segmented injection [7]. Low sample
consumption is an integral property of CE system, which is beneficial when dealing with
amount- and volume-limited samples. Moreover,
if the sample is rather diluted, numerous in-line
and on-line preconcentration techniques will help
to leverage the sensitivity of the analysis. It is
also worth mentioning that, in contrast to RPLC,
CE allows simultaneous analysis of very short or
long peptides [8]. Another advantage is the low
flow rate that helps to overcome ionization suppression phenomena [9] and allows the use of
highly aqueous solutions. As hydrophilic molecules are commonly separated in aqueous buffers, that becomes a valuable characteristic of the
method [10].
CE-MS for Proteomics and Intact Protein Analysis
When combined with the high sensitivity of
MS, CE indeed becomes a lucrative analytical
solution. However, CE and RPLC in proteomics
are not competitive but rather complementary
techniques. This complementarity of the techniques has already been demonstrated for both
peptides and proteins [11, 12], and the combination is even being referred to as “Swiss knife” for
proteomics investigations [13]. Additionally,
with the growing popularity of two-dimensional
(2D) separation systems, it is not surprising to
find hyphenated LC-CE-MS approaches being
used for protein analysis [14].
This chapter aims to explore various parameters involved in achieving high-end CE-MS analysis of proteins, peptides, and their
posttranslational modifications. The structure of
the topics discussed in this book chapter can be
conveniently mapped on the scheme of the system itself (Fig. 1), starting from sample preconcentration and injection techniques and finishing
with mass analyzer considerations. After going
through the technical considerations, a variety of
relevant applications for this analytical approach
are presented.
2
Technological Considerations
A CE-MS setup for the analysis of peptides and
proteins does not conceptually differ from any
other type of CE-MS application; however, a vast
53
majority of the research for these analytes is done
using capillary zone electrophoresis (CZE) as a
separation mode. Electrospray ionization (ESI) is
the most used option for the hyphenation with
MS. Therefore, in this chapter we will be mainly
focusing on this setup. Whereas CZE-ESI-MS is
most frequently used, in every step of the analytical workflow (Fig. 1), specific considerations can
make or break its success in peptide and protein
analysis. Below, these issues are sequentially
discussed.
2.1
Sample Introduction
and Preconcentration
Hydrodynamic injection (HDI) is the most
straightforward and the most used way of introducing any sample in the capillary. Here, the
sample plug is pressure-pushed from the injection end of the capillary or is moved due to
induced vacuum at the exit end of the capillary or
is introduced with so-called siphoning by raising
the inlet vial to a certain level above the outlet
vial. The analyte portion will be injected as a
fraction of the sample, and the amount of analyte
will be proportional to the injection volume,
independently on the charge of the analyte and
the sample matrix. Alternatively, electrokinetic
injection (EKI) is performed by applying a certain voltage over the capillary, introducing analytes based on their mobility and (potentially) the
Background electrolyte
Application:
• PTM analysis
• Biopharmaceuticals
• Biomarker discovery and
clinical application
Sample pre-concentration
PEP
PEP
Preventing analyte-wall
interactions
CE-MS interfacing
Sample introduction
Mass analyzers
Fig. 1 Schematic representation of a CE-ESI-MS setup with the essential processes/components mapped
54
generated electroosmotic flow (EOF). The electrokinetic injection procedure is intrinsically
more selective than hydrodynamic injection;
however, it requires careful fit-for-purpose evaluation. It will not perform successfully on weakly
charged analytes or in case of a highly charged
matrix components. A more detailed discussion
on the advantages and limitations for the two
injection types for various applications from a
mathematical modelling perspective can be found
in the review of Breadmore [15]. He demonstrates the capacity of EKI to increase the signal
intensity (Fig. 2) [15] and argues that this injection technique is superior over conventional HDI.
V. O. Kuzyk et al.
Electrophoretic Sample
Concentration
One of the abovementioned advantages of
CE-MS is the very small volume of sample
needed for the analysis. However, that may
become a serious drawback if the sample is at a
very low concentration and/or is available in limited quantity. To overcome this issue, several
electrophoretic preconcentration approaches
have been developed. All of them make use of a
difference between the composition of the sample solution and the bracketing solutions in the
capillary, primarily background electrolyte
(BGE), and additives. The concentration of the
analyte takes place on the boundary of solution
with different chemical properties. When the
stacking event finishes, the analytes enter the
BGE and undergo electrophoretic separation. To
integrate these principles in CE-MS of proteins
and peptides, one should consider two major
restrictions. First, the stacking CE solutions must
be MS-compatible. Second, the solution chemistry must be tuned to favor the analyte solubility
and proper conformation. This is often crucial for
intact protein analysis. However; it must not be
neglected for the (glyco)peptide analysis either,
since carbohydrates may shift the chemical properties of the peptide. Wet chemistry suitability for
CE-MS is further covered in Sect. 2.3.
Isotachophoresis (ITP) is one of the most used
techniques for increasing the sample loadability.
It operates with discontinuous buffer systems of
high ionic mobility leading electrolyte (LE) and
low ionic mobility terminating electrolyte (TE).
These two solutions bracket the sample plug that
has an intermediate ionic mobility. LE and TE
co-ions should have higher and lower effective
mobilities than those of analytes, respectively.
Subsequent application of an electric potential
results in analytes being focused at the LE/TE
interface, and the concentration of all the ions in
the sample plug will adhere to the Kohlrausch
adjustment of concentration. A form of isotacho-
Fig. 2 Simulations of hydrodynamic and electrokinetic
injections of a mixture of cations and anions, showing the
difference in sensitivity and potential bias towards certain
analytes. Separation conditions with no EOF, BGE
20 mM Tris-HEPES. Hydrodynamic injection occupies
1% of the capillary; electrokinetic injection is performed
at 10 kV for 12 s (matrix volume occupies 1% of the capillary). Sample contains 1 μM of each analyte [15]
2.1.1
CE-MS for Proteomics and Intact Protein Analysis
55
phoresis, that is frequently used for stacking in MS-compatible. It is best suited for amphiprotic
CE-MS, is transient isotachophoresis (t-ITP), peptides but also weakly acidic or weakly basic
where the concentration step is directly followed ones since there is an ionization difference in
by an electrophoretic separation of ionizable ana- loading buffer and BGE [20, 21]. Fine tuning of
lytes [16]. In that case a sample is dissolved in the buffers may increase the stacking effect even
either TE or LE (the choice depends on the ion further. If the analyte of interest is well-studied
mobilities of the analytes) and is bracketed by the and its isoelectric point is known, electrolytes
counterpart electrolyte. When the ITP focusing can be selected for their buffering capacity; it
step is finished, ions of the TE are already mixed should be negligible at the peptide’s or protein’s
with the BGE; hence the electrophoretic separa- pI but sufficient in the basic and acidic regions.
tion is initiated. Typically this method has no This will allow the pH junction to exist for a lonadverse effect on separation efficiency and even ger period and leads to even higher signal output
offers an improvement on it [17]. Integration of [22]. As an example, Hasan et al. tested the applit-ITP might increase the limit of detection (LOD) cability of pH junction concentration method
up to two orders of magnitude [18] and allows to (Fig. 3A) for four model proteins with different
inject up to 25% of the total capillary volume isoelectric point and reported 1000- to 10,000-­
instead of standard 1–2% [19]. However, its fold enhancement without compromising the
application is limited by the choice of suitable peak shape (Fig. 3B) [23].
electrolytes and buffers. Acetic acid is a well-­
Approaches like field-amplified sample stackknown TE that is most often used for CE-MS of ing (FASS) and field-enhanced sample injection
peptides and proteins. A great advantage of t-ITP (FESI) can also be used to significantly improve
is overcoming CE-MS incompatibility with non- the detection sensitivity. FASS and FESI events
volatile buffers: the ITP event can be used to are achieved by creating a conductivity discontiseparate the analytes in the nonvolatile condi- nuity in the CE capillary. Here the target analyte
tions, and the following CZE separation will is dissolved in a low-concentration and low-­
immanently transfer them to the volatile BGE, conductivity loading buffer (LB) and injected in a
which does not interfere in the ESI ionization high-conductivity and high-concentration backprocess.
ground electrolyte (BGE). The difference
The dynamic pH junction preconcentration between FASS and FESI is the way the sample is
mechanism is based on a large pH difference introduced, via hydrodynamic vs electrokinetic
between the sample plug and the adjacent injection, respectively. When a high voltage is
BGE. The most widely used buffer system for pH applied, the electrical field in the LB will be
junction in CE-MS is basic leading buffer (LB) higher than the one in BGE. As a consequence,
with the sample plug sandwiched between acidic analytes will have a higher electrophoretic
BGE (e.g., acetic acid). Peptides and proteins ­velocity in the sample plug as compared to the
often bear a negative charge in a basic environ- BGE. When analytes stumble upon sample plug/
ment; therefore, they migrate backwards to the BGE interface, they will migrate slower and will
anode until encountering the acidic BGE and be effectively stacked on this boundary. The
hence acquire a positive charge when they pass anticipated sample concentration factor for FASS
the pH boundary. This makes them migrate back is in range of one to two orders of magnitude. To
to the cathode, and, as a consequence, analytes exemplify, integrating FASS in the CE-MS workget concentrated on this pH boundary. As soon as flow allowed to quantitatively analyze single-cell
the boundary gets neutralized, the stacked ana- proteome of Xenopus laevis embryo, reaching a
lytes migrate to the detector. The use of the 11 nmol/L LOD [24]. FESI typically delivers
inverse buffer system (base-acid-base) is also higher sensitivity enhancement factors. Monton
possible, albeit, less common. The technique and Terabe reported 3000-fold signal improvegained popularity in the proteomics field, since ment reaching fmol/μL LODs when using FESI
even high-concentration acidic BGEs are for peptide mapping of protein tryptic digests
56
Fig. 3 Principle and application of pH-mediated stacking. (a) Schematic illustration of the pH-mediated sample
stacking process. A short plug of strong base is injected
into acidic BGE and is followed by a long hydrodynamic
injection of a sample in alkaline matrix. Peptides gain
positive charge from the protons in the BGE or loose
charge to the hydroxyl ions in the basic plug and are con-
V. O. Kuzyk et al.
sequently concentrated on the pH boundary upon application of the separation voltage. (b) Comparison of CE-MS
electropherograms obtained for the analysis of two peptides by conventional pressure injection (left) and the pH
junction stacking method (right). A clear increase in
signal-­
to-noise ratio is observed when applying pH-­
mediated stacking [23]
[25]. They also mention FESI-bound acidic con- it is directly followed by a plug of strong acid or
ditions as an additional benefit, since it lowers the base (for analyzing cations or anions, respecpeptide adsorption on the silica surface. Some tively). When the voltage is applied, the sample
years later, Pourhaghighi et al. used FESI for zone gets fully titrated, thus creating a low-­
intact protein analysis and demonstrated 3200- conductivity region and initiating a FASS event.
and 4800-fold improvement in terms of peak The zwitterionic properties of peptide sequences
height and peak area, respectively [26]. Despite allow to effectively use the technique in proFASS and FESI appear to be well-established teomics. For CE-MS analysis of a protein digest,
preconcentration methods, the intrinsic medium the concentration sensitivity of tens of fmol/mL
to high conductivity of biological samples may can be reached without losing the separation effidemand prior purifications and sample pre-­ ciency [6]. The method was also successfully
treatment before the techniques become applica- used for separation of site-specific phosphopepble.
Additionally,
they
also
require tide isomers, where sample concentrations are
high-conductivity BGEs, which might affect the typically low [29].
electrospray ionization process and spray stabilAnother way to use electrokinetic injection is
ity. Moreover, one should consider limited pro- exploited by the recently introduced electrokitein or peptide solubility in the loading buffer and netic supercharging (EKS). It is a combination of
still limited injection volumes [27]. On the other a long field-amplified EKI with a t-ITP refocushand, the techniques appear promising due to ing step that combines the selectivity of t-ITP
their simplicity, not requiring careful design of with concentration power of field-amplified samelectrolyte systems.
ple injection resulting in several orders of magniAs mentioned above, biological samples are tude enhancement in sensitivity. It gained notable
rarely low-conductive. To circumvent the extra popularity in microchip-based CE, since EKS
sample pre-treatment steps, which often result in exhibits flexibility not only towards different
sample loss, the mechanism of pH-mediated designs and geometries but also to various detecstacking was suggested [28]. In this technique, a tors and with many types of microchips [30, 31].
sample of high ionic strength is injected, and then Currently, EKS-CE with MS detection is not
CE-MS for Proteomics and Intact Protein Analysis
widely used, mainly due to a limited amount of
available MS-compatible electrolytes. However,
initial tests have shown that sensitivity enhancement factors between 10 and 100 can be obtained.
It was also reported to result in an improved separation when tested on a tryptic digest of beta-­
lactoglobulin [32]. EKS used in analysis of
parathyroid hormone splice variants lowered limits of quantification (LOQs) to 10 pg/mL [33].
However, as for any stacking technique, buffer
compatibility remains a challenge. That, along
with relatively recent implementation of EKS in
the field, may explain the limited amount of publications on the topic. However, the work of
Busnel et al. demonstrated that efficient tryptic
digest sensitivity with enhancement factors up to
10,000 can be obtained with full MS compatibility [32]. Unlike some other techniques, the EKS
stacking mechanism is successfully explained
with computer modelling approaches. Many
experimental factors influencing the concentration efficiency can be integrated in mathematical
models to ensure the best and the most controlled
performance [34].
Injection manipulations may also be used to
speed up the analysis, increase the throughput,
and gain extra sample information. Comparing to
RPLC for bottom-up proteomics applications,
CZE is known to produce less peptide identifications per time unit. That is attributed to the dead
time between the sample injection and the first
compound reaching the detector that ranges
around 25% of the total analysis time [35].
Multisegment injection (MSI) allows several
analyses to be performed in a single run through
introducing several sample plugs alternating with
the BGE spacers. In that manner, the dead time
for n + 1th injection is used to detect peaks of the
nth injection. Being initially developed for metabolite analysis, it also proved useful for proteomics
applications. Dovichi’s group demonstrated that
by doing a triple injection of a yeast protein
digest within a single injection time frame, the
peptide identification rate doubled, and the separation profile remained the same for all the injections, as demonstrated in Fig. 4 [7]. MSI was
used to evaluate B-type natriuretic peptide bioavailability after therapeutic infusion [36]. This
57
peptide is a heart failure biomarker that gets
catabolized by plasma proteases. Its dynamic
generation and breakdown were monitored in
time by means of MSI; up to seven injection
plugs were used in this study.
2.1.2
Chromatographic Sample
Concentration
Increase in sample loadability is effectively
achieved by manipulating the injection process.
However, chromatography-based techniques can
be annexed to CE for the same purpose. An
offline preconcentration, e.g., solid-phase extraction (SPE), is sometimes considered as a separate
step in the protocol; however, a CE-MS analysis
often cannot go without it. Apart from increasing
the analyte concentration and hence sensitivity of
its detection, SPE is often necessary for sample
pre-treatment and cleanup. Analytes in proteomics often come as complex mixtures with
physical (viscosity) and chemical (pH, salts)
properties unsuitable for a high-performance
CE-MS analysis.
The offline preconcentration and pre-­treatment
methods are bound to a simple rule: separation of
the analyte from other components and its elution
in a suitable volume of the appropriate buffer.
Accordingly, liquid-liquid extraction, centrifugation, precipitation, membrane-assisted filtration
and dialysis, SPE, and immunoaffinity approaches
may be used prior to CE-MS analysis of proteins
and peptides. The latter two techniques have also
developed towards in-line and on-line setups for
time-efficient analysis with minimal sample loss.
Integrating SPE directly in the CE-MS platform
is often referred to as chromatographic sample
concentration.
A SPE step can be coupled on-line to the
CE-MS as a separate entity via a switching valve
[37], or it can be introduced as a segment (in-­
line) of the capillary (Fig. 5) [38]. Various stationary phases (usually based on silica-based and
polymer sorbents) and separation principles have
been used in both setups, such as monolith-­
bracketed reversed-phase pre-columns [39],
immunoaffinity chromatography using magnetic
beads [40], reversed-phase [41, 42], and ion
exchange [43]. For miniaturization of the setup,
58
V. O. Kuzyk et al.
Fig. 4 Increase of CE-MS throughput by using multisegment injections as shown for a tryptic digest of yeast. (a)
Three sequential injections are done with different separation time periods between them. Time between injections
are 32.5 min (top), 47 min (2nd), 66.5 min (3rd), and
90 min (bottom). (b) Peptide identification rates obtained
during the runs shown in (a). Average number of IDs is
calculated by dividing the total number of IDs by the total
separation time [7]
Fig. 5 Schematic representation of an integrated immunoaffinity SPE design for serum transthyretin analysis by
CE-MS. Magnetic particles are either trapped in (a) a
microcartridge body (using a large particle size) or are (b)
retained by a magnet at the inlet of the capillary [38]
CE-MS for Proteomics and Intact Protein Analysis
59
the standard capillary may be replaced by a tein groups and 3381 peptides from only 50 ng of
microcartridge. This has been shown for the anal- the digest.
ysis of opioid peptides, where a C18 SPE carTo summarize, CE-MS supports numerous
tridge was combined with a sheathless CZE ways of signal enhancement, and the choice of
setup, resulting in a 5000-fold decrease in LOD the sample preconcentration methods would
when compared to the same CE-MS setup with- depend on both analyte and matrix properties. To
out preconcentration [43]. The dead volume at ease the choice of the technique, we provide a
the sleeve region is reported as a frequent limita- decision flowchart (Fig. 6) [50] for the initial
tion of that arrangement, but a seamless interface guidance.
design solution has been already reported to fix
the issue [44].
Immunoaffinity approaches often reinforce the 2.2
Background Electrolyte
conventional SPE setups discussed above with a
target-specific enrichment. For that, antibodies or BGE pH is the key factor to the overall electromiantibody fragments are immobilized on a solid gration process, since both ionic/effective mobilsupport comprised of monoliths or particles. The ities of the analytes and EOF strongly depend on
technique has been largely applied by the group pH and buffering capacity of the electrolyte.
of Sanz-Nebot for the detection of erythropoietin When selecting a suitable BGE, one should take
[45], opioid peptides [46], serum transthyretin into account the properties of analytes in a sam[46, 47], and other large proteins in complex bio- ple: the ideal BGE pH should lie in between
logical samples [46]. The method was also suc- anionic and cationic species pKas, maybe slightly
cessfully applied to detecting well-known cancer shifted to the cationic value [51]. This will yield
biomarkers: alpha-1-acid glycoprotein (AGP) optimal and stable separation accompanied with
[40] and C-reactive protein [48]. As for medical good resolution due to a constant and stable ionapplication, immunoaffinity CE separations are ization of the analyte. Tuning the pH of the BGE
already used in clinical laboratories [49]; how- can increase the stacking effect of t-ITP.
ever, the addition of MS detection is still in devel- Furthermore, it is not uncommon to improve the
opment since the implementation costs of the separation efficiency and peak shape by increasinstrument still remain rather high.
ing the ionic strength of the BGE. This results in
SPE does offer a great improvement for sam- analyte stacking and lowering of the EOF velocple complexity reduction and preconcentration; ity. However, these approaches are rather limited
however, average elution volumes of both in- and by additional requirements for the electrolyte
on-line SPE are larger than 1–2% of the capillary systems in CE-MS of proteins and peptides. The
volume used for injection. Therefore, additional buffer components have to be volatile to avoid
stacking techniques are often still required for salt-induced signal suppression and high backappropriate sensitivity enhancement. To exem- ground signals.
plify, the recent work of Dovichi’s group shows
These requirements limit the buffer systems
possibility of doing large-scale proteomics analy- options. Potential suitable ionic species with their
sis with an enrichment factor of 3000 compared ionic mobility and pKa values were evaluated in
to conventional electrokinetic injection [43]. the review from Pantuckova and colleagues [52].
They implemented an in-capillary cation-­ They also used a mathematical model of electroexchange monolith for solid-phase extraction lyte migration in free solution using different
with pH gradient elution. That allowed for inte- BGE compositions [53]. The number of comgral sample stacking and pH junction events, monly used BGEs for CE-MS in proteomics is
since the elution buffers series had lower concen- even smaller than this estimation [8]. Nonetheless,
tration but higher pH values than the BGE. This the set of volatile BGE systems often used in
unique setup coupled to a high-resolution LTQ CZE covers almost the entire pH range and
Orbitrap Velos allowed identification of 799 pro- enables the direct coupling of CZE to MS using
60
V. O. Kuzyk et al.
Fig. 6 Decision flowchart to select the most suitable CE sample preconcentration technique. (Modified and reproduced
from Breadmore and Sänger-van de Griend [50])
ESI (Fig. 7) [52]. Free acetic acid [54], formic
acid [55, 56], and the ammonium salts of both
[57] are the first choice BGE buffers for peptides
and protein separations in CE-MS. Sometimes,
the use of carbonate as BGE component may be
favorable, albeit it comes with a drawback of
instability and gas bubbles formation, which can
severely compromise the analysis. For particular
analytes – highly hydrophobic peptides – a nonaqueous buffer system may be considered. Using
a nonaqueous BGE proved to reliably allow the
separation of temporin peptides in less than
12 min of analysis time, thus offering a good
alternative to RPLC-MS systems [58].
Contemplating the complexity of protein
sequence and structure, it is also important to
ensure proper analyte solubility. Mathematically
this was modelled by Ruckenstein and Shulgin,
who focused on salts and organic additives affecting the protein solubilization in aqueous solvents
[59]. The basic principles may be brought down
as follows: proteins denature in high alcohol concentrations, high pH, and high salt concentration.
Therefore, an investigator may want to be
extremely cautious when considering, for example, isopropyl alcohol in the BGE or using
untreated biological matrices as samples.
CE-MS for Proteomics and Intact Protein Analysis
61
9.25
3.75 9.25 3.75
(NH4)2CO3
HFo +NH4Fo
-56.6 76.2 -56.6
76.2
3.75
HFo
4.76
9.25
6.35
76.2
-46.1
HAc +NH4Ac
-42.4 76.2 -42.4
3
76.2 -202.05
NH4Ac +NH4OH
76.2 -42.4 76.2 -202.05
-42.4
2
9.25
9.25 4.76 9.25
HAc
pH
-71.8
NH4HCO3 +NH4OH
4.76 9.25 4.76
-56.6
10.33
4
5
6
7
8
9
10
11
Fig. 7 Commonly used buffers and their operational pH
regions recommended for on-line CZE–ESI-MS. Upper
number represents pKas and lower numbers represent
ionic mobilities of the corresponding ionic species.
(Modified and reproduced from Pantuckova et al. [52])
The use of nonvolatile BGEs in CE-MS is
reported sporadically [60, 61]. Although this
might work under specific choices of the system
design, it is unlikely to become a commonly used
arrangement. One of the main issues with nonvolatile components is the ion pairing phenomenon, where salts form complexes with analytes,
leading to a decreased response in the MS. In
case additives are used in the buffer – such as
dynamic coatings, discussed later in this chapter – their compatibility with MS detection has to
be ensured as well. Unfortunately, in general,
BGE additives that effectively interact with proteins and peptides also result in a significant
decrease of MS detection sensitivity. A way to
circumvent it is to minimize the concentration of
the MS-interfering substances or to dilute the
unwanted compounds with the sheath liquid.
This variant integrally comes as a sensitivity and/
or CE performance trade-off, as Tang et al. has
demonstrated for protein capillary isoelectric
focusing coupled to ESI-MS [62] before the
MS-friendly ampholytes were introduced [63].
One could also use alternatives to ESI as an ionization technique. The use of matrix-assisted
laser desorption ionization (MALDI) has shown
great tolerance to different CE additives as well
as strong advantages for CE-MS analysis of proteomics samples (see corresponding paragraph
on interfacing). However, since it is an offline
hyphenation, it rarely performs as a high-­
throughput method.
Hyphenating incompatible methods is analytical challenging and requires novel solutions.
Two-dimensional (2D) CE systems are widely
used to tackle that problem. The ESI-interfering
components are used in the first dimension to
obtain optimal separation performance and are
either removed from the fractions or separated
from the analytes of interest in the second dimension. This approach was demonstrated by the
group of Neusüß with a heart-cut 2D-CE separation system equipped with an isolated mechanical valve [64]. With the help of UV/Vis detection,
in the first dimension, an analyte of interest was
cut and sent for the second CE separation. The
system feasibility was tested on a BSA digest,
revealing minor peak broadening. Two years later
they have published a review of the current trends
in CE-CE-MS and their applications [65]. As an
example, the ESI interference of ε-aminocaproic
acid (EACA)-based BGE system (routinely used
for pharmaceutical analysis of mAb charge variants by CZE [66]) was circumvented by using it
as a first dimension in a CE-CE-MS setup [67].
When the target analytes reach the UV detector
in the first separation system, analytes are being
pressure-transferred into the mechanical valve
62
sample loop to be then injected to the second
dimension CZE coupled to ESI-MS. Essentially
the charge variant separation happens in the first
dimension, while the second one aims to separate
the mAb from the MS-interfering compounds
(EACA), thus actually serving as a cleanup
measure.
V. O. Kuzyk et al.
charged groups on the analyte surface [69]. On
the downside, those BGE alterations may have
severe adverse effects on analyte ionization, MS
detection, and protein/peptide stability and solubility. Another way to prevent protein-wall interactions is the addition of organic solvents to the
BGE [70]. It is generally assumed that only electrostatic interactions cause protein adsorption,
but hydrophobic interactions and/or protein con2.3
Preventing Analyte-Wall
formational changes can also be involved in this
Interactions
process. For this approach it is difficult to develop
a generic guideline, so the effect of the organic
The main point of concern when dealing with solvent has to be investigated on a case-by-case
high-throughput and comparative CE-MS analy- basis. In general rigid proteins benefit from the
sis of biological samples is keeping the procedure addition of organic solvent to the BGE, whereas
repeatable, reproducible, and robust. To maintain flexible proteins are negatively affected [70]. In
that, the attention should be primarily paid to other studies, organic solvents in the BGE have
migration time stability and EOF consistency, positively affected the separation efficiency of
and both can be easily compromised due to intact proteins [71, 72] and peptides [73]. Organic
adverse interactions of analytes with the (bare-­ solvent addition to the BGE has other advantage,
fused silica) capillary inner wall. Easily put, the such as increasing separation selectivity, reducsilica surface bears a negative charge, which ing Joule heating, shortening analysis time, and,
attracts basic components [68]. If an analyte is in certain cases, enhancing the analyte solubility.
prone to interact with the silica, it may be However, some researchers argue whether these
retarded, and the migration time may be increased benefits are correctly estimated and relied upon
or stretched (thus promoting band broadening). [74].
In a worst-case scenario, adsorption may even be
The most successful and versatile strategy to
irreversible, preventing proper analyte quantita- prevent protein adsorption to the capillary wall in
tion and incapacitating the capillary for further CE-MS is utilizing capillary coatings. Excellent
use. Due to buffer composition inconsistency, the reviews describing and discussing these coatings
EOF stability also gets vulnerable; therefore are available, and the reader is referred to those
sample matrix components should not interact for more in-depth information [69, 75, 76].
with the wall as well.
Coatings may effectively protect the adverse
To prevent protein adsorption, several interactions by creating either steric hindrance or
approaches may be considered. The easiest one is surface inertness (preventing either electrostatic
to use BGEs with an extremely low or high pH, and/or hydrophobic interactions). Another way to
which ensures that the silica is not charged (low achieve that is mounting opposite (positive or
pH) or that both the silica and protein and pep- negative) charges on the analyte of interest and
tides are fully negatively charged (high pH). the capillary wall to create electrostatic
However, proteins tend to be unstable at these pH repulsion.
values due to their complex structure.
The types of coatings and how they adhere to
Furthermore, it is desirable to still have the pos- the surface could be split into three major groups.
sibility of tuning the BGE pH so that separations Dynamic coatings, composed by surfactant-like
can be optimized in CZE mode. Increasing ionic BGE additives, compete with the analyte for the
strength of the BGE or adding surfactants and/or silanol groups of the capillary wall. Since they
ion pairing reagents can also reduce the analyte-­ are added in excess, they will nearly always win
wall interactions by protonating, neutralizing, or the competition. Structurally they are mostly
creating steric hindrance towards the positively polymers (polyamines or polysaccharides) and
CE-MS for Proteomics and Intact Protein Analysis
may be either cationic or neutral. These coatings
are very easy to use, and they are frequently
applied for CE analysis of proteins [75]. However,
they may cause notable background noise and
MS contamination and disturb the ESI process.
Theoretically, there is a potential for their use in
offline CE-MS, but that has not been reported so
far. Capillaries can also be permanently coated
with polymers, otherwise known as static coatings. They are attached covalently based on silane
chemistry and polymerization reactions. Most of
these coatings bear no charge (neutral coating)
which results in little to absence of EOF. This can
impair the ESI spray stability; however, it may be
advantageous for increasing the separation window. These coatings have long-term stability but
are impossible to regenerate if worn-out. The
third way to craft a coating is by adsorption.
Often the capillary is rinsed with a chemical
agent solution prior to analysis and, if a refresher
is necessary, in between the runs. These coatings
may form neutral, cationic, or even anionic [77]
layers on the capillary wall. Their adherence to
the capillary wall is based on hydrogen bonding
and hydrophobic interactions (neutral coatings)
or ionic interactions (cationic and anionic coatings). Another interesting technique is to use a
mixed interaction approach, where coating is first
adsorbed to the capillary surface, but the electric
field transforms it into a permanent one via silicate polymerization [78]. This kind of coating is
relatively easy to apply (simple rinsing steps) and
to strip off the capillary, and the reagent consumption is very moderate (microliter range).
One disadvantage of adsorbed coatings is the
bleeding effect that is sometimes observed at
high BGE pH values [69] that often causes MS
signal quenching.
While it is important to minimize the analyte
migration distortion during CE-MS analysis in
proteomics and protein analysis, coatings are also
vital for the overall method development since
they can modify the EOF strength and direction.
As a short example, if a neutral coating is used,
(almost) no EOF is generated, regardless of the
BGE conditions. Thus, the CE polarity has to be
set considering the net charge of the analyte of
interest in the BGE, since it has to migrate
63
towards the outlet. By making use of a charged
coating, the direction and magnitude of the EOF
may be tuned according to the separation purpose
(see Fig. 8 [76] for the example of coating affecting the peptide separation and Fig. 9 [76] on glycopeptide separation). The advantages and
limitations of various coatings for cation and
anion analysis with respect to CE polarity and
generated EOF were comprehensively reviewed
by Huhn et al., and the reader is referred there for
more details [69]. Besides dictating separation
conditions, coatings also have an impact on the
MS hyphenation by keeping the EOF stable. The
stability of EOF is much more important for
CE-MS than for conventional CE, since no outlet
vial is present and sheath liquid and/or air can get
in the capillary and ruin the separation.
A large variety of polymeric compounds are
used as coatings, some being extensively used
and commercialized, others being newly explored
and shown as a proof of principle. The most popular coatings currently used for proteomics and
intact protein analysis in CE-MS are shown in
Table 1. Most of these coatings were initially
developed with CE application in mind, and not
all of them are straightforwardly compatible with
MS ionization and detection. It must be also
noted that very complex mixtures (real biological
samples) may not be easily analyzed even with
the use of appropriate coatings. Numerous reports
showing high plate numbers and sharp peaks are
reported only for a set of carefully selected,
highly purified standards. However, when complex biological matrices come into play, the picture would most likely change, and therefore
proper tests are to be conducted before any coating can be used on a newly generated sample.
2.4
CE-MS Interfacing
To hyphenate a liquid phase CE separation to
MS, the analyte-carrying solvent needs to be
transferred to the gas phase via an ionization
source. Although MS systems can be equipped
with a variety of ion sources [96], ESI holds
absolute dominance. In ESI the analyte molecule
is transferred directly to the MS and is prone to
64
V. O. Kuzyk et al.
Fig. 8 CZE-ESI-MS analysis of a tryptic bovine serum
albumin (BSA) digest using a capillary with (a) a neutral
coating, (b) a high-density positively charged coating, and
(c) a lower-density positively charged coating. Analytes
were injected for 5 s at 50 mbar. The separation voltage
was +30 kV for the neutral and −30 kV for the cationic
coatings. Traces represent the base peak electropherogram
constituted of the masses of all major BSA signals [76]
acquiring multiple positive or negative charges.
Multiple charging is beneficial for proteomics
applications, since it allows for detection of large
peptides and full-size proteins, as well as give
better results in protein/peptide fragmentation.
The purpose of the ion source or interface is not
only to facilitate ionization but also to complete
and (preferably) decouple the electrical circuits
of the CE and ESI-MS.
Generally, interfacing of a CE system with
MS via an ESI source can be performed by a
sheath-liquid or a sheathless approach (more
details can be found in chapter “Capillary
Electrophoresis-­
Mass
Spectrometry
for
Metabolomics: Possibilities and Perspectives” of
this book). The sheath liquid provides the electrical connection on the tip and outside of the separation capillary. The main advantage of this
CE-MS for Proteomics and Intact Protein Analysis
65
Fig. 9 CZE-ESI-MS separation of glycopeptides present
in a human immunoglobulin G tryptic digest. Separations
were performed on (a) a 60-cm capillary modified with a
high-density positively charged coating, and (b) 60-cm or
(c) 80-cm capillaries modified with a lower-density positively charged coating. Note the increase in migration
time and resolution in the latter case. A further increase of
separation efficiency could be achieved by switching from
buffer A – 3:1 acetic acid/formic acid, 1 mol/L each –
(A–C) to buffer B (d), which has the same composition
but at 2 mol/L each [76]
approach is that the composition of the sheath
liquid can be tuned to modify the ionization without changing CE selectivity and efficiency. For
both peptide and protein analysis, the typical
composition of a sheath liquid is a mixture of
water and volatile organic modifier (methanol,
acetonitrile, or isopropanol), often with addition
of a volatile acid or base to improve the ionization efficiency. These parameters have shown to
influence the signal intensity as well as the shape
and position of the charge envelop. Therefore,
pH, electrolyte concentration, and type and concentration of organic modifier used for the sheath
liquid are utterly important variables.
Additionally, unwanted CE buffer properties
(described in the Sects. 2.2 and 2.3 of this chapter) could be balanced out by sheath liquid incorporation, although that results in sensitivity
trade-off due to the sample dilution (CE effluent
flows are in the nL/min, whereas sheath liquids
are commonly applied in the μL/min range [97,
98]). Nonetheless, due to the robustness of the
V. O. Kuzyk et al.
66
Table 1 Overview of most commonly used capillary coatings for peptide and protein analysis by CE-MS
Charge
Mechanism
of crafting
Features
Adsorbed
followed by
covalent
attachment
Covalent
attachment
Covalent
attachment
Adsorbed
pH-independent
EOF
Very high
reversed EOF that
decreases over
time, short
lifetime
Generates very
high EOF, may
lead to poor
resolution
High reverse EOF
at low pH, often
used in
multilayered
systems
High and
p-independent
EOF
Stable reverse
EOF
Polyvinyl alcohol
Neutral
Linear polyacrylamide
Neutral
Hydroxypropyl
methylcellulose
Low of high normal linear
polyacrylamide coating
Neutral
3-(aminopropyl)
trimethoxysilane
Cationic
Covalent
attachment
3-(methacryloylaminopropyl)
trimethylammonium chloride
(MAPTAC)
Cationic
Covalent
attachment
polyamine coatings
(polyE-­323, poly-LA 313,
polybrene)
Cationic
Adsorbed
Polybrene-poly(vinyl
sulfonate) bilayer coating
Anionic
Adsorbed
Omega-iodoalkylammonium
salts
Cationic
Covalent
attachment
Neutral
setup, it became widely acknowledged for
CE-MS hyphenation. As an example, using this
approach, LODs as low as 1 nmol/L were reached
in protein digest analysis while having up to 12
times greater throughput than with nano-LC-MS
[6]. Besides, extremely fast ( 40,000 for small proteins) offering high-­
quality isotope distributions [136]. Additionally,
as for most CE separations, a capillary coating
might be considered to prevent protein adsorption on silica (see Sect. 2.3 of this chapter). With
these prerequisites, multiple deamidation studies
of intact proteins with CE-MS(/MS) have been
successfully conducted. Isoform and charge variant characterization (also encompassing glycosylation heterogeneity) were established for
interferon-β1 [152], human growth hormone
[153], human erythropoietin [154], ricin toxin
[155], and last but not least monoclonal antibodies (mAbs) [150, 156]. Even a 2D CE-MS separation was reported for mAb charge variants with
V. O. Kuzyk et al.
highly efficient (albeit MS-incompatible) EACA-­
based separation in the first dimension and a
“cleanup” MS-coupled second CE dimension.
These developments are not only valuable from
analytical point of view: aforementioned targets
are of a great significance in biopharmaceutical
industry QC and QA processes.
Glycosylation is probably the most abundant
and complex PTM. It gained a lot of research
spotlight over the last two decades due to development of technologies that allowed its rapid
characterization. CE-MS(/MS) has become an
attractive system for glycoproteomics due to
multiple reasons. CE separates analytes based on
charge, size, and shape, thereby allowing to separate positional and linkage isomers of
­glycoconjugates (Fig. 11) [157]. Moreover, sialic
acids that are often present in the N-glycan structures additionally modify the electrophoretic
mobility of the molecules. CE is uniquely useful
for monitoring small and hydrophilic glycopeptides which are troublesome to ionize in LC-MSbased proteomics [158]. A lot of work has been
done in this area, which cannot all be covered in
here. Therefore, the reader is referred to a recent
comprehensive review of methods and applications for sialoglycosylation analysis [159]. The
authors cover both glycopeptide and intact protein levels of analysis and highlight the added
value of CE separation system. The majority of
glycoprofiling studies were performed on total
enzymatically released N-glycan pool of a certain sample, the so-called released glycan
approach. In that case, the information on the
microheterogeneity – location of a certain glycan
subset on a certain asparagine residue – gets lost.
That is often considered to be a vital piece of
data, for example, in biomarker research [160],
antibody quality control assurance [161], and
structure-function relationships of glycoproteins
[162]. Alongside with that, if the sample is complex (i.e., harbors more than one glycoprotein),
released glycans cannot be correlated to a particular protein. Here, a glycopeptide-based approach
comes of a great use, allowing to map glycan
structures on a protein of interest. Since the physicochemical properties of a given glycopeptide
depend on both glycan and peptide portion,
CE-MS for Proteomics and Intact Protein Analysis
73
Fig. 11 CE-ESI-MS analysis of prostate-specific antigen
(PSA) tryptic (glyco)peptides enabling the differentiation
between α2,3- and α2,6-sialylated glycopeptides without
prior derivatization. (a) Representative base peak electropherogram observed for a tryptic digest of PSA. (b)
Separation of non-sialylated glycopeptides. (c) Separation
of differentially linked mono-sialylated glycopeptides. (d)
Separation of differentially linked di-sialylated glycopeptides. A total of 75 different glycopeptides and differentially linked variants were identified. The “PEP” label
illustrates the tryptic peptide sequence N 69K to which the
glycan is attached [157]
CE-MS is better than conventional reversedphase LC-MS, which is common for proteomics
analyses. Besides, the latter may result in broad
peaks [81] and also requires larger sample
amounts.
Glycopeptide analysis poses a challenge for
three main reasons. First, it tremendously
increases the complexity of the sample on separation and data analysis steps. The latter is based on
the fact that the majority of proteomics software
is peptide-centric and is not well-designed to
annotate complex, sometimes chemically modified, PTM attachments. Only recently program
solutions started to emerge, namely, GlycoQuest
(Bruker Daltonics) and Byonic (Protein Metrics);
however, manual curation is still a must for the
majority of complex samples. For more information on current state-of-art strategies for interpretation of glycopeptide tandem mass spectral data,
the reader is referred to a review article by Hu
and colleagues [163]. Second, a glycopeptide
approach results in more diversion of analyzed
molecules, therefore complicating the identification of low-abundance species “hidden in the
grass.” Attempts to tackle this limitation involve
increasing of CE-MS sensitivity and lowering the
signal-to-noise ratio. A way to go about this issue
was reported by Kammeijer and colleagues
74
V. O. Kuzyk et al.
[114], who demonstrated the benefits of using a signal-to-noise ratios, thereby allowing low-­
coaxial nitrogen gas flow enriched with organic abundance glycopeptide identifications [114].
dopant. It enhanced the LOD of low-abundance
Application-wise, CE-MS of glycopeptides
glycan species and also improved ESI spray sta- also steps into the clinical setting, particularly the
bility and ionization efficiency. The third chal- biomarker research. A large-scale urinary glycolenge is the optimization of simultaneous glycan proteome study was conducted to establish a
and peptide fragmentation in tandem MS analy- “normal” urinary proteome signature – for fursis. Standard CID fragmentation energies easily ther comparisons of deviations – and to develop a
break glycosidic bonds of glycan fragments and diagnostic peptide marker model for pancreatic
rarely yield sufficient peptide fragmentation. In cancer [166]. The instrumental setup and conditurn, higher CID energies are capable of breaking tions were developed and adapted for human
down the peptide bond, but not glycosidic ones. urine and cerebrospinal fluid (biofluids with
ETD fragmentation could be an addition to CID moderate protein content). They were able to
to break down the peptide part, but it shows poor resolve around 1000 native polypeptides within
performance for typical glycopeptide masses 60 min [167]. Glycopeptide approaches also
(higher than 900 Da). A solution of stepping demonstrated their power for biotechnology
mode CID was demonstrated by Hinnenburg applications and particularly for antibody characet al., where collision energies increase gradually terization. Full sequence coverage with simultato fragment both structural entities of the glyco- neous PTM mapping was achieved in one
peptide [164]. Nonetheless, it is not a universal injection using therapeutic antibodies as a model
solution for any sample and instrument, thereby analyte [19]. The analysis demonstrated suffifragmentation parameters should be carefully cient robustness and speed to be further impleoptimized for the analyte of interest.
mented in the industrial setting.
Overall, it is yet early to name CE-MS(/MS)
Intact protein glycosylation analysis currently
the trailblazer of the glycopeptide analysis, but it starts to rapidly advance as well. This stems from
has a potential to become one since the number the simplicity of the sample preparation (no
of published manuscripts has been continuously enzymatic pre-treatment is needed) and is fed by
increasing every year. The standardized general hardware technical developments of high-­
conditions for the CZE-ESI-MS and CZE-­ accuracy and high-resolution MS, as well as good
MALDI-­MS analysis of glycopeptides described electrophoretic resolution in CE. Glycotyping
in detail by Amon and colleagues paved the way involves characterization of the number and relafor controlled and efficient separations, focusing tive abundancies of glycoforms, as well as charon coatings influencing the EOF control [165]. acterization of associated oligosaccharide
The current state-of-the-art method is proposed structures. Whereas the latter is better performed
by Dovichi’s group, offering CZE-ESI-MS anal- in glycopeptide-based manner, the former is betysis with LODs in the low ng/μL range, minimal ter investigated at the intact glycoprotein level as
sample consumption, and impressively short it does not introduce the sample preparation bias.
analysis time of 9 min [105]. The developments That is of a great importance for biopharmaceutifor the in-depth analysis go alongside with better cals, mainly for recombinant antibodies and
separation approaches. Where initially only main antibody-­based therapeutics (as will be discussed
glycoforms were separated, now even positional in the next section). For example, Han et al. preglycan isomers can be separated on the glycopep- sented a proof-of-concept CE-MS method to anatide level. Kammeijer et al. demonstrated a base- lyze degradation products and glycovariants of
line separation method for α2,3- and non-reduced antibodies, which is robust, effiα2,6-sialylated glycopeptides without pre-­cient, and durable enough to be used in routine
derivatization of the sample [157]. This method monitoring. In parallel, Haselberg with colalso provided increased sensitivity and lowered leagues also delivered a low-flow sheathless
CE-MS for Proteomics and Intact Protein Analysis
CE-MS method for stability and purity assessment of antibody-derived therapeutics. The
method showed sufficient robustness (overall
migration time RSDs below 2.2%), capacity to
separate isomeric deamidated products, as well
as reliably resolved sialylated glycoforms from
their non-sialylated counterparts [168]. With
increasing resolution and accuracy of the CE-MS
instrumentation, intact protein level analysis of
glycoprotein samples certainly has a potential to
be the main technique for routine analyses, albeit
yet is credible only for mildly glycosylated
proteins.
75
suppression resulted in very high separation efficiencies of closely related recombinant human
EPO glycoforms on the intact protein level [113,
154]. The separation was achieved mainly due to
differences in amount of sialic acid residues.
Differences in the hexose-N-acetyl-hexoseamine
content also lead to small shifts in electrophoretic
mobility and thus partial separation. Overall,
more than 250 different isoforms, including glycosylation, oxidation, and acetylation products,
could be distinguished in one CE-MS run of EPO
[136]. The same protein has also been characterized on the peptide level, demonstrating the site-­
specific microheterogeneity of the glycosylation
[170].
3.2
Biopharmaceutical
As just described the peptide-based approach
Development and QC
still provides the most comprehensive, deep, and
detailed analysis of a protein. The overall workTo develop a protein biopharmaceutical possess- flow time is significantly prolonged with sample
ing certain physical, chemical, and biological pre-treatment steps but gives the reward of site-­
properties and to ensure its stability in storage specific modification assignment. In that manner,
and use, one should first focus on the consistency Gahoual et al. have achieved full antibody pepof its amino acid sequence. That should be done tide mapping simultaneously with microvariant
in a quick and robust manner, and the possibility characterization for four therapeutic monoclonal
to get the separation within minutes, as would be antibodies (mAbs) in a single injection [19]. To
feasible in CE, seems ideal. However, its use still achieve that, they used a modified (surfactant-­
remains limited, since more development was assisted) proteolytic scheme and analyzed the
initially put into LC-MS systems, due to its initial digest using sheathless CE-ESI-MS system with
coupling straightforwardness and better stability t-ITP preconcentration. This way they managed
of operation. Nonetheless, more and more to lower down the required sample amount to
attempts towards CE-MS application in QC of 200 fmol of the digest. CE-MS of peptides also
biopharmaceuticals have been undertaken. As offers an advantage over LC-MS with the oppormentioned in the previous paragraph, alongside tunity to fully separate isomers of aspartic acid (a
with resolving protein sequence consistency, marker of protein degradation) [82]. Without an
CE-MS offers a possibility to simultaneously efficient separation, these isomers could not have
assess modifications, such as asparagine deami- been discriminated since this modification does
dation, methionine oxidation, C-terminal glu- not change the net mass of the peptide.
tamic acid cyclization and aspartic acid
A combination of an intact, middle-up, and
isomerization. In each case, the modified pep- bottom-up techniques shows great promise to
tides could be baseline separated from the intact characterize the total amount of modifications
counterpart, detecting modification levels as low and its location concomitantly [171]. Such an
as 2% [169]. The evaluation of these PTMs is approach is wholesome for biopharmaceuticals
essential, as they do influence the stability, half-­ like antibody-drug conjugate (ADC) and serves
life, and performance of the biopharmaceutical.
as a requirement for their proper characterization
Erythropoietin (EPO) was the first biopharma- [172]. The information about average molecular
ceutical extensively analyzed by CZE-­weight of ADC, drug to antibody ratio, and drug
ESI-­MS. This protein is heavily glycosylated and distribution is obtained time-efficiently by anatherefore intrinsically hard to characterize. EOF lyzing them on the intact level. Middle-up
V. O. Kuzyk et al.
76
approaches establish the co-occurrence of drug
attachment on a certain fragment. And last but
not least, a bottom-up approach gives the information of the site-specific distribution alongside
with peptide mapping of the protein backbone.
This pipeline is essential for the development and
quality assurance phases but might be too time
and labor consuming for the routine quality control and monitoring. To gain throughput on that
phase, the analysis of intact biopharmaceutical
molecules comes into play. Alongside with the
mass analysis to resolve truncated forms of the
protein molecule [173, 174], intact mAbs may be
resolved on the basics of their glycosylation profile and isomeric deamidation [168, 175].
Microfluidic integrated platforms also start to
emerge to increase the throughput. The use of
such a platform was demonstrated by Redman
and colleagues to characterize lysine-linked
ADCs [156]. The minimal sample preparation
and short analysis time allowed drug load assessment, charge variant determination, and characterization of glycan heterogeneity.
To conclude, the intrinsic technical challenge
of robust CE-MS coupling and analysis is being
more and more addressed by analytical scientists,
paving the CE-MS way to the biopharmaceutical
industry. Nowadays, commercial solutions are
being offered for antibody and ADC characterization, such as Thermo Scientific Q Exactive
hybrid quadrupole-Orbitrap mass spectrometers
with ZipChip system. The analytical method
development, exploiting more and better hardware solutions, does not stop either, being mostly
directed towards getting more valuable information from the intact level analysis. In light of all
of this, a collaborative study on the robustness
and portability of CE-MS can be mentioned as a
notable undertaking. It was performed by an
international team, consisting of 13 independent
laboratories from both academia and industry
[176]. All participants analyzed the samples from
the same batch and used identical reagents and
coated capillaries to run their assays; however,
the analysis itself was performed on the equipment available in their laboratories. Migration
time, peak height and peak area of ten representative target peptides of trypsin-digested bovine
serum albumin were determined by every laboratory on two consecutive days. The results demonstrated that CE-MS is robust enough to allow a
method transfer across multiple laboratories and
should promote a more widespread use of peptide mapping and other CE-MS applications in
biopharmaceutical analysis and related fields.
3.3
Biomarker Discovery
and Clinical Application
As CE-MS is a powerful analytical technique, its
appearance on the biomarker research scene is
not surprising. It allows for identification of
broader peptide range (if compared to the LC
separations) and exhibits almost no sample carryover phenomena. Also, the reliability of the
platform combined with worked-through data
processing and mining methods, to date, makes
CE-MS the most advanced technique for biomarker discovery of clinical significance. Most
of the developments currently address the urinary
proteome as a source of new biomarkers. Protein
concentrations in urine are very moderate (when
compared to serum); thus it does not require
complex pre-purification steps and can be almost
directly analyzed by CE-MS. Additionally, urine
can be sampled in large quantities and in noninvasive manner, thus allowing enough material for
hitting the concentration limit.
A big portion of studies focuses on the
assessment of the urinary peptidome. Typical
sample preparation involves a short pre-cleanup
to eliminate large proteins, accompanied by buffer exchange. The remaining fraction is subsequently analyzed by CE-MS. Studies centering
around kidney malfunctions are the most obvious area to find urinary biomarkers. For example, a multimarker model built to assess kidney
injury and based on peptides assessed by CE-MS
was proven viable by Metzger et al., revealing
the intensity of only two peptides, which
allowed for early and accurate prediction of
acute kidney injury [177]. The same condition
appeared to produce another set of peptidome
signatures that were shown to predict kidney
function improvement [178]. Urinary pepti-
CE-MS for Proteomics and Intact Protein Analysis
77
dome studies have also demonstrated their use 4
Conclusions and Future
for the diagnostics and patient stratification in
Perspectives
other diseases, since 30% of the urinary proteins
are derived from serum [179]. To exemplify, CE-MS is masterfully climbing towards its rightsystematic lupus erythematosus [180] and vari- ful analytical niche in proteomics and intact proous types of cancer [181] have been diagnosed tein analysis, being not a rival but a helping friend
using a peptide marker panel measured by of LC-MS. Recalling CE advantageous features –
CE-MS. One of the peptide biomarker panels, high sensitivity and resolving power, low sample
termed CKD273, has been successfully trans- and solvent consumption, short analysis times,
lated into the clinic and received an FDA and many options to tune the separation – makes
approval for management of chronic kidney dis- CE-MS a solid choice for protein samples charease [182]. Despite being more challenging to acterization on multiple levels of complexity.
handle, the urinary proteome also gained quite CE-MS offers superb performance for PTM charsome attention as a protein biomarker source. acterizations of therapeutic proteins, and it has
The applications range from total protein frac- already proven to be a promising tool in biotion evaluation in patients with heart failure marker discovery and validation (as amply
[183] to in-depth study of urinary PSA isoforms described in Sect. 3.3 of this chapter). The necesin prostate cancer [184].
sary step towards vast clinical implementation is
The spectrum of other biofluids to be used as a the demonstration of robustness and validity of a
protein/peptide biomarker source is very broad, certain analytical method, and CE-MS seems to
namely, cerebrospinal fluid [185], saliva [186], already pass this milestone, being recognized for
seminal plasma [187], bile [188], and certainly its reproducibility, stability, sensitivity and interblood plasma [38]. Some body fluids catalogued laboratory applicability and well-established data
as “less-conventional” (cerumen, breast milk, processing pipelines. We expect further developand more) were also recently reviewed for their ments of the system robustness, especially for
biomarker potential [189]. Typically, the CE-MS complex setups with sample enrichment and/or
analysis itself remains relatively standardized for cleanup and multidimensional separations.
all the fluids, but the sample preparation differs Ongoing implementation of microchip-based CE
dramatically. Plasma and serum pose the biggest (MCE) separations presents a lucrative opportuchallenge for their high dynamic range between nity for commercialization, by lowering costs
high- and low-abundance proteins, which no con- and improving user experience. Market-available
temporary instrument can handle directly. MCE devices yet avoid MS coupling but already
Ironically, the low-abundance proteins frequently show potential in clinical setting. To bring an
hold the biggest biomarker potential. Therefore, example, the 2100 Bioanalyzer MCGE system
strategies for abundant protein depletion are vital has been utilized for therapeutic protein analysis,
for plasma proteins [190] and other biofluids, including glycoprotein heterogeneity assessment
alongside with other strategies of sample pre-­ [192]. The same platform has been implemented
treatment. The latter issue has been recently for C-reactive protein (CRP) detection as a sepsis
­protocolled by Mischak’s research group, along- marker on clinical serum samples [193]. The
side with a roadmap and considerations for the implementation of MS would help resolve issues
analysis of proteomics and peptidomics biomark- with complex sample preparation or low accuers in biofluids [191]. For the plasma proteome racy of MW determination. Furthermore,
biomarker studies, it is often important to com- researchers do highlight the problem of protein
bine background protein depletion with target adsorption in CE/MCE systems and expect more
protein enrichment. Here immunoaffinity and research to be devoted towards novel coating
lectin enrichment SPE techniques may be materials development [194].
engaged either in on-line manner [38] or perAs a purely analytical tool for proteomics,
formed offline [158].
CE-MS has plenty of high-end workflows, for
V. O. Kuzyk et al.
78
example, those on intact protein (structural) analysis and single-cell “omics,” that are yet presented only as a proof of concept with all the
analyses performed on test proteins or peptides
[117, 174, 195–197]. Given the impressive outcomes, we hope to see and possibly contribute to
their development and use for real biological
samples analyses.
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