The technique of charge reduction electrospray mass spectrometry (CREMS), which can reduce the charge state complexity produced in electrospray ionization (ESI), is discussed

Smith, Lloyd M.

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CRITICAL INSIGHT
Is Charge Reduction in ESI Really Necessary?
Lloyd M. Smith
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA
The technique of charge reduction electrospray mass spectrometry (CREMS), which can
reduce the charge state complexity produced in electrospray ionization (ESI), is
discussed. (J Am Soc Mass Spectrom 2008, 19, 629–631) © 2008 American Society for Mass
SpectrometryCharge reduction in the gas-phase occurs when-ever oppositely charged ions combine. It hasbeen in routine use in the atmospheric science
community for particle analysis applications for de-
cades [1]. In the last decade it has been developed in the
field of biological mass spectrometry in two primary
forms: at reduced pressure, typically inside of an ion
trap mass spectrometer [2], and at atmospheric pres-
sure, to reduce the charge of electrospray-generated
ions before their entry into a mass spectrometer [3–6].
The latter approach has been developed primarily by
our group at the University of Wisconsin-Madison, and
it will serve as the principal subject of the present
discussion.
We coined the acronym CREMS, for charge reduc-
tion electrospray mass spectrometry, for the approach.
The nominal rationale for developing CREMS was to
achieve complexity reduction in mass analyzing mix-
tures of many different molecules. The two main meth-
ods for producing gas-phase ions from macromolecules
(e.g., proteins, nucleic acids, synthetic polymers) are
matrix-assisted laser desorption/ionization (MALDI)
and electrospray ionization (ESI). For reasons that are
not yet entirely clear [7], MALDI produces primarily
singly charged ions, whereas ESI produces a distribu-
tion of multiply charged ions in various charge states.
Thus, whereas MALDI produces relatively simple mass
spectra from mixtures of different molecules, with one
major peak per analyte species, an ESI analysis of the
same mixture may give an unresolved composite of
many overlapping peaks. Charge reduction offered the
potential of being able to have the simplicity of MALDI
spectra while maintaining the advantages of ESI with
respect to its real-time capability and gentler ionization.
We have developed two approaches to CREMS,
differing in how reagent ions are generated. In the first
approach [3, 4], we employed a 210Po source of -par-
ticles to create “air ions” in a chamber placed between
the ESI capillary and the mass spectrometer inlet. We
Address reprint requests to Dr. L. M. Smith, Department of Chemistry,
University of Wisconsin, 1101 University Avenue, Madison, WI 53706-1396,
USA. E-mail: smith@chem.wisc.edu
© 2008 American Society for Mass Spectrometry. Published by Elsevie
1044-0305/08/$32.00
doi:10.1016/j.jasms.2008.02.002were able to show effective charge reduction of
ESI-generated ions down to the singly charged state,
with the desired reduction in charge state complexity
and concomitant expansion of the m/z range (lower
z  greater m/z). This yields “MALDI-like” ESI mass
spectra from mixtures of proteins or nucleic acids.
Figure 1 shows an example of the spectral simplification
afforded by CREMS on a mixture of seven proteins
(reproduced from reference [4] with permission). In
subsequent work, we were able to employ a corona
discharge source to produce reagent ions in place of the
210Po source [5, 6]. This eliminated the regulatory and
practical issues associated with obtaining and using
radioactive materials, and in our most recent design the
corona reduction chamber is quite small, efficient, and
easy to put together [6].
Now, that is all a very nice background, but it does
not address the question posed in the title of this article:
“Is Charge Reduction Necessary?” This begs the ques-
tion “Necessary for what?” What would one like to do
with charge reduction that is not otherwise possible?
Charge reduction might arguably find utility anytime
one seeks to determine the masses of high molecular
weight components in a mixture. Nucleic acids, syn-
thetic polymers, and proteins are all compound classes
of interest. Let us focus on proteins, as proteome
analysis (a.k.a. proteomics) is the most active emerging
area in mass spectrometry.
Proteomic analyses fall into two major bins: identifi-
cation and quantification. For identification, the ques-
tion is “What proteins are present in this sample?” For
quantification, the question is “How much of each is
present?” Both questions are usually addressed by
means of peptide intermediaries. To find out what
proteins are present in a sample, one typically digests
the sample with trypsin to obtain the tryptic peptides,
separates them by LC if necessary, and then either from
the peptide masses or, more commonly, from the
MS/MS fragmentation patterns, identifies the peptide
[8]. The presence of the corresponding protein is in-
ferred from the presence of peptides present in the
protein sequence, and the more such peptides are
identified for a given protein, the greater the confidence
Published online February 23, 2008
r Inc. Received January 15, 2008
Revised February 7, 2008
Accepted February 7, 2008
uctio
630 SMITH J Am Soc Mass Spectrom 2008, 19, 629–631that the protein was in fact present. Quantification
proceeds similarly, in that the amount of a given protein
of interest is determined either from the absolute signal
intensities for peptides of interest [9], or from their
intensities relative to isotopically-tagged peptides used
either as standards [10], or using heavy-light isotopic
tagging chemistries [11].
This is all well and good, and these strategies con-
stitute the major paradigm of our day for proteome
analyses. However, there are some embarrassingly im-
portant aspects of proteomics that are not captured by
these approaches. The most notable issue has to do with
protein variation.
The ability to determine the extent and nature of
protein variation is a critical missing piece in proteom-
ics today. A surprise revealed by the success of the
human genome project was the much lower than antic-
ipated number of genes present in humans, in the range
of30,000 rather than the predicted100,000 [12]. This
fact has led to the general recognition that much of the
complexity and sophistication afforded by our biologi-
cal machinery is at the level of protein variation rather
than just resulting from a large number of distinct genes
[13]. These protein variations occur at three levels at
least: alternative splicing of the RNA transcript, codon
substitutions, and a wide variety of post-translational
modifications. Such modified proteins play central roles
in a wide variety of biological processes, from cell-
signaling and signal transduction to gene regulation to
controlling the activity of complex protein machines
such as the ribosome or proteasome. For example,
recent work in the identification of the isoforms of
histone H3.2 has shown the existence of over 150
300 4240 8180
(a)
(b)
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+1
dim er
+1
+1
+2
+1
+2
+2
+2
+1
+2
2455
634
In
te
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si
ty
 
(ar
bi
tra
ry
 
u
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its
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In
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ns
ity
 
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Figure 1. ESI spectra of an equimolar mixture o
acetic acid) without (a) and with (b) charge reddifferent post-translationally modified forms of theprotein [14], and a similar analysis of histone H4 has
revealed 74 different isoforms [15]. The levels of the
many protein variants are dynamically regulated dur-
ing the cell cycle and control the patterns of gene
expression during development and differentiation.
Today, there is no technology available that can
rapidly determine the nature and amounts of such
protein variants present in a sample of interest. It is
clear, however, that one is needed. In principle, charge
reduction could play an important role in such an
analysis. A plausible scenario is as follows: a sample of
interest (imagine a nuclear extract) is subjected to an
enrichment step for a set of proteins of interest (imagine
immunoprecipitating histone H3 proteins). This mod-
erate complexity sample is either directly infused into a
mass spectrometer via electrospray and charge reduc-
tion, or subjected to a prior chromatographic or electro-
phoretic separation for further complexity reduction,
followed by electrospray and charge reduction. In a
wonderful world, every protein form would be repre-
sented by a peak in the mass spectrum, the intensity of
that peak would reflect the relative amount of that
species, and the width of the peak would be sufficiently
narrow to allow that isoform to be identified (of course,
such simple mass analysis would not provide any
information on isomeric variants with identical masses).
This might arguably also provide a useful tool for
studying variations in protein complexes, as in low
charge states there is much less electrostatic repulsion
between the protein components, providing greater
stability to the complex in the gas-phase.
This is a nice idea. What are the obstacles standing in
the way of its implementation? There are quite a few.
12120 16060 20000
s (m /z)
+1
aceta te adduct
acetate  adduct
+1
dim er
+1
neurotensin
m elittin
glucagon
insulin
ubiquitin
cytochrom e c
m yoglobin
en proteins (5 M each in 1:1 H2O/CH3CN, 1%
n. Adapted from reference [4] with permission.M as
f sevVariations in ionization efficiency and ionization sup-
631J Am Soc Mass Spectrom 2008, 19, 629–631 IS CHARGE REDUCTION IN ESI REALLY NECESSARY?pression effects compromise the desired quantitative
nature of the analysis [16]. Singly charged ions are more
prone to adduction than highly charged ions, and such
adducts can compromise mass analysis. The mass accu-
racy that can be obtained for large singly charged
proteins is poor; the high m/z range necessitates a
time-of-flight (TOF) mass analyzer, and neither
MALDI-TOF nor ESI-TOF are currently able to give
resolutions in the dalton range (sufficient to discern the
isotopic envelope) for mixtures of proteins in the 10,000
to 100,000 Da range. Ion detectors used in TOF analysis
are generally based on secondary electron generation
[electron multipliers, microchannel plates (MCPs)], the
efficiency of which falls off strongly with increasing m/z
[17]. Thus, much work is needed on all aspects of large
molecule mass spectrometry, including ionization pro-
cesses, mass analyzer design, and detector perfor-
mance, to make this scenario into a reality. Although
clearly a difficult and long-term problem, I would argue
that the importance of being able to open a window into
this critical domain of proteomics is such that develop-
ing approaches to tackle these issues is an exciting
challenge for the entire community. Of course, the
approach outlined above is just one possible strategy,
and doubtless many others could be conceived that
would also merit exploration.
Are there any less daunting applications for charge
reduction? The answer is likely yes. One interesting
idea is to try to concentrate ions into single higher
charge states, an approach pioneered by McLuckey
using ion–ion reactions in ion traps, and termed “ion
parking” [18]. If one were able to do this under the
atmospheric pressure conditions employed in CREMS,
there would be fewer issues with adduction (due to the
higher charge states, which are easier to free of ad-
ducts), and the m/z range would be reduced, possibly
permitting other mass analyzers such as FTICR to be
employed, with their concomitant advantages [19]. It
would also help with ion detection, as highly charged
ions are more easily detected, either directly (the greater
number of charges leads to higher signal in Faraday cup
or inductive detectors), or indirectly (as more highly
charged ions acquire greater kinetic energy when accel-
erated by an electric field, and thus are more efficient in
the generation of secondary electrons in electron mul-
tipliers or MCPs). One approach to this might be to
employ labeling chemistry that converts specific protein
functional groups (e.g., carboxylic acids) to fixed
charges, as we and others have shown for metabolomics
applications [20–22]. This could increase ionization
efficiency, ideally to near unity, which would not only
increase detection sensitivity, but would also greatly
facilitate quantitation.
So, what is the response to the question “Is charge
reduction in ESI really necessary?” Charge reduction
has not yet enabled a critical application in mass
spectrometric analysis. Exploiting its full potential will
require significant advances in several other aspects ofmass spectrometry, which may yet be many years from
reality. However, the overriding problem it seeks to
address, of deciphering the complexity of the proteome
at the whole protein level, is a crucial unaddressed
problem in biomolecular analysis. Whether by charge
reduction or by some other approach, the development
of solutions to this interesting and complex analytical
challenge is an important priority for the community to
address.
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Is Charge Reduction in ESI Really Necessary? 

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Is Charge Reduction in ESI Really Necessary?

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