immobilized enzyme reactors in proteomics 2011 trac trends in analytical chemistry

8/18/2019 Immobilized Enzyme Reactors in Proteomics 2011 TrAC Trends in Analytical Chemistry

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Immobilized enzyme reactors

in proteomics Junfeng Ma, Lihua Zhang, Zhen Liang, Yichu Shan, Yukui ZhangFast, efficient characterization of proteins is becoming one of the hottest topics in the bioanalytical community, especially for

large-scale proteomic studies. As an attractive approach, protein digestion by enzymes supported on various matrices (referred to

as immobilized enzyme reactors, IMERs) has recently attracted much attention.

In this article, we present a critical overview of some highly efficient IMERs and related analytical systems. We give major

coverage to applications of IMERs in proteomic analysis, including protein-expression profiling, characterization of proteins with

post-translational modifications, and protein quantification. We also comment on promising trends for IMERs in proteomics.

ª 2011 Elsevier Ltd. All rights reserved.

Keywords: Enzyme; Glycoprotein; Immobilized enzyme reactor; Phosphoprotein; Protein characterization; Protein digestion; Protein profiling;

Protein quantification; Proteome; Proteomics

Abbreviations: APTES, 3-Aminopropyltriethoxysilane; BMA, Butyl methacrylate; BSA, Bovine serum albumin; CE, Capillary electrophoresis; EDMA,

Ethylene dimethacrylate; ESI-MS, Electrospray ionization-mass spectrometry; GMA, Glycidyl methacrylate; HPLC, High-performance liquid

chromatography; IgG, Immunoglobulin G; IMER, Immobilized enzyme reactor; MALDI-MS, Matrix-assisted laser desorption/ionization-mass

spectrometry; MBA, Methylenebisacrylamide; PET, Poly(ethylene terephthalate); PNGase F, peptide-N-glycosidase F; RNase B, Ribonuclease B;

RPLC, Reversed-phase liquid chromatography; SCX, Strong cation exchange; TEOS, Tetraethoxysilane; VAL, 2-vinyl-4,4-dimethylazlactone; WAX,

Weak anion exchange; WCX, Weak cation exchange

1. Introduction

As an important research paradigm in

post-genomic era, technology-driven pro-teomics has provided huge opportunities

as well as challenges to the analytical

community. Different from classical pro-

tein chemistry, proteomics aims to study

all the proteins expressed by cells, tissues,

and organisms. According to current

estimates, the human serum and plasma

proteome contains at least 100,000 pro-

teins (including isoforms) with up to

20,000 proteins expressed at any given

time. Due to the extreme complexity of

proteomic samples, identification, charac-

terization, quantitation and mapping of

post-translational modifications (PTMs) of

proteins are among the most significant

challenges in proteomics [1].

With the advent of two ionization

techniques [i.e. electrospray ionization

(ESI) and matrix-assisted laser desorption/

ionization (MALDI)], mass spectrometry

(MS) has evolved to be a central tool in

almost all proteomic workflows [2].

Sample-pretreatment procedures prior to

MS detection are often tedious and time-

consuming, greatly restricting the analyt-

ical throughput for protein identification

and characterization [3]. As a key element

in efficient sample pretreatment,proteolytic digestion is traditionally per-

formed in solution by free enzymes (typi-

cally trypsin). With this approach, a small

amount of enzymes is often added

into protein solutions [enzyme/sub-

strate = 1:20–1:100 (w/w)], leading to a

long incubation time (typically 5–24 h)

and even inefficient digestion for low-

abundance proteins and diluted protein

samples.

As an interesting alternative to the in-

solution method, proteolytic digestion by

enzymes immobilized on solid supports

(referred to as immobilized enzyme reac-

tors, IMERs) has gained in popularity in

recent years. Since enzymes are immobi-

lized in a narrow space (often with lL/nL

volume), high enzyme-to-substrate ratio

can be achieved, resulting in substantially

improved digestion capacity, short diges-

tion time, little auto-digestion of enzymes,

and efficient digestion, even for low-abun-

dance proteins and minute proteomic

samples. In addition, the repeatability of

Junfeng Ma 1, Lihua Zhang*,

Zhen Liang, Yichu Shan,

Yukui Zhang,

Key Laboratory of Separation

Science for Analytical

Chemistry,

National Chromatographic

Research and Analysis Center,

Dalian Institute of Chemical

Physics,

Chinese Academy of Sciences,

Dalian 116023, China

*Corresponding author.

E-mail: [email protected] address: Depart-

ment of Biological Chemis-

try, The Johns Hopkins

University School of Medi-

cine, Baltimore, MD 21205,

USA

Trends in Analytical Chemistry, Vol. 30, No. 5, 2011 Trends

0165-9936/$ - see front matter ª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2010.12.008 691

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immobilized enzymes, which could provide reliable iden-

tification of proteins, is of ultimate importance especially

for some studies (e.g., discovery of disease-specific bio-

markers for diagnostic and prognostic purposes). More

importantly, some IMERs can be readily coupled to sepa-

ration and identification systems, enabling fast, efficient,

high-throughput and automated proteome analysis.Herein, we do not describe all the supporting materials

and immobilization techniques that have been developed

for the preparation of IMERs, as seen in several excellent

reviews published recently [4–10]. Instead, this article

concerns the evaluation and applications of IMERs from

a proteomic view, covering only the latest reports

(mainly focusing on the literature since 2005, but also

including some earlier important publications). Specifi-

cally, the main aims are:

(i) to present some newly developed IMERs and

IMER-related analytical systems for protein-

expression profiling;

(ii) to introduce some IMERs for the characterizationof proteins with PTMs (i.e. phosphorylation and

glycosylation);

(iii) to report on the application of IMERs for protein

quantification; and,

(iv) to comment on the prospects of IMERs in proteo-

mics.

2. IMERs for protein-expression profiling

The main application of IMERs is the proteolytic

digestion by endoproteases (typically trypsin) for

protein-expression profiling. In general, IMERs can be

used batch-wise (mainly for those based on micro-/

nano-spheres) or flow-through (for those packed or

in-situ synthesized in microcolumns, capillaries, and

microfluidic channels). Concerning the coupling to

separation and identification systems, IMERs can be

utilized in two ways: off-line and on-line, as illustratedin Fig. 1.

2.1. Performance evaluation and off-line application

Monolithic materials, which possess unique porous

structures and large surface areas, are quite advanta-

geous for enzyme immobilization. Monolith-based IMERs

often have excellent permeability, fast mass transfer, and

high digestion efficiency.

With 2-hydroxyethyl methacrylate (HEMA), ethylene

dimethacrylate (EDMA), and 2-vinyl-4,4-dimethyl-

azlactone (VAL) as active monomers, Svecs group [11]

prepared a poly(VAL-EDMA-HEMA) monolithic supportin microfluidic channels for trypsin immobilization. Eight

proteins with molecular mass in the range 2.8–77.8 kDa

were efficiently digested after a residence time of less

than 1 min within the IMER.

Foret and co-workers [12] fabricated a poly(ethylene

dimethacrylate-glycidyl methacrylate, EDMA-GMA)

monolith within a capillary (75 lm i.d. · 2 cm), result-

ing in up to 35-lg trypsin being immobilized. With such

a trypsin-based IMER (trypsin-IMER), cytochrome c was

digested in less than 30 s at 25C with the sequence

coverage of 80%, comparable to 3-h digestion in solution

at 37

C.

Figure 1. Typical configurations of coupling IMERs with separation and identification systems. It should be pointed out that, in most cases, HPLCin these configurations represents analytical column for separation, but it may also be replaced by others, such as trap/capture columns and solid-phase-extraction columns, especially when some specific fractions (e.g., phospho-/glyco-proteins and low-abundance proteins) are to be ana-lyzed.

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Zhangs group [13] prepared a trypsin-IMER sup-

ported on porous poly(GMA-EDMA-acrylamide) mono-

lith in capillaries. The digestion efficiency of immobilized

trypsin was found to be over 230 times greater than

when in-solution digestion was performed.

Sakai-Kato et al. [14] encapsulated trypsin in a tet-

ramethoxysilane (TEOS)-based hydrogel matrix. Theresultant trypsin-IMER showed excellent enzymatic

activity, which was 700 times higher than that in free

solution. Later, Zares group [15] developed a trypsin-

IMER by preparing a reactive hydrophilic macroporous

poly(ethylene glycol)-modified photo-polymerized sol-gel

monolith, followed by functionalization with trimeth-

oxysilylbutyraldehyde and immobilization of trypsin via

covalent bonding. The proteolytic activity of the trypsin-

IMER was enhanced more than 2000 times compared to

that performed in solution. However, no applications of

such a trypsin-IMER for the complex sample analysis

have been reported so far.

Recently, Zhangs group [16] introduced an organic-inorganic hybrid silica monolith for the preparation of

trypsin-IMER. The monolithic support bearing amine

groups was activated with glutaraldehyde, and trypsin

was then covalently immobilized. With a decapeptide

C-myc (EQKLISEEDL) as the substrate, the apparent

maximum velocity (V max) of the trypsin-IMER was

nearly 6600-fold greater than that of the free trypsin.

The sequence coverage of 200-ng myoglobin for IMER

digestion was 92%, the same as that obtained from in-

solution digestion. While the residence time in the

IMER was a mere 30 s, it was about 1500 times

shorter than the time required to achieve the samedegree of in-solution digestion (ca. 12 h). Moreover, a

minute amount of myoglobin (0.1 lg/mL, 10 fmol)

was efficiently digested and then positively recognized.

The applicability to proteome analysis was demon-

strated by the digestion of 20 lg of Escherichia coli

extract. A total of 208 proteins were identified from

microflow reversed-phase liquid chromatography

(lRPLC) coupled with ESI tandem MS (ESI-MS2) after a

150-s residence time in the trypsin-IMER, while only

176 proteins were recognized after 24-h of in-solution

digestion.

Due to striking features (e.g., large surface areas,

tunable surface properties, and high dispersability in

both aqueous and organic solutions), micro/

nano-spheres also represent favorable supports for

enzyme immobilization. Qiao et al. [17] exploited

cyano-functionalized mesoporous silicate nanoparticles

for the immobilization of trypsin via adsorption. The

performance of such a trypsin-IMER was evaluated by

digesting 5 lg of the cytoplasm extract of human-liver

tissue for 20 min, and 165 proteins were unambiguously

identified by RPLC and MALDI-time-of-flight-tandem MS

(MALDI-TOF-MS2). Considering the excellent micro-

wave-absorption capacity of magnetic particles, the

usage of microwaves might improve the digestion effi-

ciency of magnetic particle-based IMERs.

Deng and co-workers [18] synthesized core/shell-

structured magnetic zeolite microspheres for trypsin

adsorption. Sequence coverages of 77% for cytochrome

c, 89% for myoglobin, and 25% for BSA were achieved

using the trypsin-IMER with the assistance of micro-waves for a short period of 15 s. The applicability of

such a microwave-assisted IMER digestion approach to

proteomics was tested by Lin et al. [19] with a trypsin-

IMER supported on silica gel-coated magnetic Fe3O4microspheres. When 10 lg of rat-liver extract was

digested with the trypsin-IMER upon microwave

irradiation for 15 s, 364 proteins were identified by

RPLC-ESI- MS2.

Lius group [20] proposed a self-assembly method to

embed enzymes on a poly(ethylene terephthalate) (PET)

microfluidic chip. Chitosan/hyaluronic acid multilayer

films coated on the PET surface by electrostatic interac-

tion supplied a biocompatible, hydrophilic microenvi-ronment to accommodate a large amount of trypsin

while preserving bioactivity. The value of V max of ad-

sorbed trypsin was 600 mM/min/lg, thousands of

times faster than that in solution (0.2 mM/min/lg).

Even 15 fmol (0.25 ng) of myoglobin was positively

recognized when digested by the trypsin-IMER with a

residence time


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digestion. It is claimed that the complete digestion of a

standard protein mixture can be achieved in 15 min,

resulting in higher sequence coverages than those ob-

tained by the overnight in-solution digestion.

ProteoGen Bio Srl [25] has produced DigesTip by

equipping a standard pipette tip with a cartridge con-

taining immobilized trypsin (patent pending). After1 min digestion of a five-protein mixture with DigesTip,

more peptides and higher sequence coverage for most

proteins were obtained than those reported with a

trypsin spin column with digestion time of 15 min.

Recently, Veuthey et al. [26] developed a trypsin-

based micro-IMER prepared on a monolithic ethylene-

diamine BIA Separations CIM (convective interaction

media) minidisk. Interestingly, in comparison to the

Poroszyme immobilized trypsin beads, even higher cov-

erages were yielded when a five-protein mixture was

digested by the minidisk-supported trypsin-IMER within

10 min.

It should be noted that the wide acceptance andapplications of these commercial IMERs for protein

analysis, especially for large-scale proteome profiling, are

still to be investigated.

2.2. IMER coupling techniques

One of the most striking features of IMERs is the ease in

coupling to separation and identification systems, en-

abling high-throughput, automated proteome profiling,

which is urgently required in current MS-based proteo-

mic workflows. In general, IMERs can be coupled with

capillary electrophoresis (CE), HPLC, and other modes

(e.g., microextraction) for on-line analysis.

2.2.1. Coupling IMER to CE. To improve the compati-

bility for proteolytic digestion by IMERs and protein and

peptide separation by CE, some aspects, including buffer

exchange and pH adjustment, should be considered, in

particular when the IMER and the separation channel

are to be integrated in a single capillary.

At the beginning of 1990s, Kuhr and Amankwa [27]

explored the possibility of coupling trypsin-mediated

digestion with CE for peptide mapping via a solution gap.

Ye et al. [28] coupled the trypsin-IMER and the peptide-

separation column via a fluid joint. With a-lactalbumin, a

large globular protein, as the model protein, more than

20 peaks were resolved, and the column efficiencies formost peaks were over 120,000 theoretic plates/m, while

the entire analysis was within 16 min. Since acidic run-

ning buffers could not only suppress the adsorption of

proteins or peptides on the inner surface of capillaries, but

also render the direct coupling of CE with MS, Sakai Kato

and co-workers [29] developed an IMER inside a fused-

silica capillary by coating pepsin on a porous silica

monolith formed by photo-initiated polymerization. The

resulting peptides were directly separated in the remain-

ing part of the capillary, which was free of monolith, and

then detected by ESI-MS2. By such an IMER-ESI-MS2

system, the sequence coverages obtained for insulin chain

b and lysozyme were 100% and 73%, respectively.More recently, Dovichis group [30] constructed a fully

automated CE-IMER-CE-MS2 platform (Fig. 2). A pepsin-

IMER based on monolithic poly(GMA-EDMA) support

was prepared at the distal end of the CE column used for

peptide separation. By using a finely-machined interface,

one CE column for protein separation was coupled with

IMER-CE-ESI/MS2. When a two-protein mixture was

analyzed by this system, the sequence coverages ob-

tained for cytochrome c and myoglobin were 48% and

22%, respectively, showing multiple advantages (e.g.,

fully automated operation, fast speed and high efficiency)

for protein analysis.It is noteworthy that, although CE has demonstrated

quite promising potential in many proteomics applica-

tions due to the inherently fast analysis time, high sep-

aration efficiency, low sample and reagent consumption,

and a number of separation modes, several challenges

have limited its applications in large-scale MS-based

proteomics study, including:

Figure 2. CE-IMER-CE-MS2 system (with permission from [30]).




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(i) most 2-D CE separation conditions (e.g., buffers

from the second dimension) are not currently

MS compatible;

(ii) the sensitivity necessary for coupling low-mass

2-D CE to MS has not been reached;

(iii) the reproducibility and the robustness of CE sepa-

rations and the interfaces need to be further im-proved [31].

The coupling of IMER with CE has therefore only been

successfully used for profiling of standard proteins or

protein mixtures. Until the problems mentioned above

are solved, analytical systems involving IMER and CE

cannot be used to their full potential in proteomics.

2.2.2. Coupling IMER to HPLC. In comparison with CE,

1D or 2D HPLC has displayed excellent compatibility

with MS (especially ESI-MS2), high resolution for protein

and peptide separation, and satisfactory robustness and

reproducibility, dominating current MS-based proteomic

studies. The coupling of IMERs to modes of HPLC sepa-ration has therefore attracted much attention in recent

years.

By using a switching valve and a trap column, IMER

can be easily assembled before an analytical column.

With a trypsin-IMER supported on an epoxy-modified

silica monolith, specially prepared by Merck KGaA

(Darmstadt, Germany), Calleri and co-workers [32] at-

tempted to couple IMER with HPLC-ESI-MS2. In their

approach, proteins were first digested by a trypsin-IMER,

and the digests were subsequently collected by a trap

column. After desalting and concentration, the peptides

were flushed from the trap column, eluted onto the C18analytical column and separated, followed by the iden-

tification with MS2. With such an IMER-HPLC-ESI-MS2

system, the variants of transthyretin in human serum

were successfully recognized.

By contrast, Feng et al. [33] developed a semi-auto-

mated platform by coupling a monolith-based nL tryp-

sin-IMER with lRPLC-ESI-MS2 system. The protein

sample was injected and digested in the trypsin-IMER.

After a certain time of digestion, the IMER was con-

nected directly to the separation column to load the

digests. Then, the IMER was detached from the flow line

while the digests were analyzed by RPLC-ESI-MS2.

Compared with the 16-h in-solution digestion of 590-ng

cell lysate of Saccharomyces cerevisiae, slightly fewer

proteins were identified by on-line digestion with an

incubation time of 1 min (541 versus 624).

Unlike the IMER-HPLC-MS2 systems, few reports about

HPLC-IMER-MS2 have been published, probably due to

several challenges, e.g.:

(1) the need to adjust the separation conditions (e.g.,

solvents and pH) to meet the requirements for pro-

teolytic digestion and MS identification; and,

(2) band broadening (e.g., caused by post-column

volumes, and connections).

Slysz et al. [34] integrated C4 RP protein separation

with trypsin-IMER digestion and MS2 identification for

on-line, real-time analysis. By adopting pH adjustment

and make-up flow, excellent compatibility between each

unit was achieved. Although the performance of such asystem was successfully tested by a four-protein mixture,

the potential application for complex sample profiling

remains unclear because of the less efficient protein-

separation technique and the absence of a peptide-

separation step.

Recently, Zhangs group [35] established a highly

integrated platform involving protein separation on a

mixed weak anion exchange and weak cation exchange

(WAX/WCX) microcolumn, on-line digestion by a tryp-

sin-IMER, and peptide separation and identification by

lRPLC-ESI-MS2 (Fig. 3). The WAX/WCX column yielded

good separation for proteins under a pH value of 8.3, an

optimum condition for the downstream tryptic digestion,thus enabling largely improved compatibility between

protein fractionation and IMER-lRPLC-ESI-MS2. In

comparison to the off-line method, the whole analytical

time for the on-line system was shortened from 30 h to

5 h. When a 30-lg extract from human lung-cancer

cells was analyzed by such a platform, 284 proteins were

positively recognized, demonstrating that it might pro-

vide an attractive tool for large-scale proteome profiling.

2.2.3. Other coupling modes. A dual-function microde-

vice integrating solid-phase extraction (SPE) and IMER

digestion was developed by Svecs group [36]. The device

was fabricated from a long porous poly(BMA-EDMA)

monolith prepared within a capillary. One portion of the

monolith was selectively functionalized for trypsin

immobilization. The other portion of unmodified hydro-

phobic monolith was served as a micro solid-phase

extractor (l-SPE). When digested by the dual-function

device in two different flow directions, SPE-IMER and

IMER-SPE, almost equal sequence coverages of myoglo-

bin were obtained. Compared with a single function

IMER operating without the pre-concentration step, the

dual-function device produced higher sequence coverage

of proteins.

Recently, Zhangs group [37] developed an integrated

sample-treatment device, which comprised a membrane

interface and monolith-based trypsin-IMER, for simul-

taneous sample-buffer exchange, protein enrichment

and on-line digestion. With such a device, acetonitrile

content in the sample buffer was reduced to one-tenth of

the initial value, and the pH value was adjusted from

3.0 to 8.0, compatible with on-line tryptic digestion.

Furthermore, the signal intensity of protein digests was

improved by over 10-fold. These features render such a


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device great potential for the analysis of complex protein

samples.

A typical sample preparation for proteolytic digestion

involves multiple steps (e.g., denaturation, reduction,

and alkylation), which are regarded as a necessary

means to enhance the efficiency of digestion and iden-

tification, especially for complex sample profiling. Al-

though many efforts have been devoted to accelerate the

digestion steps, as mentioned above, little has been done

to speed the whole procedure.

Figeyss group [38] developed a microdevice, termed

the proteomic reactor, by which protein adsorption,

reduction, alkylation, digestion and elution were per-

formed on the SCX resin. With their device, trypsin and

proteins were loaded onto the reactor at pH 3.0, and thevast majority of them were absorbed onto the SCX resin

and then digested by activating trypsin with an increase

of the pH to 8.0. The whole procedure for protein-sample

pretreatment took only 3 h (including digestion time of

2 h). Totally, 205 unique proteins were identified from

10 lg of mouse P19 cell lysate by nanoRPLC-ESI-MS2.

Recently, inspired by the boiling-assisted denaturation

and reduction method in sample preparation for gel-based

proteomics, Zhangs group [39] integrated multiple

sample pretreatment steps involving on-line thermal dena-

turation, reduction, and digestion with lRPLC-ESI-MS2

for high-throughput, gel-free, proteome profiling. In their

approach, native proteins were denatured on-line and

reduced within a heater, digested with a monolith-based

trypsin-IMER, and then analyzed by lRPLC-ESI-MS2. In

comparison to the traditional off-line sample-preparation

method, cysteine alkylation, termination of the digestion

reaction and peptide desalting could be avoided, and,

more importantly, the whole procedure could be per-

formed automatically without the risk of sample loss or

contamination. With such a platform, the sample-pre-

treatment time was substantially shortened from typically

several hours or even one day to 7 min, including 2 min

of thermal denaturation and reduction, and 5 min of

IMER digestion. When 18.3 lg of the soluble fraction from

mouse-liver extract was analyzed by the on-line system intriplicate runs, 244 unique proteins were confidently

identified. The integrated platform might provide a

promising avenue for high-throughput treatment and

analysis of proteome samples.

3. IMERs for characterization of proteins with

PTMs

Proteins with PTMs (e.g., phosphorylation and glyco-

sylation) play important roles in most cellular events and

Figure 3. On-line system integrating protein separation by WAX/WCX, on-line digestion by trypsin-IMER, and peptide separation andidentification by lRPLC-ESI-MS2 (with permission from [35]).




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control many biological processes (e.g., proliferation,

differentiation, and apoptosis) [40]. The last few years

have witnessed significant advances in methodologies for

characterizing protein PTMs in almost all aspects,

including sample preparation, purification and enrich-

ment of modified proteins and/or peptides, MS identifi-

cation techniques, and bioinformatic tools for theverification of the sites of PTMs. As an indispensable step

in sample preparation, digestion has also gained some

attention, so IMERs have found their applications in

characterization of proteins with PTMs.

3.1. IMERs for phosphoprotein characterization

Two kinds of enzymes are mainly used for the charac-

terization of phosphoproteins:

(1) endoproteases (e.g., trypsin and pronase) (for prote-

olytic digestion of proteins); and,

(2) alkaline phosphatase (for dephosphorylation of

phosphoproteins and phosphopeptides).

To date, several IMERs with such enzymes have beendeveloped and exploited for analyzing phosphoproteins.

Temporini et al. [41] proposed an integrated chro-

matographic system for phosphoprotein analysis, by

which phosphoproteins were on-line digested by mono-

lith-based trypsin-/pronase-IMER for 50 min, and the

phosphopeptides in the digests were then selectively

enriched with a TiO2-trap column, followed by lRPLC-

ESI-MS2. Its potential was tested on a mono-phosphor-

ylated fraction of insulin-like growth factor-binding

protein 1 (IGFBP-1) purified from amniotic fluid. Three

phosphopeptides, each containing one phosphorylation

site, were unambiguously assigned with such a system.Dovichis group [42] developed an automated enzyme-

based diagonal CE system for phosphopeptide character-

ization. In their design, a superparamagnetic

microsphere-based alkaline phosphatase-IMER was pre-

pared at the distal end of the first capillary column.

Unphosphorylated peptides fell on the diagonal of the

reconstructed electropherogram, while peptides that

underwent dephosphorylation fell off the diagonal. The

performance of the system was demonstrated by charac-

terizing phosphopeptides in the tryptic digest of a-casein.

By integrating the proteomic reactor [38] with a

phosphopeptide-enrichment reactor (fritted fused silica

tubes packed with TiO2 beads), Figeyss group [43] de-

signed a microfluidic phosphoproteomic reactor for ana-

lyzing phosphoproteins in complex samples. Combining

the phosphoproteomic reactor with sub-cellular fractio-

nations, they identified over 1000 phosphopeptides from

621 phosphoproteins that were localized in 15 different

sub-cellular fractions from human HUH7 cell lines.

3.2. IMERs for characterization of glycoprotein

Two kinds of enzymes are commonly used for the

characterization of glycoproteins:

(1) endoproteases (e.g., trypsin and pronase), which

can be used for specific or non-specific proteolytic

digestion of glycoproteins. In general, due to the

presence of glycans adjacent to tryptic cleavage

sites, the digestion efficacy of trypsin is often largely

reduced, producing heterogeneous population of

glycopeptides with high-molecular-weight peptidemoieties containing more than one glycosylation

site. In contrast, non-specific cleavage of glycopro-

teins with proteases (e.g., pronase) can be used to

produce glycopeptides with significantly smaller

peptide moieties, facilitating simultaneous determi-

nation of glycan composition and peptide structure

by MS2; and,

(2) exo-/endo-glycosidases, among which peptide-N-

glycosidase F (PNGase F) is widely used, since it

can specifically remove N-glycans from glycopro-

teins and glycopeptides to identify glycosylation

sites and oligosaccharide heterogeneity in glycopro-

teins. With these enzymes, some IMERs and relatedsystems have been reported so far.

Based on a pronase-IMER supported on silica mono-

lith, Temporini et al. [44] proposed an automated ana-

lytical approach for simultaneous characterization of

glycans and peptide moieties in pronase-generated gly-

copeptides. The IMER was integrated with a porous

graphite-carbon trap column and a normal-phase LC-

MS2 system, enabling rapid digestion of glycoproteins,

selective enrichment, and fast identification of produced

glycopetides to determine the glycosylation sites as well

as the heterogeneity. With a model protein ribonuclease

B as the substrate, rapid (20 min), reproducible proteo-lytic digestion by the IMER was achieved. Compared

with most of the traditional off-line methods, which re-

quired 3 d, such a system reduced the glycoprotein

analysis time to 1 h.

With polyacrylamide monolith as the support, Palm

et al. [45] prepared a PNGase F-IMER to remove N-gly-

cans from small and medium-sized glycoproteins [e.g.,

ribonuclease B (RNase B), asialofetuin, a1-acid glyco-

protein, and ovalbumin]. Compared to the deglycosyla-

tion conducted in solution, which took about 12 h, a

short residence time of only 3.5 min was required by

IMER.

Svecs group [46] developed another PNGase F-IMER

supported on poly(GMA-EDMA) monolith. The release of

the five glycan structures from RNase B using this IMER

within 3.3 min at room temperature was comparable to

that achieved with free PNGase F in solution for 24 h at

37C. A large glycoprotein, human immunoglobulin G

(hIgG), was also deglycosylated with the IMER in a

slightly longer time of 5.5 min. The PNGase F-IMER was

then integrated into a system comprising on-line glycan

release and separation via hydrophilic interaction LC

followed by ESI-MS2 detection. The performance of such




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a system was demonstrated by the characterization of

hIgG, showing great potential for the analysis of various

N-linked glycoproteins.

With their previous success in developing the proteo-

mic reactor [38], Figeyss group [47] designed a glyco-

proteomic reactor combined protein concentration and

purification (by a concanavalin A affinity chromatog-raphy column), disulfide-bond reduction, PNGase

F-mediated 18O-labeling and deglycosylation, alkylation,

tryptic digestion and peptide fractionation in a device

(with a reaction volume of 1 lL). The glycoproteomic

reactor decreased the sample processing time to less than

1.5 h, and reduced the reagent consumption, while

providing over 1000-fold concentration of the sample. In

addition, both glycopeptides and non-glycosylated tryp-

tic peptides were analyzed by nanoRPLC-ESI-MS2, lead-

ing to higher protein coverage and more reliable

identification. In total, 82 unique glycopeptides repre-

senting 41 unique glycoproteins were positively recog-

nized with as little as 5 lL of human plasma.

4. IMERs for protein quantification

After the initial protein identification and characteriza-

tion, a remaining challenge in proteomics is accurate

quantification of proteins. To date, various methods for

protein quantification have been developed. Of all the

current relative quantification methods by isotopic

labeling in vitro, enzyme-mediated proteolytic 18O-label-

ing has been shown to be simple, specific, cost effective

and applicable to a wide range of analyses. In a typical18O-labeling protocol, proteins are first digested with

enzymes, dried, and then labeled overnight by incubation

with H218O in the presence of enzymes. After inactivating

or quenching the enzymatic activity, peptides are sub-

jected to LC-ESI-MS2 to identify and to quantify the pro-

teins from which the peptides originated. Such a protocol

suffers from several drawbacks (e.g., long incubation

times, low labeling efficacy due to limited amount of

enzymes added, enzyme auto-digestion, and possible back

exchange of 18O with 16O atoms) [48]. To address these

problems, several IMERs have been presented for enzyme-

mediated 18O-labeling in quantitative proteomics.

The residual protease activity is mainly responsible for

the back exchange from 18O to 16O at C-termini of the

labeled peptides, leading to significantly decreased

quantitative accuracy. This is most readily eliminated

using immobilized proteases, which can be physically

removed from the reaction. Sevinsky et al. [49] employed

Poroszyme-immobilized trypsin beads (Applied Biosys-

tems, MA, USA) in the protein-digestion step. By contrast

to the in-solution digestion for 18 h at 37C, near-ideal

labeling (ratio 18O/16O = 0.99) was achieved for peptides

produced by protein samples when digested with trypsin-

IMER overnight at 25C. Moreover, the C-terminal 18O

label was preserved well throughout the downstream

separation process (i.e. immobilized pH gradient iso-

electric focusing).

The 18O-labeling method also focuses on the time-

consuming digestion and labeling steps. Using trypsin

spin columns (Sigma-Aldrich, MO, USA), Mirza and co-

workers [50] digested and labeled samples within15 min, yielding even higher labeling efficiency com-

pared to the conventional overnight incubation method.

In addition, since the protein samples were readily di-

gested by the spin columns with septa, the resulting

tryptic digests were eluted into a clean tube without any

contamination from active trypsin into the eluent, fur-

ther minimizing undesired back exchange.

Recently, Smiths group [51] reported Poroszyme-

immobilized trypsin beads (Applied Biosystems) con-

comitant with an ultrasonic irradiation method for

quantitative proteomic applications. With such a

method, efficient digestion of extracts from Shewanella

onedensis and mouse plasma was completed within1 min. Moreover, thorough 18O-labeling was achieved,

even with a time of only 30 s, and more than 90% of the

quantified pairs had a labeling efficiency of >90%,

allowing for fast, accurate peptide quantification.

5. Some considerations concerning IMERs

Although significant advances have already been made

in recent years, several issues of IMERs need to be ad-

dressed.

5.1. Kinetics

As exemplified above, proteolytic digestion by proteases

supported on various matrices, especially the porous

materials, is a very fast process, where proteins can be

digested in minutes compared to the conventional in-

solution digestion by free proteases taking place over

hours. To investigate this surprising phenomenon, many

researchers employed the Michaelis-Menten equation to

determine the kinetic parameters (i.e. K m and V max). Of

note is that, although the activity of some enzyme mol-

ecules may be damaged during the immobilization pro-

cess, highly concentrated enzymes are often supported

within a confined space, acting collectively to enhance

catalytic capacity. It would therefore be better to describe

the kinetic parameters calculated from this equation as

apparent, due to the possible differences from true values.

It is also noteworthy that although the Michaelis-Menten

equation works well for homogeneous catalysis by free

enzymes in solution, to some extent, it is not suitable for

IMERs due to their heterogeneous nature. Even though

the mobility of immobilized enzymes is severely restricted

and the solid matrix backbone is often viewed as a dif-

fusion-restricted media, these are of only a minor degree

for porous supporting materials, which possess excellent




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permeability, leading to increased mass transport of

substrate over the enzymatic sites to which solvent is

more freely accessible in IMERs (e.g., convective trans-

port in monoliths). This is much superior to restricted

diffusional mass transfer, which is predominant in the

free enzyme-digestion method. Thus, the matrix charac-

teristics (e.g., permeability factor and mass-transfer ef-fect) need to be taken into account for IMERs.

Recently, Liu and co-workers [52] developed a

modified kinetics model on the basis of a sequential

mechanism to explain and to simulate the kinetics of

catalytic reaction in the nanopores of macroporous

ordered siliceous foam (MOSF) entrapped with trypsin.

Their simulation showed that the major factor for very

fast digestion kinetics observed stemmed from a pep-

tide-confinement effect, where the generated peptides

were trapped within a confined space for further pro-

teolysis to the final products. In addition, the entry

and the diffusion of the proteins into the porous cavity

could accelerate or limit the first proteolytic step,which required the encounter between substrates and

enzymes. Although the authors claimed that their

model can be widely applied to different enzyme-cata-

lyzed reactions, the applicability to IMERs supported

on other kinds of porous materials with different

structures (e.g., monolithic materials) is unclear, and

perhaps more appropriate models of the kinetics for

highly efficient proteolysis with these IMERs remain to

be established.

5.2. Non-specific adsorption

Non-specific adsorption of proteins and peptides on

supporting materials is a common problem for most

IMERs. Reduction or elimination of undesired non-

specific adsorption on IMERs is an important issue,

especially for the analysis of minute proteomic samples

(e.g., some clinical samples). To this end, two general

approaches have been exploited:

* a straightforward approach is the addition of a cer-

tain amount of organic solvents into highly aqueous

digestion buffers. It is found that the presence of 20%

methanol [12] or 20% acetonitrile [13] could effectively

reduce non-specific interactions between hydrophobic

peptides and supporting materials while maintaining

excellent digestion efficacy of IMERs [unpublished datafrom Prof. Lihua Zhangs laboratory]; and,

* another approach is the development of IMERs

supported on materials formed by hydrophilic compo-

nents {e.g., polysaccharides [20], acrylamide [53,54],

and poly(ethylene glycol) dimethacrylate [15]} or

modified with hydrophilic coatings {e.g. poly(ethylene

glycol) methacrylate) [55]}.

5.3. Comparison of IMERs

Although comparison of the performance of several self-

made IMERs was described in some reports [12,26,56],

the aims of these publications focused on investigating

the effect of different supporting materials and immobi-

lization techniques on the enzymatic activity of IMERs.

Moreover, it is difficult to compare IMERs from different

laboratories, since no unified parameters are available.

The value of protein-sequence coverage is now widely

used to evaluate the digestion performance of IMERs, butwe should keep in mind that many factors can affect the

resultant sequence coverage values (e.g., type of stan-

dard protein(s) digested, protein concentration, injected

quantity of the digests, type and settings of the mass

spectrometer used, and database-search conditions).

Only when experiments are performed under identical

conditions, can comparison of the performance of IMERs

from different laboratories become meaningful and

convincing. Moreover, besides the sequence coverage,

the number of unique peptides matched, and the missed

cleavage sites in identified peptides should also be pro-

vided to describe the performance of IMERs in detail

[11,12,26]. Of note is that some other characteristics of IMERs, including the digestion yield, stability (storage

and operational stability), reproducibility, and non-spe-

cific adsorption capacity are still to be addressed, in

particular for those applied to large-scale proteomic

studies.

5.4. Comparison between IMERs and in-solution

digestion

Comparison with in-solution digestion by free enzymes is

a generally recognized method to evaluate the perfor-

mance of IMERs. In contrast to the in-solution coun-

terpart, IMERs often show high proteolytic efficacy,yielding equal or more peptides within a substantially

shorter digestion time. It is noteworthy that some dif-

ferent peptides might be produced, mainly resulting from

the slightly altered digestion specificity of immobilized

enzymes. This phenomenon is sometimes negligible

when analyzing standard proteins [16]. However, a

significant number of different peptides, and thus differ-

ent corresponding proteins, can be obtained, showing

some exclusive identifications from the same proteomic

sample [16,33,57].

5.5. Other IMERsSince each protease has its own specificity for the

proteolysis of proteins, enzymatic cleavage using

different enzymes can provide complementary

structure information, leading to enhanced coverage

of proteins or proteomes. Besides the commonly

preferred trypsin-IMERs, other IMERs, including

chymotrypsin-IMER [58], Glu-C-IMER [59], and Lys-

C-IMER [55], have been developed in recent years.

Their potential usage individually, or in combina-

tion, for proteomic studies is to be highlighted in

future work.




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5.6. Membrane-protein profiling

Although membrane proteins have unique, important

functions, analysis of these proteins is still a challenging

task due to their hydrophobic nature. Even though

many IMERs have shown success in analyzing complex

samples (mainly soluble fractions), as mentioned above,

a few applications for membrane-protein profiling havebeen reported. With the assistance of efficient solubili-

zation solvents, specially-designed IMERs might provide

solutions to this problem.

Very recently, Zhangs group made the first attempt to

construct an IMER-based integrated platform to profile

integral membrane proteins, as illustrated in Fig. 4 [60].

In their approach, membrane proteins were extracted

and solubilized by formic acid, on-line digested by apepsin-IMER, and then analyzed by SCX-lRPLC-ESI-

Figure 4. Membrane-proteome profiling with an on-line system involving pepsin-IMER and SCX-RPLC-ESI-MS2 (with permission from [60]).

Table 1. Representative immobilized enzyme reactors (IMERs) applied for the analysis of proteins

Analytical task IMERs Analytical strategies/Comments Ref.

Protein-expressionprofiling

Trypsin-IMER Complex samples were digested off-line within several minutes oreven seconds, yielding comparable identifications with thetraditional in-solution digestion for 12–24 h.

[16,17,19,22]

Trypsin-IMER IMERs were integrated with HPLC and MS2 for on-line digestion,separation and identification of complex samples.

[33,35,54,55]

Trypsin-IMER Multiple sample-treatment steps (e.g., enrichment, denaturation,reduction, digestion, and/or desalting) were performed onanalytical platforms integrated with IMERs.

[37–39]

Pepsin-IMER IMERs were integrated with CE or HPLC and MS2 for on-line

digestion, separation and identification of proteins.

[29–30,60]

Characterization of proteins with PTMs

Trypsin-IMER Phosphoproteins were on-line digested within reduced incubationtimes.

[41,43]

Alkaline phosphatase-IMER Real-time de-phosphorylation from phosphopeptides wasachieved.

[42]

pronase-IMER Efficient digestion of standard glycoproteins was obtained,producing glycopeptides with small peptide moieties (1–8 aminoacids).

[44]

PNGase F-IMER N-glycans were removed from standard glycoproteins withinseveral minutes.

[45,46]

Protein quantification Trypsin-IMER Commercial IMERs were used for efficient proteolytic digestion of proteins and 18O-labeling at C-termini of the resulting peptides.

[49–51]



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MS2. After a few minutes digestion within the pepsin-

IMER, 235 unique proteins were positively identified

from 100 lg of rat-liver microsomal extract, in which

39% (91/235) were annotated as membrane proteins

with one or more transmembrane domains, showing

promising potential for efficient sample treatment and

on-line automated profiling of membrane proteomes.

6. Conclusions and perspectives

In recent years, numerous IMERs have been successfully

prepared and employed in many aspects of proteomic

studies (e.g., protein-expression profiling, phospho-/

glyco-protein characterization and protein quantifica-

tion), as summarized in Table 1. Among these, some

IMERs have demonstrated high digestion efficiency and

excellent compatibility with sample processing (e.g.,

enrichment and desalting), protein and peptide separa-

tion and identification systems, largely accelerating the

current analytical workflows for proteomic research.

Of note is that, although many publications so far

have mainly focused on the development of IMER

methodology, the applications of IMERs and related

platforms in proteomic research are still tentative and

preliminary. As far as IMERs themselves are concerned,

robustness, repeatability, and hydrophilicity are still to

be improved and validated, especially for the study of

protein quantification and biomarker discovery. More-

over, novel IMERs remain to be designed for membrane-

protein profiling. For analytical platforms integrated

with IMERs, more efficient separation techniques (atprotein and/or peptide level), sophisticated mass spec-

trometers, and reliable bioinformatics tools should be

utilized together to provide more convincing and com-

prehensive protein-data sets of given samples. We have

confidence that, with more efforts in IMERs and related

coupling systems, more integrated and automated

workflows with higher-throughput may be anticipated,

further advancing large-scale, in-depth proteomic anal-

ysis.

AcknowledgementsWe apologize to authors whose original publications

could not be cited or discussed in this review due to space

limitations. Financial supports from the National Basic

Research Program of China (2007CB914100), the

National Natural Science Foundation (20935004), the

National Key Technology R. & D. Program

(2008BAK41B02), the creative research group project

by NSFC (21021004), and the Project of the Chinese

Academy of Sciences (KJCX2YW.H09) are gratefully

acknowledged.

References[1] M.R. Wilkins, R.D. Appel, K.L. Williams, D.F. HochstrasserProte-

ome Research Concepts, Technology and Application, second ed.,

Springer, Heidelberg, Germany, 2007.

[2] R. Aebersold, M. Mann, Nature (London) 422 (2003) 198.

[3] D. Lopez-Ferrer, B. Canas, J. Vazquez, C. Lodeiro, R. Rial-Otero, I.

Moura, J.L. Capelo, Trends Anal. Chem. 25 (2006) 996.

[4] J. Krenkova, F. Foret, Electrophoresis 25 (2004) 3550.

[5] G. Massolini, E. Calleri, J. Sep. Sci. 28 (2005) 7.

[6] J. Krenkova, F. Svec, J. Sep. Sci. 32 (2009) 706.

[7] J. Ma, L. Zhang, Z. Liang, W. Zhang, Y. Zhang, Anal. Chem. Acta

632 (2009) 1.

[8] A. Monzo, E. Sperling, A. Guttman, Trends Anal. Chem. (2009)

28.

[9] J. Kim, B.C. Kim, D. Lopez-Ferrer, K. Petritis, R.D. Smith,

Proteomics 10 (2010) 687.

[10] X. Zhang, B. Liu, L. Zhang, H. Zou, J. Cao, M. Gao, J. Tang, Y. Liu,

P. Yang, Y. Zhang, Sci. China Chem. 53 (2010) 685.

[11] D.S. Peterson, T. Rohr, F. Svec, J.M.J. Frechet, J. Proteome Res. 1

(2002) 563.

[12] J. Krenkova, Z. Bilkova, F. Foret, J. Sep. Sci. 28 (2005) 1675.

[13] J. Duan, Z. Liang, C. Yang, J. Zhang, L. Zhang, W. Zhang, Y.

Zhang, Proteomics 6 (2006) 412.

[14] K. Sakai-Kato, M. Kato, T. Toyooka, Anal. Chem. 74 (2002)

2943.

[15] M.T. Dulay, Q.J. Baca, R.N. Zare, Anal. Chem. 77 (2005) 4604.

[16] J. Ma, Z. Liang, X. Qiao, Q. Deng, D. Tao, L. Zhang, Y. Zhang,

Anal. Chem. 80 (2008) 2949.

[17] L. Qiao, Y. Liu, H.S.P. Hudson, P. Yang, E. Magner, B. Liu, Chem.

Eur. J. 14 (2008) 151.

[18] Y. Deng, C. Deng, D. Qi, C. Liu, J. Liu, X. Zhang, D. Zhao, Adv.

Mater. 21 (2009) 1.

[19] S. Lin, G. Yao, D. Qi, Y. Li, C. Deng, P. Yang, X. Zhang, Anal.

Chem. 80 (2008) 3655.

[20] Y. Liu, H. Lu, W. Zhong, P. Song, J. Kong, P. Yang, H.H. Girault,

B. Liu, Anal. Chem. 78 (2006) 801.

[21] Y. Liu, W. Zhong, S. Meng, J. Kong, H. Lu, P. Yang, H.H. Girault,

B. Liu, Chem. Eur. J. 12 (2006) 6585.[22] Y. Liu, Y. Xue, J. Ji, X. Chen, J. Kong, P. Yang, H.H. Girault, B. Liu,

Mol. Cell. Proteomics 6 (2007) 1428.

[23] G.W. Slysz, D.S. Schriemer, Rapid Commun. Mass Spectrom. 17

(2003) 1044.

[24] Website, last accessed 1 September 2010: http://www.sigmaald-

rich.com/etc/medialib/docs/Sigma/General Information/immob-

trypsin-spin.Par.0001.File.tmp/immobtrypsinspin.pdf .

[25] R. Marangoni, R. Chiarini, G. Iannone, M. Salerno, Proteomics 8

(2008) 2165.

[26] R. Nicoli, S. Rudaz, C. Stella, J.-L. Veuthey, J. Chromatogr., A

1216 (2009) 2695.

[27] L.N. Amankwa, W.G. Kuhr, Anal. Chem. 64 (1992) 1610.

[28] M. Ye, S. Hu, R.M. Schoenherr, N.J. Dovichi, Electrophoresis 25

(2004) 1319.

[29] M. Kato, K. Sakai-Kato, H.M. Jin, K. Kubota, H. Miyano, T.Toyooka, M.T. Dulay, R.N. Zare, Anal. Chem. 76 (2004) 1896.

[30] R.M. Schoenherr, M. Ye, M. Vannatta, N.J. Dovichi, Anal. Chem.

79 (2007) 2230.

[31] B.R. Fonslow, J.R. Yates III, J. Sep. Sci. 32 (2009) 1175.

[32] E. Calleri, C. Temporini, E. Perani, A.D. Palma, D. Lubda, G.

Mellerio, A. Sala, M. Galliano, G. Caccialanza, G. Massolini, J.

Proteome Res. 4 (2005) 481.

[33] S. Feng, M. Ye, X. Jiang, W. Jin, H. Zou, J. Proteome Res. 5 (2006)

422.

[34] G.W. Slysz, D.S. Schriemer, Anal. Chem. 77 (2005) 1572.

[35] H. Yuan, L. Zhang, C. Hou, G. Zhu, D. Tao, Z. Liang, Y. Zhang,

Anal. Chem. 81 (2009) 8708.



http://www.sigmaaldrich.com/http://www.sigmaaldrich.com/http://www.sigmaaldrich.com/http://www.sigmaaldrich.com/http://www.sigmaaldrich.com/http://www.sigmaaldrich.com/


12/12

[36] D.S. Peterson, T. Rohr, F. Svec, J.M. Frechet, Anal. Chem. 75

(2003) 5328.

[37] L. Sun, J. Ma, X. Qiao, Y. Liang, G. Zhu, Y. Shan, Z. Liang, L.

Zhang, Y. Zhang, Anal. Chem. 82 (2010) 2574.

[38] M. Ethier, W. Hou, H.S. Duewel, D. Figeys, J. Proteome Res. 5

(2006) 2754.

[39] J. Ma, J. Liu, L. Sun, L. Gao, Z. Liang, L. Zhang, Y. Zhang, Anal.

Chem. 81 (2009) 6534.

[40] M. Mann, O.N. Jensen, Nat. Biotechnol. 21 (2003) 255.

[41] C. Temporini, L. Dolcini, A. Abee, E. Calleri, M. Galliano, G.

Caccialanza, G. Massolini, J. Chromatogr., A 1183 (2008) 65.

[42] R. Wojcik, M. Vannatta, N.J. Dovichi, Anal. Chem. 82 (2010)

1564.

[43] H. Zhou, F. Elisma, N.J. Denis, T.G. Wright, R. Tian, H. Zhou, W.

Hou, H. Zou, D. Figeys, J. Proteome Res. 9 (2010) 1279.

[44] C. Temporini, E. Perani, E. Calleri, L. Dolcini, D. Lubda, G.

Caccialanza, G. Massolini, Anal. Chem. 79 (2007) 355.

[45] A.K. Palm, M.V. Novotny, Rapid Commun. Mass Spectrom. 19

(2005) 1730.

[46] J. Krenkova, N.A. Lacher, F. Svec, J. Chromatogr., A 1216 (2009)

3252.

[47] H. Zhou, W. Hou, N.J. Denis, J. Vasilescu, H. Zou, D. Figeys, J.

Proteome Res. 8 (2009) 556.

[48] C. Fenselau, X. Yao, J. Proteome Res. 8 (2009) 2140.

[49] J.R. Sevinsky, K.J. Brown, B.J. Cargile, J.L. Bundy, J.L. Stephenson

Jr., Anal. Chem. 79 (2007) 2158.

[50] S.P. Mirza, A.S. Greene, M. Oliver, J. Proteome Res. 7 (2008)

3042.

[51] D. Lopez-Ferrer, K.K. Hixson, H. Smallwood, T.C. Squier, K.

Petritis, R.D. Smith, Anal. Chem. 81 (2009) 6272.

[52] H. Bi, L. Qiao, J.-M. Busnel, B. Liu, H.H. Girault, J. Proteome Res. 8

(2009) 4685.

[53] A.K. Palm, M.V. Novotny, Rapid Commun. Mass Spectrom. 18

(2004) 1374.

[54] J. Duan, L. Sun, Z. Liang, J. Zhang, H. Wang, L. Zhang, W. Zhang,

Y. Zhang, J. Chromatogr., A 1106 (2006) 65.

[55] J. Krenkova, N.A. Lacher, F. Svec, Anal. Chem. 81 (2009) 2004.

[56] A. Cingoz, F. Hugon-Chapuis, V. Pichon, J. Chromatogr., A 1209

(2008) 95.

[57] A.A. Klammer, M.J. MacCoss, J. Proteome Res. 5 (2006)

695.

[58] H. Yamaguchi, M. Miyazaki, T. Honda, M.P. Briones-Nagata, K.

Arima, H. Maeda, Electrophoresis 30 (2009) 3257.

[59] W. Lin, C.D. Skinner, J. Sep. Sci. 32 (2009) 2642.

[60] J. Ma, C. Hou, L. Sun, D. Tao, Y. Zhang, Y. Shan, Z. Liang, L.

Zhang, L. Yang, Y. Zhang, Anal. Chem. 82 (2010) 9622.



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