Capillary zone electrophoresis-mass spectrometry for bottom-up proteomics

Accepted Manuscript Capillary zone electrophoresis-mass spectrometry for bottom-up proteomics Zhenbin Zhang, Yanyan Qu, Norman J. Dovichi PII:

S0165-9936(18)30265-6

DOI:

10.1016/j.trac.2018.08.008

Reference:

TRAC 15218

To appear in:

Trends in Analytical Chemistry

Received Date: 31 May 2018 Revised Date:

7 August 2018

Accepted Date: 16 August 2018

Please cite this article as: Z. Zhang, Y. Qu, N.J. Dovichi, Capillary zone electrophoresis-mass spectrometry for bottom-up proteomics, Trends in Analytical Chemistry (2018), doi: 10.1016/ j.trac.2018.08.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Capillary zone electrophoresis-mass spectrometry for bottom-up proteomics Zhenbin Zhang, Yanyan Qu, Norman J. Dovichi* Department of Chemistry and Biochemistry

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Notre Dame, IN 46556 USA

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University of Notre Dame

CZE-Proteomics – pg 1

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Highlights Development of CZE-MS based bottom-up proteomics is reviewed.

Different sample preconcentration techniques are discussed.

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Different techniques for coating the capillaries are presented.

Different applications of CZE-MS based proteomics are reviewed.

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Future perspectives for CZE-MS based proteomics are considered.

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Abstract Bottom-up proteomics characterizes proteins by analysis of peptides generated through proteolysis. Bottom-up analysis of a complex proteome inevitably generates

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tens of thousands of peptides, and the analysis of these peptides is a serious challenge. Capillary zone electrophoresis (CZE) generates separations that are orthogonal to

reversed phase liquid chromatography, which has led to consideration of CZE as an alternative separation technology in proteomic analysis. The steady improvement in mass spectrometer (MS) technology coupled with improvements in capillary coatings

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and the development of robust CZE-MS interfaces have contributed to the rapid advancement of CZE’s identification performance in bottom-up proteomics analysis. In

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this review, we focus on recent advances of CZE-MS based bottom-up proteomics, including optimization of CZE and MS conditions, and the application of CZE-MS in phosphoproteomics, glycoproteomics, clinical diagnosis, host cell protein analysis, ultrasensitive proteomics, and quantitative proteomics. Finally, we outline future

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opportunities and challenges in this field.

CZE-Proteomics – pg 3

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Keywords: capillary zone electrophoresis; bottom-up proteomics; glycomics; post-

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translational modifications; applications in clinical chemistry

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Abbreviations: MS, Mass spectrometry; TOF, Time-of-flight; RPLC, Reverse phase liquid chromatograph; MS/MS, Tandem mass spectrometry; CZE, Capillary zone electrophoresis; ESI, Electrospray ionization; CHO, Chinese hamster ovary; HCPs, host cell proteins; MSPE, Solid-phase microextraction; APS, 3-aminopropyl trimethoxysilane; EOF, Electroosmotic flow; TEMED, N,N,N’,N’-tetramethylethylenediamine; LPA, Linear polyacrylamide; ATRP, atom transfer radical polymerization polymerization; RAFT, reversible addition-fragmentation chain transfer polymerization; CEC, Capillary electrochromatograpy; SCARAFT, Surface-confined aqueous reversible additionfragmentation chain transfer polymerization; CTA, Chain transfer reagent; tITP, Transient isotachophoresis; SPE, Solid phase extraction; ACN, acetonitrile; FA, Formic acid; BGE, background electrolyte; LE, Leading electrolyte; TE, Terminating buffer; APD, Advanced peptide determination algorithm; SCX, Strong cation-exchange; CKD, Chronic kidney disease; DN, diabetic nephropathy; i.d., inner diameter; IDs, identifications; HRMS, Ultrahigh-resolution mass spectrometer.

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1. Introduction Mass spectrometry (MS) based bottom-up proteomics analyzes proteolytically derived peptides. This analysis is used to identify, quantitate, and characterize proteins

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and their interaction, and it is an essential tool for biologists and biochemists in their efforts to understand the molecular mechanisms regulating cellular systems [1-7]. Reverse phase liquid chromatograph (RPLC) coupled to tandem mass

spectrometry (MS/MS) analysis has dominated proteomics research [8]. Capillary zone

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electrophoresis (CZE) is a complementary separation method to RPLC with a nearly orthogonal separation mechanism [9], which can improve identification numbers and protein coverage from a biological sample. Recent reports showed that CZE

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consistently outperforms RPLC for the analysis of mid-nanogram protein samples [1013]. The low sample consumption and simple geometry of the CZE separation capillary allows detection of trace levels of peptides and peptides with extreme sizes or pI values that are otherwise lost when using chromatographic separations. Furthermore, the simple separation mechanism, which is related only to the size and charge of the

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peptide, allows remarkably accurate prediction of the separation [14]. The development of reliable CZE-MS interfaces has taken a surprisingly long time since the first reports of CZE-MS instrumentation in 1987 [15]. A number of CZEMS interfaces have been developed since that first report [16-21], and these interfaces

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can be divided into two strategies. One type uses a sheath liquid to make up the flow required to maintain stable electrospray ionization (ESI) [20, 22, 23]. Another set of

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interfaces is based on a sheathless design in which the electrical connection to the capillary outlet is achieved through a porous segment near or at the capillary outlet [10, 24-26]. The interested reader is directed to a set of comprehensive reviews of the development of CZE-MS interfaces [27-32]. A number of reviews that focus on CZE-MS based bottom-up proteomics have

also appeared in last few years [33-39]. In our review, we attempt to evaluate comprehensively the contribution of CZE-MS to bottom-up proteomics and focus on recent advances of CZE-MS in the field, including optimization of CZE and MS conditions, and the application of CZE-MS in phosphoproteomics, glycoproteomics, CZE-Proteomics – pg 6

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clinical diagnosis, host cell protein analysis, ultrasensitive proteomics, and quantitative proteomics analysis. Finally, we outline future perspectives and remaining challenges in this field.

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2. CZE-MS based bottom-up proteomics A typical workflow for CZE-MS based bottom-up proteomics is shown in Figure 1. Proteins are extracted from cell culture or tissues and digested, typically with trypsin. Peptides are then analyzed directly or subjected to pre-fractionation, typically with

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reversed-phase or ion-exchange chromatography. The peptides are characterized by tandem mass spectrometry, and the resulting mass spectrometry data are analyzed

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with various software tools.

Figure 1. Typical workflow for CZE-MS based bottom-up proteomics.

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In hindsight, the initial applications of CZE to proteomics were disappointing.

Early mass spectrometers were unable to capture the narrow peaks produced by CZE, and early CZE-MS interfaces suffered from poor sensitivity and limited stability. As a result, these early studies were limited to characterization of the analytical system using a handful of standard peptides and proteins [40-50]. The situation changed with a pioneering example of CZE-ESI-MS that considered the analysis of a proteolytically digested, biologically derived protein mixture. In this first study, Tong et al. used a solid-phase microextraction system (SPME) at the

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proximal end of the separation capillary, and coupled the capillary to a Finnigan LCQ ion trap mass spectrometer with a sheathless ESI interface [51]. In an innovation that is reminiscent of multidimensional protein identification technology [52], Tong and colleagues employed a multistep elution of the peptides from a positively charged film

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(3-aminopropyl trimethoxysilane, APS), which produced relatively large electroosmotic flow (EOF) and a concomitant short separation window. Since that pioneering work, remarkable improvements in the performance of capillary coatings, electrospray

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based proteomics, as shown in Figure 2 and Table 1.

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interfaces, and mass spectrometers have resulted in a growing literature on CZE-MS

Figure 2. Citation report from the Web of Science Core Collection between 1998 and 2018 (searched on 2018 April 4). TOPIC: (capillary electrophoresis) AND TOPIC: (mass

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spectrometry) AND TOPIC: (proteome).

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Table 1. Recent published research on CZE-ESI-MS/MS based bottom-up proteomics. Sample

Sample

Fraction

Sample

Separation

Mass

Run

Identification

amounts1

method and

preconcentration

conditions2

spectrometer

time /

results

numbers

min

Protein

Reference

peptides

groups 250 ng

Online RP SPE,

Stacking

11

digest

62 cm×25 µm

Finnigan LCQ ion

i.d. APS coated

trap

330

66

136

[51]

23

1

73

[53]

165

140

344

[9]

15

49

-

[54]

50

312 ±

1,377 ±

[12]

29

128

350

871

4,902

[55]

Q-Exactive

60

283

1,159

[56]

Orbitrap Fusion

90

956

4,741

[57]

105

1,529

5,961

capillary, 0.5% acetic acid with 10% methanol, 20 to -8 kV 0.27 ng, (4

digest

fmol)

Unfractionated

tITP

88.5 cm×30 µm

Ultrahigh

i.d. uncoated

resolution-

capillary, 10%

TOF maXis from

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BSA tryptic

acetic acid, -0.8

Bruker

kV, 1.5 psi at the inlet. Secretome of

-

Offline RPLC, 11

Stacking

30 cm × 50 µm

LTQ-Orbitrap

i.d. uncoated

Velos

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M. marinum

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Yeast ribosome

capillary, 10 mM ammonium

acetate(pH5.7), 5.5 kV

Yeast tryptic

-

-

-

digest

30 cm × 50 µm

LTQ-Orbitrap

i.d. uncoated

Velos

capillary, 10 mM ammonium

acetate (pH 7), 9 kV

100 ng

-

coli digest

Stacking

60 cm × 50 µm

LTQ-Orbitrap

i.d. LPA coated

Velos

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Escherichia

capillary,

0.1%(v/v) formic acid, 15 kV

Escherichia

350 µg

coli digest

Offline RP SPE, 7

Stacking

60 cm × 50 µm

LTQ-Orbitrap

i.d. LPA coated

Velos

capillary,

0.1%(v/v) formic

Escherichia coli digest

Yeast tryptic digest

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-

-

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MCF7 digest

250 ng

400 ng

-

Stacking

400 ng

-

Escherichia

50 ng

Online SCX

62 cm × 50 µm i.d. LPA coated capillary, 0.12%(v/v) formic acid, 15 kV.

Stacking

90 cm × 50 µm i.d. LPA coated capillary,

-

HeLa digest

coli digest

acid, 15 kV.

5%(v/v) acetic acid, 15 kV. Dynamic pH junction

SPE, 5

105

2,149

10,274

60 cm × 50 µm

LTQ-Orbitrap

150

799

3,381

i.d. LPA coated

Velos

(2.5 h)

LTQ Orbitrap XL

>11,040

3,272

28,538

capillary, 50 mM formic acid, 18 kV. Yeast tryptic digest

1.4 mg

Offline RPLC, 182

Dynamic pH junction

100 cm × 30 µm i.d. neutrally coated

CZE-Proteomics – pg 9

(184 h)

[58]

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capillary,10%(v/) acetic acid, 30 kV, 1 psi at inlet

Xenopus

50 ng

Online digestion

Dynamic pH junction

protein

100 cm × 50 µm

Q-Exactive HF

100

975

3,749

[59]

i.d. LPA coated capillary,

20 kV. Xenopus

20 ng

-

-

85 cm × 40 µm

digest

Orbitrap Fusion

50

-

1,127

[60]

250

-

1,794

[61]

750

6,100

62,000

[62]

LTQ-XL

180

497

1,400

[63]

Q-Exactive HF

4,200

8,193

64,951

[64]

Q-Exactive HF

100

1,653

7,218

[13]

100 cm × 50 µm

Orbitrap Fusion

<120

4,397

27,113

Unpublished

i.d. LPA coated

with APD

i.d. uncoated capillary, 25%ACN with 1 M formic acid(pH2.3)

-

Dynamic pH junction

digest

60 cm × 50 µm i.d. LPA coated capillary, 5%(v/v) acetic acid, 12 kV.

2 mg

Offline RPLC, 60

-

90 cm × 30 µm

Q-Exactive HF

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human

LTQ XL

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Yeast tryptic

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1 M acetic acid,

lymphoblastic

i.d. neutrally

T-cells digest

coated

capillary,10%(v/) acetic acid, 30

kV, 2 psi at inlet

Mouse liver

500 ng

Single shot

Dynamic pH junction

protein digest

96 cm × 50 µm

i.d. LPA coated capillary,

5%(v/v) acetic acid, 26 kV.

500 µg

protein digest

Offline SCXRPLC, 40

Dynamic pH junction

94 cm × 50 µm

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Mouse brain

i.d. LPA coated capillary,

5%(v/v) acetic acid, 30 kV.

Xenopus

200 ng

Single shot

digest

Dynamic pH junction

99 cm × 50 µm

i.d. LPA coated

digest

220 ng

Single shot

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K562 cell

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capillary,

1 M acetic acid, 20 kV.

Dynamic pH junction

capillary, 1 M acetic acid, 22 kV.

Note: 1. For single shot CZE-MS analysis, sample amount is the injection amount. For fractionation methods, sample amount is the amount of the starting materials. 2. Separation conditions are described in the following order: length × inner diameter and type of the separation capillary, BGE, separation voltage, other conditions.

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3. Optimization of CZE-MS conditions There are three important disadvantages in CZE that can limit its performance. First, non-specific adsorption of proteins and peptides on the silanol groups at the inner

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surface of the capillary leads to sample loss, peak broadening and tailing, and poor reproducibility. Second, high EOF produced by the silanol groups results in a relatively short separation window, which limits the number of MS/MS spectra that can be

generated during proteomic analysis and the number of peptide identifications per run. Third, the concentration detection limit for CZE-MS is relatively poor due to the small

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injection volume that is typically used, which often makes it difficult to analyze dilute biological samples.

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This section will focus on the advancement on the optimization of CZE conditions

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to overcome these limitations, as shown in Figure 3.

Figure 3. Recent advancements in the optimization of CZE-MS conditions. 3.1. Coating types and techniques Currently, the most widely used method for coating the interior of the CZE capillary is based on a free radical polymerization reaction that was developed by CZE-Proteomics – pg 11

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Hjerten [65]. In this method, the inner wall of the fused capillary is pretreated by reaction with γ-methacryloxypropyltrimethoxysilane. The pretreated capillary is then filled with a de-aerated polymerization mixture, typically composed of the acrylamide, N,N,N’,N’tetramethylethylenediamine (TEMED), and potassium persulphate. This procedure

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forms a layer of linear polyacrylamide (LPA) at the inner surface of the capillary.

However, this polymerization is difficult to control because oxygen cannot be completely removed during the TEMED addition; oxygen acts as a free-radical scavenger that

irreproducibly quenches the reaction [66]. To make the process more controllable, a

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simple protocol has been reported that relies on a thermally-initiated free-radical

polymerization reaction [67]. In this case, the capillary is filled with a mixture of the

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monomer and an ammonium persulfate initiator. This mixture is relatively stable at room temperature. Heating in a water bath decomposes the ammonium persulfate and controllably initiates polymerization.

Despite these advances, there are several inherent disadvantages for coating methods based on free radical polymerization reaction. First, as noted above, free radical scavengers can be present at trace levels, leading to irreproducible coatings. In

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addition, the acrylamide monomer not only reacts with the functional group on the inner wall of the capillary but also reacts with other acrylamide monomers in solution, and the polymer formed in solution can clog the capillary, leading to the failure of the coating

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process.

To overcome these problems, coating protocols based on living radical polymerization techniques have been reported. These protocols include atom transfer

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radical polymerization (ATRP) [68] and reversible addition-fragmentation chain transfer (RAFT) polymerization [69-71]. Unfortunately, traditional ATRP systems require high (transition metal) catalyst concentrations to maintain activity throughout the polymerization [72], and removal of the transition-metal ions after the polymerization reaction can be difficult. The presence of trace amounts of the metal catalyst in polymers often raises concerns in biomedical applications [73], and bipyridine adsorption during ATRP would result in anodic EOF during CZE at low pH [74]. In contrast, the RAFT method avoids use of metal catalysts, and is tolerant of a wide

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variety of reaction conditions and functionalities [75-78]. Ali and Cheong reported a method for the immobilization of an N-phenylacrylamide-styrene copolymer on the inner surface of capillaries with RAFT polymerization for applications in capillary electrochromatograpy (CEC). A ligand with a terminal halogen (4-chloromethylphenyl

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isocyanate) or 4-(trifluoromethoxy) phenyl isocyanate was bound to the inner surface of a pretreated silica capillary in the presence of dibutyltin dichloride as a catalyst through an isocyanate-hydroxyl reaction. Attachment of initiator (sodium diethyl dithiocarbamate) to the bound ligand was carried out and followed by in situ polymerization. The coated

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capillary showed good separation performance for derivatized saccharide isomers and tryptic digest of cytochrome C in CEC with UV detection. Unfortunately, the coated

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column has not been evaluated for proteomics applications in CZE-MS. Moreover, the structure of the prepared copolymer is not clear because all the monomers were added to the polymerization mixture before the RAFT polymerization reaction. In addition, the reaction was carried out in organic solvents (toluene or p-xylene), which are not environment-friendly.

Recently, we reported an environmentally friendly coating method based on

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surface-confined aqueous reversible addition-fragmentation chain transfer (SCARAFT) polymerization reaction for covalently bonding polymers to capillary inner surfaces [79]. The schematic diagram for preparation of the coated capillary by SCARAFT method is shown in Figure 4. The capillary wall is first treated with a bifunctional chain transfer

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reagent (CTA), cyanomethyl [3-(trimethoxysilyl)propyl] trithiocarbonate. For preparation of LPA coated capillaries, a polymerization mixture, which was stable under nitrogen at

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room temperature, is prepared by mixing acrylamide (monomer) and 4,4’-azobis(4cyanovaleric acid) (initiator) in an acetate buffer and stirring to form a homogeneous solution. The mixture is then introduced into the pretreated capillary and incubated at 60 °C for RAFT polymerization on the inner surface of the CTA pretreated capillary. SCARAFT polymerization primarily takes place on the inner surface of the capillary instead of in solution, which greatly improves the homogeneity of the coating. Capillaries treated with this coating produced an electroosmotic mobility of 2.8 ± 0.2 × 10-6 cm2·V 1·s-1 (N=3), which is roughly an order of magnitude lower than that of commercial LPA coated capillaries. These coated capillaries have been employed for

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bottom-up proteomic analysis using CZE-ESI-MS/MS. The very low electroosmotic mobility produced a 200-minute separation window and improved identification performance for single-shot CZE-MS analysis. An average of 977 protein groups and 5,605 unique peptides were identified from 50 ng of an E. coli digest and 2,158 protein

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groups and 10,005 peptides were identified from 25 ng of a HeLa digest using singleshot CZE-ESI-MS/MS analysis with a SCARAFT-LPA coated capillary coupled to a QExactive HF mass spectrometer. A single capillary was used for over 200 hours (8.4 days) of continuous operation, and the relative standard deviation in migration time for

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six ions in that separation was between 2 and 3%. Besides the improved homogeneity of the coating, another advantage of the SCARAFT technique is that various coatings

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can be prepared by simply changing the functional vinyl monomers in the polymerization mixture. Moreover, a block-copolymer can be easily prepared, which further minimizes the effect of the silanol groups and improves the reproducibility of

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CZE analysis.

Figure 4. Schematic diagram for preparation of a coated capillary by SCARAFT polymerization reaction [79].

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3.2. Sample preconcentration techniques The narrow inner diameter (i.d.) of the separation capillary, typically 50-µm, requires small injection volumes to minimize band-broadening. The small injection

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volume, in turn, tends to result in mediocre concentration limits of detection for CZE, which has discouraged its use in bottom-up proteomics. Fortunately, this limitation has been eliminated by use of various sample preconcentration techniques, as described in several reviews [80, 81]. Four types of sample preconcentration techniques are now widely used in CZE-MS based bottom-up proteomics: sample stacking, transient

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isotachophoresis (tITP), dynamic pH junction, and solid phase extraction (SPE).

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3.2.1. Sample stacking

Sample stacking is based on a simple principle: if the conductivity of the sample plug is lower than that of the CZE background electrolyte, the electric field strength in the sample plug is higher than that in the background electrolyte. Consequently, the ions in the sample plug migrate faster than in the background electrolyte, and the sample ions are focused at the boundary between the sample plug and the background

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electrolyte. For a better sample stacking performance, the sample plug often contains organic solvent to reduce its conductance.

Sample stacking was used for the single shot CZE-ESI-MS/MS analysis of E. coli protein digests with an electrokinetically pumped nanospray interface [12]. Typically, the

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protein digests were dissolved in 50% (v/v) acetonitrile (ACN) with 0.05% (v/v) formic acid (FA) for CZE-ESI-MS/MS analysis with the 0.1% (v/v) FA as background electrolyte

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(BGE). Over 1,250 peptides and 300 protein groups were identified with 100 ng digest loaded and 50 min analysis time. CZE generated many more peptide identifications than a nanoACQUITY UltraPerformance LCH system (nanoUPLC) (627 ± 38 vs 342 ± 113) when the loaded digest was mass limited (1 ng). In addition, CZE and nanoUPLC provided complementary peptide identifications. The identification numbers could be further improved when coupled to a faster mass spectrometer (an Orbitrap Fusion mass spectrometer) and with 5% (v/v) acetic acid as BGE [57]. In this work, the sample was dissolved in 0.03-0.04% (v/v) FA containing 30-40% (v/v) ACN; over 10,000 peptides and 2,100 proteins were identified from 400 ng of a HeLa cell digest in approximately CZE-Proteomics – pg 15

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100 min. However, RPLC generated more than 5,000 protein and 40,000 peptide identifications (IDs) in 90 min from 300 ng of the HeLa cell digest on the same platform. The poorer performance of CZE is due to an average peak width roughly 2.5 times larger than that for RPLC, which is due to the large injection volume (>5% of the

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capillary volume) used in the CZE experiment.

Addition of an organic solvent to the sample solution gives rise to several

problems. First, the organic solvent evaporates during analysis of fractionated samples, which can lead to the change of its percentage in the sample as well as the sample

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concentration, which reduces reproducibility. Second, if the injection amount exceeded a threshold, precipitation and bubbles are observed within the separation capillary,

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resulting in unstable currents and poor separation performance [82]. The injection plug length has to decrease to alleviate this instability, limiting the injection volume. 3.2.2. Transient isotachophoresis (tITP)

tITP consists of three steps. In the first step, the sample is dissolved in a leading electrolyte (LE), which containing ions with a higher mobility than the sample; this

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mixture is introduced in the separation capillary using hydrodynamic pressure. In the second step, the inlet of the CZE capillary is placed in a vial containing the terminating electrolyte (TE), which contains ions with a lower mobility than the sample. Voltage is applied across the capillary, which forces the LE, the sample, and the TE to migrate

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towards the cathode at distal end of the capillary. Because of the differences in mobility, the sample is focused and concentrated between the LE and the TE. In the third step,

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the inlet of the CZE capillary is switched back to LE as the background electrolyte and normal CZE is performed. tITP can produce a sample concentration factor up to ca. three orders of

magnitude. The technique has been used for the determination of both peptides and proteins [83]. Larsson et al. applied tITP focusing in the on-line analysis of peptides by CZE-MS with a sheath liquid electrospray interface and a single quadrupole mass analyzer [84]. The system allows the injection volumes of up to 0.9 µL. The technique was applied to the qualitative analysis of a tryptic digest of cytochrome c, resulting in low background, high quality spectra. Busnel et al. integrated tITP with a sheathless CZE-Proteomics – pg 16

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CZE-MS interface making use of a porous tip [53]. Concentration limits of detection in the subnanomolar range for peptide were achieved with tITP and the peak capacity as high as 327 can be achieved. However, additional application of positive pressure at the separation capillary inlet is required to provide a stable spray during CZE separation

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with a neutral coated capillary and a sheathless interface. Due to the limited choice of LE and TE in CZE-MS, which requires the volatile buffer, tITP usually is combined with other technique (e.g., SPE) to improve its identification performance for CZE-MS based bottom-up proteomics [11].

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3.2.3. Dynamic pH junction

In a dynamic pH junction, samples are usually dissolved in a basic solution and

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analyzed using CZE in an acidic BGE. Analytes (e.g. peptides in case of bottom-up proteomics) are negatively charged in the basic buffer and move towards the positive (injection) electrode. The sample is neutralized as it comes in contact with the acidic background electrolyte, and is concentrated at the proximal end of the elution plug. Once the elution buffer is neutralized, analyte take on a positive charge in the acidic

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background electrolyte and normal CZE separation is carried out. Two mechanisms have been proposed for sample preconcentration by the dynamic pH junction. The first mechanism is based on stacking in the low-conductivity region that is created in the capillary by the neutralization reaction between H+ and OH-

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ions migrating against one another [85]. Another mechanism is velocity-difference induced focusing and does not depend on the conductivity [86]. Both factors likely

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contribute to sample preconcentration. A comprehensive review on the fundamental discussion of the mechanisms, buffer, and sample conditions required to concentrate various analytes by using dynamic pH junction and a list of published works between the years 1990-2010 is available [87]. Since the dynamic pH junction is based on the change of the charge state in the multisection electrolyte system, it is widely used for optimization of the separation of the analytes with known chemical properties (e.g. pKa) [88-90]. The pioneering work for on-line preconcentration of peptides by this method was reported by Aebersold et al. in 1990 [91]. The pH of the sample solution was raised to pH > 10 by adding small volumes of ammonia to ensure the peptides were negatively

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charged; CZE was performed in 10 mM citrate buffer at pH 2.5. The combination allowed the formation of the dynamic pH junction and increased sample volumes without a breakdown in resolution, which permitted the analysis of peptide solutions of an at least five times lower concentration. Its application in proteomics was also

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reviewed more recently [92]. The dynamic pH junction based CZE-ESI-MS system has been widely used for bottom-up proteomics analysis. Zhu et al. demonstrated that the pH junction is valuable and efficient for analysis of a low concentration, complex

proteome digest [93]. We recently coupled a SCARAFT polymerization method coated

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LPA capillary and dynamic pH junction based CZE to an Orbitrap Fusion Lumos Tribrid platform with advanced peptide determination (APD) algorithm [94]. More than 27,000

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peptides and approximately 4,400 protein groups were identified from 220 ng of K562 cell tryptic digest in a single injection (unpublished data), which represents by far the largest number of peptide identifications produced in single-shot bottom-up proteomics using CZE.

3.2.4. Solid phase microextraction (SPME) based combination method

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SPME preconcentration dramatically increases the sample loading volume in CZE and greatly improves its concentration limit of detection. This technique is usually combined with tITP or dynamic pH junction to further improve the preconcentration performance. Wang and coworkers combined a C18-phase SPME with tITP for the

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proteomic analysis of a moderately complex protein mixture by using CZE-ESI-MS/MS with a sheathless interface [11]. In a comparison of SPME-tITP-CZE with direct injection CZE, they found that SPME-tITP was three times more effective at identifying proteins

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with mass-limited amounts (5 ng) of a Pyrococcus furiosus tryptic digest. The authors attribute the gains in sensitivity to lower noise levels with the porous sheathless CZEMS interface as illustrated by better signal-to-noise ratios of peptide precursor ions and associated higher XCorr values of identified peptides. Complementary peptide and protein identifications were found with larger sample loading quantities (100 ng). Fabrication of a SPME concentrator with particulate stationary phases usually requires a frit to hold the material in place [95-97], and its formation is time-consuming

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and irreproducible. Moreover, the frits can cause bubble formation or introduce unwanted dead volume during CZE. Alternatively, a sulfonate-silica hybrid strong cation-exchange (SCX) monolith

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has been synthesized at the proximal end of a CZE column and used for on-line SPME based sample preconcentration [98]. Sample was prepared in an acidic solution and loaded onto the SCX monolith and eluted using a basic buffer (ammonium bicarbonate, pH ~8.2). CZE separation was performed in 1 M acetic acid. This combination results in formation of a dynamic pH junction in the eluted fraction, which allows use of relatively

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large elution volume while maintaining peak efficiency and resolution. By loading 21 µL of a 1 x 10-7 M angiotensin II solution, an enrichment factor of 3,000 compared to direct

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CZE was achieved while retaining moderately efficient electrophoretic performance (N=44,000 plates). The loading capacity of the sulfonate SCX hybrid monolith was determined to be ~ 15 pmol. The system was also applied to the analysis of a 10-4 mg/mL bovine serum albumin (BSA) tryptic digest; the protein coverage was 12% and 11 peptides were identified. Finally, by loading 5.5 µL of a 10-3 mg/mL E. coli digest, 109

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proteins and 271 peptides were identified in a 20 min separation. To further improve the identification performance for complex biological sample, a sulfonate-silica hybrid SCX monolith was synthesized in a fused silica capillary and coupled to a LPA coated separation capillary through a zero-dead volume connection,

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as shown in Figure 5. A series of five pH bumps were applied to elute E. coli tryptic peptides from the SCX monolith, followed by analysis of each fraction using CZE coupled to an LTQ-Orbitrap Velos mass spectrometer with an electrokinetically pumped

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nanospray interface. 799 protein groups and 3,381 peptides were identified from 50 ng of the digest in a 2.5 h analysis [58]. We attribute the improved numbers of peptide and protein identifications to the efficient fractionation by the online pH gradient elution, which decreased the complexity of the sample in each elution step and improved the signal intensity of low abundance peptides. Similar numbers of protein and peptide identifications were obtained on a nanoUPLC with the same sample loading amount. The results showed complementary between the two methods. Moreover, the SCX monolith in the developed platform could be used as a microreactor for online digest

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which decreased the sample loss and the time required for sample preparation(from over 24 h to less than 40 min) [59]. 975 protein groups and 3749 peptides were identified from 50 ng of Xenopus protein by coupling the microreactor to a longer

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separation capillary (∼100 cm) and a Q Exactive HF mass spectrometer.

Figure 5. Schematic diagram of the SCX-SPE CZE-ESI-MS/MS system. Provisions are made to use N2 gas pressure to pump analyte and reagents through the monolith during its synthesis. The separation is driven by the difference between the separation voltage

voltage [58].

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and the spray voltage. The sheath liquid is electrokinetically driven by the electrospray

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More recently, to eliminate the zero dead volume union, a sulfonate-silica hybrid

SCX monolith and the LPA coating were prepared sequentially in a single fused silica capillary [99]. In this work, samples were prepared in 1 M acetic acid, loaded by pressure onto the SCX SPME monolith, and eluted using 200 mM ammonium bicarbonate (pH 8). The background electrolyte for CZE was 1 M acetic acid. A CZE autosampler was coupled to a LTQ-Orbitrap Velos mass spectrometer through an electrokinetically pumped nanospray interface. By loading 50 ng of Xenopus laevis egg protein digest, 330 protein groups and 872 peptides were identified. By loading 5.5 µL of

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a 10-3 mg/mL (5.5 ng) E. coli digest, 145 protein groups and 365 peptides were identified, which is much better than the previous results obtained from SCX SPE with an uncoated separation capillary [98].

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3.3. Optimization of MS parameters As in LC-MS [100-105], mass spectrometric parameters have significant effects on the identification performance of CZE-ESI-MS/MS method for bottom-up proteomics analysis. We recently investigated the effects of MS1 injection time, MS2 injection time,

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dynamic exclusion time, intensity threshold, and isolation width on the numbers of

peptide and protein identifications for single-shot CZE-MS/MS for bottom-up proteomics analysis of a Xenopus laevis tryptic digest [13]. An electrokinetically pumped nanospray

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CZE-MS interface was used to couple a LPA coated capillary to a Q Exactive HF mass spectrometer. A sensitive method that used a 1.4 Th isolation width, 60,000 MS2 resolution, 110 ms MS2 injection time, and a top 7 fragmentation produced the largest number of identifications when the CZE loading amount was less than 100 ng. A programmable autogain control method (pAGC) that used a 1.4 Th isolation width,

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15,000 MS2 resolution, 110 ms MS2 injection time, and top 10 fragmentation produced the largest number of identifications for CZE loading amounts greater than 100 ng; 7,218 unique peptides and 1,653 protein groups were identified from 200 ng by using the pAGC method, which outperforms the identification results obtained from

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nanoUPLC-ESI-MS/MS under its optimized conditions with same loading amount (6,025 unique peptides and 1,501 protein groups). However, more peptides (11,476) and protein groups (2,378) were identified by using nanoUPLC-ESI-MS/MS when the

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sample loading amount was increased to 2 µg. 3.4. Couple offline fractionation method to CZE-MS To date, samples of high complexity cannot be analyzed in great depth in a

single shot CZE-ESI-MS/MS analysis due to the relatively low column capacity of the capillary and the large dynamic range of the proteins in the sample. To overcome this limitation, a combination of two or more separating procedures is necessary to decrease the complexity of the sample and therefore improve the detecting performance for the low abundance proteins and increase the overall number of identifications. CZE-Proteomics – pg 21

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In an early study, we demonstrated the use of CZE with an electrokinetically pumped sheath-flow electrospray interface for the analysis of a tryptic digest of a sample of intermediate protein complexity, the secreted protein fraction of Mycobacterium marinum [9]. Eleven fractions were generated from 240 µg of the

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sample using RPLC and were analyzed by CZE-ESI-MS/MS with ammonium acetate (10 mM, pH 5.7) as background electrolyte and 50% (v/v) methanol, 50% (v/v) water, and 10 mM acetic acid as the electrospray sheath flow liquid, and an LTQ-Orbitrap

Velos mass spectrometer. 334 peptides corresponding to 140 proteins were identified in

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165 min of mass spectrometer time at 95% confidence (FDR < 0.15%). In comparison, 388 peptides corresponding to 134 proteins were identified in 180 min of mass

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spectrometer time by triplicate nanoUPLC-ESI-MS/MS analyses, each using 250 ng of the unfractionated peptide mixture. Overall, 62% of peptides identified in CZE-ESIMS/MS and 67% in nanoUPLC-ESI-MS/MS were unique. CZE-ESI-MS/MS favored basic and hydrophilic peptides with low molecular masses. Combining the two data sets increased the number of unique peptides by 53%. CZE-ESI-MS/MS is a useful tool for the analysis of proteome samples of intermediate complexity. However, the uncoated

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separation capillary and the relatively high pH of the background electrolyte (pH 5.7) led to high electroosmotic flow (EOF) and a short separation window. To improve the results, a 60 cm of LPA coated capillary was used for CZE-ESIMS/MS analysis of seven fractions from 350 µg of E. coli protein digest using a C18

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SPE column [55]. Each fraction was dried and redissovled in 50% v/v ACN and 0.05% v/v FA before CZE-ESI-MS/MS analysis. 0.1% v/v FA was used as CZE background

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electrolyte, resulting in sample stacking after injection. An electrokinetic sheath-flow electrospray interface was used to couple the LPA coated capillary with an OrbitrapVelos operating in higher-energy collisional dissociation mode. A total of 4,902 peptide IDs and 871 protein group IDs were identified in 350 min. The sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 µg) was roughly four fold less than previous studies. These results demonstrated that CZE can be a useful tool for the bottom-up analysis of prokaryote proteomes.

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The performance was further improved by using RPLC instead of a C18 SPE column for fractionation. 30 fractions from 600 µg of Xenopus protein tryptic digest using RPLC were analyzed by CZE-ESI-MS/MS separations (50 h total instrument time), Each of the RPLC fractions was redissolved in 20 µL of 35% v/v ACN and 65% v/v 0.1%

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v/v formic acid. The separation background electrolyte was 5% v/v acetic acid. 4134 proteins and 22535 peptides were identified totally. 15 fractions from 400 µg of Xenopus laevis digest were also analyzed by nanoUPLC-ESI-MS/MS separations (45 h total instrument time). 5,787 proteins and 36,848 peptides were identified in total. CZE-ESI-

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MS/MS produced 70% as many protein IDs and 60% as many peptide IDs as

nanoUPLC-MS/MS with similar instrument time (50 h versus 45 h), but with 50 times

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smaller total consumed sample amount (1.5 µg versus 75 µg). CZE generated peaks that were 25% more intense than nanoUPLC for peptides that were identified by both techniques, despite the 50-fold lower loading amount; this high sensitivity reflects the efficient ionization produced by the electrokinetically pumped nanospray CZE-MS interface. The numbers of protein and peptide identifications produced by CZE-ESIMS/MS approach those produced by nanoUPLC-ESI-MS/MS, but CZE employed nearly

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two orders of magnitude lower sample amounts [82].

A SCX-RPLC based two-dimensional platform was developed for sample fractionation before CZE-MS/MS analysis [64]. 40 fractions from 500 µg of mouse brain digest using SCX-RPLC were analyzed by CZE-MS/MS. Around 8,200 protein groups

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and 65,000 unique peptides from a mouse brain proteome digest in 70 h. The results are comparable with LC-MS/MS analysis of 30 fractions from one-dimensional high pH

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RPLC (8900 protein groups, 70,000 unique peptides). However, sample fractionation before CZE-ESI-MS/MS analysis results in very long analyses; the total separation time is the separation length of the second dimension multiplied by the number of the fractions from the first dimension. In addition, the multistep operations inevitably result in sample loss. Finally, the throughput is relatively low. 3.5. Multisegment sequential injection techniques To improve peptide identification rates in CZE-MS based bottom-up proteomics, Boley et al. developed a multisegment sequential injection technique [61]. This

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technique is based on the observation of the long dead time between injection of the sample and the appearance of the fastest migrating peptides at the mass spectrometer. By shortening the time between injections, a dramatic increase in the use of the mass spectrometer’s time is observed. The shortest time between injections (time interval)

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produces a doubling in the average number of identifications per minute (12.7 vs 6.6). However, there is also a 20% decrease in the total number of identifications at the

shortest injection period due to the limited response time of the mass spectrometer (1,476 vs 1,794).

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Subsequently, Faserl et al. demonstrated the potential of sequential injection of samples in CZE-MS for rapid and sensitive proteome characterization of human

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lymphoblastic T-cells (line CCRF–CEM) [62]. The sample digest was separated into twenty fractions by RPLC and thereafter analyzed by CZE-MS within a single MS analysis. A new fraction was injected into the CZE system every 10 min. Without any rinsing or equilibration steps, they generated a continuous stream of peptides feeding the mass analyzer. Roughly 28,000 peptides and 4,800 proteins were identified in 250 min. These numbers could be increased to 62,000 peptides and more than 6,100

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proteins identified by performing three experiments analyzing a total of 60 fractions, within 12.5 h. Moreover, they found that the electrophoretic mobility of peptides can be used to trace back peptides and assign them to the fraction they originate from.

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4. Applications

During last few years, reports for CZE-MS based bottom-up proteomics have

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mainly focus on improving the identification numbers and throughput. The technique is now seeing application to a range of biological problems, as summarized in Figure 6.

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Figure 6. Published applications of CZE-MS based bottom-up proteomics. 4.1. Phosphoproteomics

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The first report for the detection of tyrosine-phosphorylated peptides and the determination of sites of protein tyrosine phosphorylation by using CZE-MS based method was published in 1995 [47]. Amankwa et al. coupled an immobilized protein tyrosine phosphatase microreactor to either CZE or LC with a single-quadrupole mass

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spectrometer equipped with an ESI interface. The platform rapidly performed enzymatic dephosphorylation, separation, and detection of the reaction products. The technique

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was able to detect low picomole amounts of a tyrosine-phosphorylated peptide in a complex peptide mixture generated by proteolysis of a protein. Moreover, the general method should be adaptable to the characterization of different posttranslational modifications as long as a specific hydrolyzing enzyme is available. A similar idea was also used in diagonal CZE which is a form of two-dimensional CZE that employs identical separation modes in each dimension and incorporates an enzyme-based microreactor at the distal end of the first dimensional capillary [106]. Mou et al. coupled an immobilized alkaline phosphatase reactor based diagonal CZE system

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to a LTQ-Orbitrap Velos mass spectrometer with an electrokinetically driven sheath-flow electrospray interface [107]. The system was used to characterize the phosphorylation status of a tryptic digest of α-casein in a background prepared from a 22-fold excess of the tryptic digest of bovine serum albumin. Peptides undergo a preliminary separation in

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the first capillary. Fractions are parked in the reactor where some peptides undergo dephosphorylation. The fractions are then periodically transferred to the second capillary and replaced by the next peptides in the sample. Peptides that are not modified by the reactor will have identical mobility in both dimensions and fall on the diagonal of a

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reconstructed two-dimensional electropherogram, while analyte that undergoes

modification will fall off the diagonal. 120 fractions underwent automated treatment in

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the alkaline phosphatase reactor and separation in the second dimension capillary for over 40 min; nine phosphorylated α-casein peptides that produced 20 different phosphorylation states were detected with high confidence. The developed platform was also used for the accurate determination of this stoichiometry, which demonstrated a linear response across nearly 2 orders of magnitude [108].

Compared to nanoUPLC-ESI-MS/MS, CZE-ESI-MS/MS is attractive for

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phosphoproteome analysis due to the significantly different migration times of phosphorylated and unphosphorylated forms of peptides [109]. 2,313 phosphorylated peptides were identified from 1 µg of enriched phosphoproteome from the MCF-10A cell line digest with single-shot CZE-ESI-MS/MS in a 100 min analysis on Q Exactive mass

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spectrometer. When the sample loading amount was lowered to 200 ng, CZE-ESIMS/MS with sample stacking injection method consistently and significantly

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outperformed nanoUPLC-MS/MS in terms of phosphorylated peptide and total peptide IDs. With 1 µg loading amount via a dynamic pH junction method, 2,313 phosphorylated peptides were identified with single-shot CZE-MS/MS in a 100 min analysis. However, nanoUPLC generated more phosphorylated peptide IDs (3,313 phosphorylated peptides) than CZE when the sample loading amount was 2 µg. We recently coupled a LPA coated capillary prepared by SCARAFT polymerization method and dynamic pH junction based CZE to an Orbitrap Fusion Lumos Tribrid platform with APD algorithm. 4,405 phosphopeptides were identified from

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220 ng of enriched phosphopeptides from mouse brain, which represents the state-ofthe-art result for single shot CZE-ESI-MS/MS based phosphoproteome (unpublished data).

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The CZE separation performance for peptides with post-translational modifications (PTMs), such as phosphorylation, acetylation, methylation, and nitration, was seriously affected by the coating types. To investigate such effects, 70 synthetic peptides with various PTMs were analyzed on different types of capillaries. Faserl and co-workers found a bare-fused silica capillary was superior in the identification of multi-

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phosphorylated peptides (12 out of 15 were identified) [110]. In contrast, RPLC-MS revealed that multi-phosphorylated peptides interact with the RP material very poorly so

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that these peptides were either washed out or elute as very broad peaks from the nano column, which results in a reduced peptide identification rate (7 out of 15). As a proof of principle, 103 fractions of phosphopeptides enriched from PC-12 pheochromocytoma cells were analyzed by CZE-MS with a fused-silica capillary in 58.4 h. 5,686 phosphopeptides were identified and 4088 were quantified. Compared to RPLC-MS, less than one third of the phosphopeptides were identical, which demonstrates the

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benefit by combining different approaches quite impressively. However, the overall throughput for CZE-MS is relatively low, less than two phosphopeptides identified per minute. Of course, the results will be improved by using a higher speed mass

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spectrometer. 4.2. Glycoproteomics

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More than 50% of proteins in nature are glycosylated [111], and glycoproteomics has developed as an important subdiscipline of proteomics [112-114]. CZE has drawn great interest in glycoscience due to its outstanding abilities in resolving carbohydrates [115-117]. Alternative to traditional glycan peak identification approaches, such as migration time indexing, enzyme or lectin profiling, and matrix assisted laser desorption/ionization-MS [117], ESI-MS/MS technique provides high-throughput and comprehensive elucidation of glycan structures such as monosaccharide composition, branching topology, and stereoisomeric linkage of the monosaccharide sequence [118]. Driven by the progress in interface development, CZE-ESI-MS/MS is now becoming a

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useful tool in glycomics analysis [119-122]. However, characterization of released glycans from proteins suffers conceptually from a lack of information about the protein carriers and the specific glycan attachment site. Instead, it is valuable to analyze intact glycopeptides, which provides insight into the connectivity between the glycan

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composition and peptide backbone. Currently, RPLC-ESI-MS/MS was commonly

utilized for intact glycopeptide characterization, however, starting with the work in

Victòria Sanz-Nebot’s laboratory [123, 124], CZE-ESI-MS/MS has been presented as an alternative powerful approach.

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Khatri et al. developed a microfluidics-based CZE-MS system for analysis of released glycans, glycopeptides and monosaccharides for comprehensive glycoprotein

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analysis [125]. An aminoxy tandem mass tag regent was used to improve electrophoretic migration of neutral saccharides and to enable multiplexing in tandemMS experiments. In addition, a sialic acid derivatization method was used to reduce interaction of the negatively charged carboxyl groups with the positively charged capillary surface coating and to discriminate sialic acid linkages. Moreover, method used the same electrophoresis solutions for all three compound classes, greatly

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simplifying the task of glycoprotein characterization. Applications in glycoproteomics emphasized that higher sensitivities are produced compared to nano-LC-ESI-MS/MS [126]. Heemskerk and colleagues coupled

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a porous sheathless interfaced CZE-ESI-MS/MS system with sample concentration using tITP in neutral capillaries to improve the sensitivity in glycopeptide analysis [127]. The limit of detection (LOD) was estimated to be 20 amol for individual IgG1 N-

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glycopeptide with an improvement by a factor of 40 as compared to nano-LC-ESIMS/MS.

To enable the comprehensive characterization of glycan heterogeneity, we

reported the use of CZE-ESI-MS/MS with an electrokinetically-pumped nanospray interface for the sensitive characterization of site-specific protein glycosylation [128]. Roughly 3 amol mass detection limit was achieved for N-glycopeptide from IgG2, which is a 300-time improvement compared with the 1 fmol LOD obtained by nano-RPLC-ESIMS/MS. Although the concentration detection limits for CZE-ESI-MS/MS were three

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times higher than for RPLC-ESI-MS/MS methods, a 200-fold smaller injection volume was used, which reflects the outstanding ionization efficiency of the CZE-MS interface. To obtain a better ESI performance, a strategy that applies a coaxial gas flow

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around ESI emitter was investigated [129], with the observation of an overall enhanced detection sensitivity by improved desolvation and ionization efficiency. Kammeijer and colleagues connected a dopant enriched nitrogen-gas supply to the commercial

sheathless CZE-ESI-MS/MS system to perform high-sensitivity and high-repeatability glycopeptide analysis [126]. Using ACN as a dopant, up to 25-fold higher sensitivities

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for model N-glycopeptides were obtained compared to conventional sheathless CZE-

MS/MS (0.9 amol vs 1.7 fmol).

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ESI-MS/MS, producing a modest LOD improvement compared with nano-RPLC-ESI-

The increased molecular complexity of intact glycopeptides (conjugated with heterogeneous glycans and diverse peptides) in MS challenges deep site-specific glycosylation characterization. Separations of glycopeptides by using RPLC are mainly based on the peptide composition, while glycopeptide separations in CZE are

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dominated by the sialic acid composition of the glycan. We reported the first combination of these two techniques to achieve superior N-glycopeptide analysis [130]. Glycopeptides were first enriched on the hydrophilic interaction liquid chromatography SPE column. Four fractions of glycopeptides were collected from nanoUPLC and

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analyzed by CZE-ESI-MS/MS; representative base peak chromatograms are shown in Figure 7. This two dimensional workflow identified 268 N-glycopetides from alpha-1-acid glycoprotein, producing ∼35% more N-glycopeptides than direct nanoUPLC-ESI-

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MS/MS analysis and ∼70% more than direct CZE-ESI-MS/MS analysis. In addition to the complementary separation to nanoUPLC, the more intense peaks generated by CZE-ESI-MS/MS for weakly detected glycopeptide species also contributed to coverage improvement.

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Figure 7. Base peak chromatograms obtained for (A) deglycosylated alpha-1-acid glycoprotein

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(AGP) peptides and (B) intact AGP glycopeptides by nanoUPLC-ESI-MS/MS analysis. (C) Base peak electropherogram of fraction 1 AGP glycopeptides collected at 6.0-15.0 min from nanoUPLC separation by CZE-ESI-MS/MS analysis [130].

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The high resolution of CZE-ESI-MS/MS has enabled the advanced isomer separation and linkage analysis of glycosylated peptides. Kammeijer et al. reported the selective analysis of α 2,3- and α 2,6-sialylated glycopeptides by using a high-resolution

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separation platform based on CZE-MS [131]. These differentially linked glycopeptides showed an identical fragmentation pattern but different electrophoretic mobilities, allowing for baseline separation of the different linkages. An internal standard approach was applied for the analysis of highly complex and heterogeneous glycopeptides from prostate specific antigen (PSA) tryptic digest. This platform appears attractive for the identification of differentially linked sialic acids that may be related to pathological conditions. That group also established a high-performance PSA glycomics assay, combing PSA affinity purification, tryptic digestion, and peptide analysis with CZE-ESI-

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MS/MS, for differentiation of α 2,6- and α 2,3-sialylated isomers in patient urine PSA, the latter isomer being suggested to be a hallmark of aggressive types of cancer [132]. An average interday RSD of 14% for detected glycopeptides was achieved, indicating

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the low technical variation of employed CZE-ESI-MS/MS detection. As a major limitation, interpretation of glycan mass spectra is mostly done

manually, which is tedious and time-consuming. The lack of a comprehensive database and efficient tool for interpretation of glycopeptide or glycan tandem mass spectral data seriously hindered the development of CZE-MS based large-scale glycoproteomics.

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Fortunately, much work has been done to improve these limitations [133].

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4.3. Clinical diagnosis

Due to its fast separation speed and small sample volume, CZE-MS has been used for disease biomarker discovery in clinical proteomics [134-139]. Analysis of microbial mixtures in complex systems, such as clinical samples, using mass spectrometry can be challenging because the specimens may contain mixtures of pathogens and nonpathogens. Hu et al. applied CZE-selective MS/MS of peptide

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marker ions to the identification of common pathogens in clinical diagnosis [140]. They identified peptides that matched a protein associated with a particular pathogen that were selected as marker ions to identify that bacterium in clinical specimens. Various clinical specimens were analyzed using both biochemical and selective MS/ MS

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methods that showed good matching rate. By taking advantage of the fact that the number of basic and neutral polar amino acids of biomarkers sequences distinctly

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correlates with their CZE-MS migration time/molecular weight coordinates, Zürbig et al demonstrated that sequence analysis of potential biomarkers by CZE-MS via MS/MS is platform-independent [141]. Good and co-workers built a human urinary peptidome database including a panel of 273 peptides that are differentially regulated in the urine of patients with chronic kidney disease (CKD) compared to healthy controls by using CZE-MS platform (The CKD273 classifier) [142]. The database aims to serve as a universal platform for definition and validation of biomarkers for a variety of diseases and (patho)-physiological changes. Zürbig evaluated CZE-MS based urinary peptidome analysis as a tool for prediction of diabetic nephropathy (DN) and found it enables

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noninvasive assessment of DN risk at an early stage via determination of specific collagen fragments [143-145]. Belczacka et al. identified a general urinary marker pattern for detection of solid tumors by targeting common systemic events associated with tumor-related inflammation [146]. 193 peptides including mucins, fibrinogen, and

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collagen fragments that differing at statistically significant levels between cases and controls were selected and combined to a multi-dimensional marker pattern using

support vector machine algorithms. Currently, most of the published reports on clinical diagnosis by using CZE-MS are still for research purpose only. The clinical application

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of CZE-MS still has many challenges, However, many key forward steps have been made in the past few years [147].

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4.4. CHO host cell protein analysis

CZE-MS was used for absolute quantification of host cell proteins (HCPs) in recombinant human monoclonal antibodies [148]. An electrokinetically pumped nanoelectrospray interface was used to couple CZE with an LTQ-Orbitrap Velos instrument (Thermo Fisher Scientific). The heavy-labeled peptides were spiked in the

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HCP digests at known concentrations. After CZE-ESI-MS/MS analysis, the absolute amounts of the three proteins present were determined to be at the picomole level in a 20 µg sample of digested HCPs.

We also compared the identification performance of different methods for

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analysis of HCP impurities in a recombinant mAb [149]. CZE-MS/MS was able to identify more protein groups (220 vs 34) and peptides (976 vs 53) from depleted HCP

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digest compared to LC-MS/MS with same limited sample loading amount (~50 ng). However, 290 protein groups and 1,011 peptides could be identified by LC-MS/MS when the sample loading amount was increased to 1 µg. Moreover, the HCP digest without depletion was also analyzed directly by CZE-MS/MS and 185 protein groups and 709 peptides were identified. These results were improved by using an on-line SCXSPE-CZE-ESI-MS/MS system with pH gradient elution, which resulted in identification of 230 protein groups and 796 peptides.

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4.5. Ultrasensitive proteomics The behavior of a single cell cannot always be reliably deduced from the average behavior of cells in a population [150]. The average of cellular responses, as generally

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generated in current proteomics experiments, can dilute responses or markers produced by rare, but important cells. Ultrasensitive proteomics analysis to generate the proteome of single cells is of increasing importance [151, 152]. As a result, the high sensitivity of CZE-MS should be of great value in single cell analysis [153]. An

ultrasensitive CZE-MS system based on an improved electro kinetic driven sheath liquid

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interface has been developed [154]. Over 100 proteins were identified from 16 pg digests with an accurate mass and time tags strategy. Blastomeres isolated from 16-cell

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Xenopus laevis embryo was used as a model for single cell proteomics by using CZEMS [60]. This experiment coupled CZE to a tribrid ultrahigh-resolution mass spectrometer (HRMS) to enable discovery proteomics with a ~25 amol mass detection limit. CZE-HRMS enabled the identification of 500-800 nonredundant protein groups from a 20 ng sample. Based on the quantification result from ~150 nonredundant protein groups between all blastomeres and replicate measurements, significant

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translational cell heterogeneity along multiple axes of the embryo were found. To enhance performance, fractions were generated using a RP column (ZipTip) before ultrasensitive CZE-HRMS to enhance identifications from protein digest amounts that approximate to a few mammalian neurons [155]. RP fractionation minimized co-isolation

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spectral interferences during MS/MS, and increased the temporal rate of peptide identification by up to ~57%. The approach was scalable to 500 pg of protein digest (~a

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single neuron), identifying 141 protein groups on average per analysis. However, the offline fractionation method could results in serious sample loss, especially for mass limited sample. Online fractionation method could overcome this limitation [58, 98]. 4.6. Quantitative Proteomics Li et al. demonstrated the use of CZE with an electrokinetic sheath-flow electrospray interface coupled to a triple-quadrupole mass spectrometer for the accurate and precise quantification of Leu-enkephalin in a complex mixture using multiple-reaction monitoring (MRM) [156]. The limit of detection was 60 pM,

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corresponding to the injection of 335 zmol of peptide, which is a 10-20-fold improvement in mass sensitivity than that obtained by nanoUPLC-MRM. Further quantification was performed in the presence of stable-isotope-labeled the peptides and the concentration

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detection limit was 240 pM. Wang et al. demonstrated the direct coupling of transient cITP-CZE with a highsensitivity triple quadrupole mass spectrometer operating in selected reaction

monitoring (SRM) mode for sample quantitation [157]. A linear dynamic range spanning four orders of magnitude was observed. An average signal-to-noise ratio of 50 was

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observed for 50 amol of targeted peptide in the presence of a complex and much more abundant BSA digest. Correlation of variation (CV) of <10% for peak area was

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measured from triplicate sample analyses at 50 pM peptide concentration, showing good reproducibility of this online cITP-CZE-SRM MS platform, and with limit of quantitation (LOQ) demonstrated to be well below 50 pM. To increase the sample loading volume, a large inner diameter separation capillary was used [158]. A linear dynamic range spanning 4.5 orders of magnitude and a 10 pM LOQ with measurement reproducibility of the CV < 22% were obtained experimentally for both targeted peptides.

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By using a new sheathless cITP/CZE-MS interface, which is based on a commercially available capillary with an integrated metal-coated ESI emitter, a LOQ below 5 attomole can be achieved [159].

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For large scale quantitative proteomics using CZE-MS, Faserl et al. combined RPLC fractionation with CZE-ESI-MS/MS for analysis of a digest mixture of stable isotope labeling by amino acids in cell culture labeled and an unlabeled yeast strain

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[160]. 182 RPLC fractions were collected from 1.4 mg of digested yeast proteins. A total of 28,538 peptides that correspond to 3,272 proteins were quantified based on CZEESI-MS/MS analysis. Moreover, 1,371 phosphopeptides were quantified without further enrichment strategies due to the ultralow flow rate of the interface (~10 nL/min) used in this work, which reduces ion suppression and improves sensitivity. However, the low flow rate also results in low throughput. Without considering the time required for RPLC fractionation, the total CZE-MS analysis time was >184 h (for each fraction, including 55 s for injection and 60 min for separation).

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tagging is a powerful tool for quantitative proteomics. As one example, CZE-MS was applied to iTRAQ-labeled PC12 cells that had been induced to differentiate [161]; the labeled sample was fractionated by RPLC and analyzed by CZE-MS. 835 proteins

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and 2,329 peptides were identified. 5. Conclusions and future prospects

The commercialization of a robust and ultra-sensitive CZE-MS interface will lead to increasing applications of CZE-MS not only for bottom-up proteomics but also in

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other fields. These applications of CZE-MS depend on improvements in mass

spectrometer performance, such as the scan rate, mass accuracy, and resolution. Currently, a high-quality art mass spectrometer can collect ~60 MS/MS spectra in one

single shot CZE-MS analysis.

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second, which provides potential for faster and deeper proteomics analysis by using

The state-of-the-art single shot CZE-MS studies is within a factor of two of LCMS system. Due to the inherent limitation of the relatively low injection capacity of CZE, it will necessary to combine other techniques for sample preseparation and

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preconcentration to improve the identification numbers for CZE-MS based bottom-up proteomics. Moreover, combination of CZE-MS and LC-MS could achieve deeper proteome mapping of the biological sample due to their good complementary. Due to the unspecific interactions between the PTM groups and the stationary

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phases in LC, CZE-MS will widely use for the identification and characterization of various PTMs in proteins, such as phosphorylation, glycosylation, acetylation, etc. CZE

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is particularly powerful for characterization of those PTMs that change the charge of a peptide, due to the dramatic change in mobility that accompanies that change in charge. As an example, reference 130 reported the detection of tetrasialylated glycans, which are very difficult to characterize by RPLC. We focus this review on bottom-up proteomics. CZE-MS may have an even brighter future for top-down proteomics. In a recent report, Sun and colleagues used chromatographic prefractionation followed by CZE-MS for the analysis of intact proteins from E. coli; over 5,700 proteoforms were identified [161].

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Acknowledgments This paper is inspired by Hanfa Zou, who was an outstanding scientist and mentor.

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We also thank Drs. William Boggess and Matthew Champion in the Notre Dame Mass Spectrometry and Proteomics Facility for their help with this project. This work was funded by the National Institutes of Health (Grant R01GM096767).

The electrokinetically pumped nanospray CZE-MS interface technology has been

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licensed to CMP Scientific.

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