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Array-Based Online Two Dimensional Liquid Chromatography System Applied to Effective Depletion of High-Abundance Proteins in Human Plasma Zhi Huang, Guoquan Yan, Mingxia Gao, and Xiangmin Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04553 • Publication Date (Web): 19 Jan 2016 Downloaded from http://pubs.acs.org on January 20, 2016

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Analytical Chemistry

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Array-Based Online Two Dimensional Liquid Chromatography System Applied to Effective Depletion of High-Abundance Proteins in Human Plasma

9 Zhi Huang,1,2 Guoquan Yan,1,2 Mingxia Gao,*1,2 Xiangmin Zhang*1,2

10 11 12 13

1

Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China 2

Department of Chemistry, Fudan University, Shanghai 200433, China;

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ACS Paragon Plus Environment


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Abstract:

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In this work, an array-based online two-dimensional liquid chromatography

3

(2D-LC) system was constructed for protein separation and effective depletion of

4

high-abundance proteins in human plasma. This system employed a strong anion

5

exchange (SAX) column in the first dimension and eight reversed-phase liquid

6

chromatographic (RPLC) columns in the second dimension. All the protein

7

components in the first dimension were enriched on the trapping columns,

8

simultaneously back-flushed and concurrently separated in the second dimension. LC

9

eluents were then collected on 96-well plates for further analysis. Compared with

10

common 2D-LC system, this system showed an eight-fold increase in throughput and

11

convenient utilization of stop-flow mode for sample separation. The RSD of retention

12

time and peak area were separately below 0.51 % and 8 %. Recovery rates of four

13

standard proteins were all above 95%. This array-based 2D-LC system was

14

subsequently applied to the analysis of proteins in human plasma. The eluents

15

containing high-abundance proteins were rapidly located according to the results of

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bicinchoninic acid (BCA) assay. In all, with the effective depletion of 84

17

high-abundance proteins, a total of 1332 proteins were identified through our system.

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The dynamic range of the identified protein concentrations covered 9 orders of

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magnitude, ranging from 10 g/L level for HSA down to 0.01 ng/mL level for the

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low-abundance proteins.

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Introduction

3

Human plasma is one of the most favorite sources for proteomic studies in

4

various diseases, since it not only contains a wealth of biomarkers but also possesses

5

the merits of low cost and convenient sample processing.1 However, human plasma is

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a challenging sample to be analyzed due to the sheer complexity of the plasma

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proteome and the huge dynamic range of protein concentrations (over 10 orders of

8

magnitude).2 To date, many disease biomarkers are predicted to be present at a level

9

of ng/mL or lower.3-6 Although the dynamic range of mass spectrometry (MS) is up to

10

4-6 orders of magnitude,7 the signals of high-abundance proteins largely suppress the

11

signals of low-abundance proteins due to the masking effect in complex samples.

12

Therefore, to reduce the sample complexity and make the identification of

13

low-abundance proteins tenable, it is essential to deplete high-abundance proteins

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prior to the biomarker discovery in human plasma.8-10

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Nowadays, immune-affinity method is one of the most popular strategies for

16

depletion of high-abundance proteins.11-13 However, most high-abundance proteins act

17

as carrier proteins for low-abundance proteins. Thus, the low-abundance proteins of

18

interest may also be simultaneously removed due to their interactions with

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high-abundance proteins, which would result in the loss of potential biomarkers.14

20

Moreover, the immune-affinity method can only deplete a limited number of known

21

proteins. Besides, based on animal immunization, the immune-affinity method may 3



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easily cause batch-to-batch variability.15

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Multidimensional liquid chromatography (MDLC), combining two or more

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separation modes together, is another effective and widely used strategy in proteomic

4

research.16-19 Previously, our group developed an offline anion exchange

5

chromatography/ reversed phase liquid chromatography (AEC/ RPLC) system for the

6

analysis of human liver proteome, with which dozens of high-abundance proteins

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were depleted.18,19

8

However, offline MDLC is often labor-intensive, time-consuming and tends to

9

introduce contamination. In contrast, online MDLC has the advantages of automation

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and easier sample processing.20,21 The most popular online MDLC approach is

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multidimensional protein identification technology (MudPIT), which utilizes

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multidimensional chromatography online coupled with mass spectrometry.22,23 This

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approach subjects peptides to the MDLC separation and MS analysis, leading to an

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increase in proteome coverage.24 On the other hand, to reduce the complexity,

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separation at protein level may be an alternative strategy.25-27 More recently, a MDLC

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strategy utilized for the protein separation achieved an improvement in resolving

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power.27

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In common online MDLC, with a single column placed in the second dimension,

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series operation mode is therefore adopted in the second dimensional separation,

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which results in the reduction of throughput for separation of complex samples.28

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Several approaches such as shorter columns29 or highly permeable monolithic 4


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columns30 have been employed in the second dimension to alleviate this problem. An

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array of columns integrated in the second dimension may be a better choice on

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account of that multiple fractions could be simultaneously separated in the second

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dimension, leading to a remarkable increase in sample throughput. In 2006, our group

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successfully constructed an online capillary column array-based two dimensional

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liquid chromatography (2D-LC) system for the analysis of proteolytic peptides.31

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In this work, to build an effective strategy for the high throughput separation of

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plasma components at protein level, we constructed an online 2D-LC system with

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eight RPLC conventional columns integrated in the second dimension for parallel

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separation. With this improved configuration, the online system allowed a concurrent

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gradient elution of the first dimensional fractions, thereby providing an increase in

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separation throughput. The conventional column configuration instead of capillary

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columns also increased the sample loading capacity. The effectiveness of the

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array-based system for high-throughput and effective depletion of high-abundance

15

proteins was demonstrated by analyzing human plasma sample.

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Experimental Section

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Chemicals and materials.

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HPLC grade acetonitrile (ACN) was purchased from Merck (Germany).

20

Trifluoroacetic acid (TFA), TPCK treated trypsin, cytochrome c (Cyto C), lysozyme,

21

bovine serum albumin (BSA), myoglobin and Tris were from Sigma (St. Louis, USA). 5



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Water was acquired by a Milli-Q system (Millipore, USA). Human plasma was from

2

the Zhongshan Hospital (Shanghai, China). Bicinchoninic acid (BCA) concentration

3

kit was obtained from Beyotime Institute of Biotechnology (Haimen, China). 96-well

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plates (polystyrene, flat-bottomed, cat. no. 3599) were from Corning Corporation

5

(New York, USA).

6 7

System assembly and operation.

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The scheme of online array-based two-dimensional liquid chromatography

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system is presented in Fig. 1. In this system, a binary gradient pump (Pump A) system

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(Wufeng Corp., Shanghai, China) was used for the first dimensional separation and a

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quaternary gradient pump (Pump B) system (Techcomp Corp., Shanghai, China) was

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used for the second dimensional separation. A ten-port electrically actuated

13

multi-position valve (Valco Instruments Co., Inc., USA) was used to control the

14

transfer of the first dimensional fractions onto the trapping columns. A home-made

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fraction collector (Anxuan Corp., Shanghai, China) was used to collect the second

16

dimensional eluents for further analysis. Two six-port two-position valves (Rohnert

17

Park, USA), eight three–way micro-splitter valves and an eight-channel flow splitter

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(Valco Instruments Co., Inc., USA) were also used in this system.

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During the first dimensional separation, Port 1 of valve B was connected to Port 6

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(denoted as position A). By switching the multi-position valve, the first dimensional

21

fractions were loaded onto the trapping columns while the mobile phase solution from 6


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pump A was directly drained to the waste through the flow splitter. During the second

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dimensional array-based separation, Port 1 of valve B was connected to Port 2, and

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valve C was turned to the blocking plug (denoted as position B). The mobile phase

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from pump B was split equally by flow splitter. Components on the trapping columns

5

were simultaneously back-flushed and concurrently separated on the array-based

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columns. The second dimensional eluents were continuously collected onto the

7

96-well plates for further analysis.

8 9

Analytical conditions for human plasma.

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In the first dimension, a ProPac™ SAX-10 column (10 µm, non-porous, 250 mm

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× 4.0 mm i.d.) from Dionex (USA) with the corresponding guard column (50 mm ×

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4.0 mm i.d.) was integrated. Human plasma was diluted seven-fold with mobile phase

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A. 100 µL plasma solution (1 mg proteins) was used for the separation. Gradient

14

elution was performed with mobile phase A (10 mM Tris-HCl, pH 8.0) and mobile

15

phase B (10 mM Tris-HCl, 500 mM NaCl, pH 8.0). The flow rate was 0.5 mL/min

16

and the UV detection was carried out at 215 nm. The gradient was set as follows: 0

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min, 100% A; 5 min, 100% A; 15 min, 10% B; 45 min, 25% B; 70 min, 40% B; 90

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min, 67% B; 91 min, 100% B; 101 min, 100% B; 102 min, 100% A; 130 min, 100%

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A. The time program for the fraction transfer was set as follows: 0-18 min, Fraction A;

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18-40 min, Fraction C; 40-58 min, Fraction A; 58-64 min, Fraction D; 64-68 min,

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Fraction E; 68-72 min, Fraction F; 72-78 min, Fraction G; 78-85 min, Fraction H; 7



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85-120 min, Fraction B.

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In the second dimension, the solvent system was composed of mobile phase A (95%

3

H2O, 5% ACN, 0.1% TFA) and mobile phase B (5% H2O, 95% ACN, 0.1% TFA).

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Eight Xtimate™ C8 columns (5 µm, 30 nm, 250 mm × 2.1 mm i.d., Welch Materials,

5

Inc., China) were integrated as the separation columns and eight guard columns (10

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mm × 2.1 mm i.d.) were used as the trapping columns. The flow rate of pump was 1.6

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mL/min and the split flow rate of column was 0.2 mL/min. The eluents were collected

8

every minute. The gradient was performed as follows: 0 min, 100% A; 5 min, 100% A;

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15 min, 25% B; 25 min, 33% B; 60 min, 40% B; 80 min, 54% B; 85 min, 67% B;

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85.1 min, 100% B; 95 min, 100% B; 95.1 min, 100% A; 110 min, 100% A.

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The BCA concentration assay was conducted according to the manufacturer's

12

instructions.32 BCA reagent and collected eluent were mixed for subsequent

13

measurement. The protein content in each well was obtained according to the results

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of BCA concentration assay. The wells with high protein content were classified as

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high-abundance protein (HAP) fractions and the rest were classified as

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low-abundance protein (LAP) fractions. All the fractions were lyophilized and then

17

reconstituted with 25 mM NH4HCO3 for the digestion. The detailed description of

18

digestion was depicted in the Supporting Information.

19 20 21

Protein depletion by the Removal Kit and in-gel digestion of HAPs. ProteoExtract Albumin/IgG Removal Kit (CalBiochem, CA, USA) was used 8


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according to the manufacturer’s instructions. Briefly, plasma solution was firstly

2

loaded onto the column. The column was then washed twice to remove

3

nonspecifically bound proteins. Afterwards, the elution buffer was used to elute the

4

bound proteins. Bound fractions were pooled for further analysis. The SDS-PAGE and

5

in-gel digestion procedures were modified according to Shevchenko’s protocol.33 The

6

detailed process was shown in the Supporting Information.

7 8

Nano-LC-MS/MS analysis and Data analysis

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Nano-LC-MS/MS analysis was performed on a nano Acquity UPLC system

10

(Waters Corporation, USA) coupled with a LTQ Orbitrap XL mass spectrometer

11

(Thermo Scientific, Germany). Detailed LC-MS/MS data analysis descriptions were

12

displayed in the Supporting Information.

13 14

Data analysis

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The Mascot Daemon software (Version 2.3.0, Matrix Science, London, UK) was

16

used for searching of the mass spectra. Trypsin was selected as the proteolytic enzyme

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and up to two missed cleavages were allowed. Carbamidomethylation of cysteines

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was set as a fixed modification and oxidation on methionine was set as a variable

19

modification. The peptide mass tolerance was 10 ppm and the fragment ion tolerance

20

was 0.8 Da. The global FDR was set at 1%. The proteins with greater than 95%

21

confidence and at least one peptide matched were accepted. Among the proteins with 9



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only one peptide matched, only those proteins with one unique peptide matched were

2

included. More data analysis descriptions were presented in the Supporting

3

Information.

4 5

Results and Discussion

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The array-based 2D-LC system.

7

In this work, as depicted in Fig. 1 and Fig. 2, we constructed a high-throughput

8

online array-based 2D-LC system to address the increasing demand for improved

9

separation at protein level. In this system, the first dimension was a SAX column and

10

the second dimension was an array of eight RPLC columns. Each fraction separated

11

through the first dimensional column was collected and enriched on the trapping

12

columns. Components on the trapping columns were then simultaneously

13

back-flushed and concurrently separated in the second dimension column array. The

14

two dimensional separations were independent, which could offer an improvement in

15

peak capacity.

10


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Figure 1. Schematic diagram of the online array-based 2D-LC system. 1: 1st HPLC

3

pump, 2 (valve A): six-port two-position injection valve, 3: SAX column, 4: UV

4

detector, 5 (valve C): ten-port electrically actuated multi-position valve, 6: three–way

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micro-splitter valve, 7: trapping column array, 8: eight-channel flow splitter, 9: 2nd

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HPLC pump, 10 (valve B): six-port two-position valve, 11: RPLC column array, 12:

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micro-fraction collector. For valve A, the bold lines represented sample injection

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status and the dotted lines represented sample load status. For valve B and valve C,

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the bold lines represented 1st SAX separation and the dotted lines represented 2nd RP

10

separation.

11

11



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Figure 2. Photograph of the online array-based 2D-LC system.

3 4

The columns utilized in this system were all conventional columns, which allowed

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a remarkable increase in the injection amount comparing with the capillary

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array-based 2D-LC system developed in our previous work.31 Moreover, the

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implement of array-based columns in the second dimension could conduct a

8

concurrent gradient elution of the first dimensional fractions. This could bring a clear

9

benefit of increase in system throughput. Another advantage was the convenient

10

utilization of stop-flow mode for sample separation. For instance, if 16 first

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dimensional fractions were adopted for sample separation by our system (Fig. S1, S2),

12

the 2D-LC separation was performed for the first eight fractions as described in

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experimental section. Afterwards, all the valves were switched to position A for the

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separation of next eight fractions. The process was analogously repeated to complete

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the separation of all the fractions. 12


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Column selection of the array-based 2D-LC system.

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The column used in the first dimension was a SAX silica-based column with a

4

high loading capacity and the pH value of mobile phase was close to the physiological

5

pH of human plasma, thus avoiding plasma constitutions precipitation.34,35 The

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loading capacity of the trapping column was about 250 µg (Fig. S3), which was

7

sufficient for the trapping of the first dimensional fractions.

8

For the second dimension, the chromatographic conditions of eight RPLC

9

analytical columns and trapping columns were carefully selected. The RSD of

10

pressure was 2 % (Table S1). The optimal flow rate for the C8 columns (2.1 mm i.d.)

11

was 0.2 mL/min. Thus the volume of collected solution at 60-s interval was 200 µL,

12

below the upper limit of well volume of 96-well plate. In addition, the smaller inner

13

diameter column could reduce sample dilution effect, thus enhancing the

14

identification performance for low-abundance proteins.

15 16

Reproducibility and recovery evaluation of the array-based 2D-LC system.

17

The chromatographic performance was evaluated to verify the effectiveness of

18

the system. The same plasma sample was separated by the online system for 3

19

consecutive runs (Fig. S4, S5). For 8 randomly selected peaks, the RSD of the

20

retention time was less than 0.51 % for both dimensions (Table S2) and the RSD of

21

the peak area was less than 8 % (Table S3). 13



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Four standard proteins (Cyto C, lysozyme, BSA and myoglobin) were used for

2

the recovery evaluation. These proteins were selected to cover a wide range of

3

molecular weight (12.4−67 kDa), pI (4.98−11.0) and polarity. After 2D-LC separation

4

of standard proteins (15 µg), the recoveries were above 95% (Table S4) and even

5

when the amount of standard proteins was as low as 90 ng the recoveries were still

6

above 92%. These results revealed a satisfactory recovery of this system for protein

7

separation. Human plasma sample was further used to assess the performance of this

8

array-based system. Almost identical separation profiles were obtained using 2D-LC

9

separation as compared to the direct injection analysis (Fig. S6).

10 11

Method development for depletion of HAPs in human plasma

12

Next, this array-based 2D-LC platform was applied for the depletion of HAPs in

13

human plasma. Eight SAX fractions were transferred to the second dimensional

14

separation. Fig. 3 showed the first dimensional separation profile of the human plasma.

15

A salt gradient was carefully optimized and a 130-min gradient was finally adopted.

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In order to reduce sample complexity, peaks with high UV intensities were classified

17

as Fractions C to H and the rest were classified as Fractions A and B. The second

18

dimensional concurrent separation profile was shown in Fig. 4A. Several peaks with

19

very high UV absorbance imply the existence of the high-abundance proteins. The

20

magnified version of the second dimensional separation profile was shown in Fig. 4B.

21

A wealth of peaks with moderate and low UV intensity also emerged, demonstrating 14


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that this system had both high peak capacity and resolving power.

2 3

Figure 3. Chromatogram of human plasma by SAX separation. Symbols A-H

4

represented the number of fractions injected into the corresponding 2nd dimension.

5

The gray line represented the change of mobile phase B in the elution gradient.

6

7 8

Figure 4. (A) The array-based RP diagram of the first dimensional SAX fractions. (B)

9

Magnified version of array-based RP diagram of 8 SAX fractions. Eight C8 columns 15



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1

were adopted for the second dimension separation.

2 3

Validation for the depletion of HAPs in human plasma.

4

UV detector routinely used in liquid chromatography is single channel.19 It is not

5

practical to utilize eight UV detectors in the second dimension of this system. In this

6

work, the strategy employed for the rapid location of high-abundance proteins was the

7

BCA concentration assay. The linear coefficient correlation of the BCA standard

8

curve for BSA dissolved in RPLC mobile phase solution was 0.996 (Fig. S7),

9

indicating that this strategy was suitable to rapidly quantitate the protein content in

10

each well. During the array-based RPLC separation, ten 96-well plates were used to

11

collect the eluents every one minute. Afterwards, all the plates proceeded BCA

12

concentration assay. Fig. 5 showed a representative photograph of the fifth 96-well

13

plate in BCA concentration assay and the depth of color represented the amount of

14

proteins in the solution.

15

16


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Figure 5. A typical photograph of the fifth 96-well plate in BCA concentration assay.

3

During the array-based RPLC separation, the 96-well plates were used to collect the

4

eluents every one minute. This plate collected the eluents eluted from 49 to 60 minute.

5

Afterwards, all the plates proceeded BCA concentration assay. The depth of color

6

represented the amount of proteins in the solution.

7 8

After the 2D-LC separation of human plasma, the wells with higher absorbance

9

value were classified as HAP fractions while the rest were classified as LAP fractions.

10

In order to further validate the effectiveness, all the HAP fractions were subsequently

11

identified by LC-MS/MS analysis. A total of 169 non-redundant proteins were

12

identified and 84 of which (Table S5) were reported to be high-abundance proteins

13

according to Hortin et al.,36 which confirmed the availability of BCA concentration

14

assay for the rapid location of HAPs. Afterwards, to further evaluate the co-elution of

15

low-abundance proteins in HAP fractions, several HAP wells and the elution solution 17



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of ProteoExtract Albumin/IgG Removal Kit were compared by SDS-PAGE analysis

2

(Fig. 6A). To alleviate the masking effect in LC/MS analysis, HAP and LAP slices

3

were excised and in-gel digested separately. For the elution of Albumin/IgG Removal

4

Kit, seven high-abundance proteins, other than HSA and IgG, were identified.

5

Meanwhile, 32 low-abundance proteins were also identified, most of which belonged

6

to the list of proteins interacting with HSA.14 For the HAP wells, the number of

7

identified HAPs in wells 5E-7, 5F-7, and 5G-8 were 2, 3 and 4, respectively.

8

Moreover, the number of LAPs identified in wells 5E-7, 5F-7, and 5G-8 were 9, 11

9

and 11, respectively. Almost all identified LAPs were not in the list of proteins

10

interacting with HSA. The weak co-elution may result from the close retention time

11

between high-abundance proteins and low-abundance proteins. The results also

12

proved that our HAP depletion strategy could achieve a low degree of LAP co-elution

13

compared with Albumin/IgG Removal Kit.

14

15 16

Figure 6. (A) SDS-PAGE image of the elution of ProteoExtract Albumin/IgG

17

Removal Kit and 2D-LC eluent solution. M: marker; Lane 1 correlates to elution of 18


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ProteoExtract Albumin/IgG Removal Kit; Lane 2 correlates to 5E-7; Lane 3 correlates

2

to 5F-7; Lane 4 correlates to 5G-8 in Fig. 5. (B) The protein identification results

3

identified by in-gel digestion analysis.

4 5

Performance evaluation for the proteomic analysis of human plasma.

6

An offline 2D-LC system developed previously for the depletion of

7

high-abundance protein needed a very long separation time19, which was not practical

8

in routine research. In this work, by integrating the array-based separation in the

9

second dimension, the total separation time was decreased to 4 h, which was

10

comparable to the analysis time of immune-affinity method. Therefore, this

11

array-based system is feasible in high-throughput proteomic research of complex

12

samples

13

The proteins in all fractions were finally identified in duplicate by LC-MS/MS

14

analysis. A total of 1332 non-redundant proteins (Table S6) were identified, which is

15

comparable to the previous results.37 Moreover, several proteins with extremely low

16

concentrations were also identified. For example, C-X-C motif chemokine 9

17

(Q07325), androgen receptor (P10275) and Inosine-5'-monophosphate dehydrogenase

18

2 (P12268) were reported at 0.01 ng/mL level in plasma.38 HSA was reported to be at

19

41 g/L in human plasma,36 which meant that the dynamic range of identified protein

20

concentrations covered 9 orders of magnitude.

21 22

Conclusions 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

In this work, we presented a strategy for effective depletion of high-abundance

2

proteins in human plasma by using an array-based 2D-LC system. With a dramatic

3

reduction in the total separation time, high resolution and good reproducibility were

4

still achieved. This system was successfully applied to the analysis of human plasma.

5

A total of 84 high-abundance proteins were depleted and the dynamic range of

6

identified protein concentrations covered 9 orders of magnitude. Therefore, this

7

array-based 2D-LC strategy has great prospect of high-throughput separation of

8

complex samples in proteomic research.

9 10

Supporting information available

11

Details of experimental procedures and additional data of performance evaluation

12

of this array-based 2D-LC system is available free of charge via the Internet at

13

http://pubs.acs.org.

14 15

Corresponding Authors

16

* Xiangmin Zhang, E-mail: [email protected]. Tel: +86-21-65641740. Fax:

17

+86-21-65641740.

18

* Mingxia Gao, E-mail: [email protected]. Tel: +86-21-65643983. Fax:

19

+86-21-65643983.

20 21

Notes 20


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1

The authors declare no competing financial conflict.

2 3

Acknowledgments

4

This work was supported by the National High-Tech R&D Program (No.

5

2012AA020202), National Basic Research Program (No. 2012CB910604) from the

6

State Ministry of Science and Technology of China, and the National Natural Science

7

Foundation Programs (No. 21175026, 21275034 and 21475027).

8 9

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