Glycan Moieties as Bait to Fish Plasma Membrane Proteins

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Glycan Moieties as Bait to Fish Plasma Membrane Proteins Fei Fang, Qun Zhao, Zhigang Sui, Yu Liang, Hao Jiang, Kaiguang Yang, Zhen Liang, Lihua Zhang, and YuKui Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01082 • Publication Date (Web): 18 Apr 2016 Downloaded from http://pubs.acs.org on April 27, 2016

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Analytical Chemistry

Glycan Moieties as Bait to Fish Plasma Membrane Proteins

1 2 3 4

Fei Fang1,2†, Qun Zhao1†, Zhigang Sui1, Yu Liang1, Hao Jiang1,2, Kaiguang Yang1,

5

Zhen Liang1, Lihua Zhang*1 and Yukui Zhang1

6 7 8

1

9

Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of

National Chromatographic R. & A. Center, Key Laboratory of Separation Science for

10

Science, Dalian 116023, China

11

2

University of Chinese Academy of Sciences, Beijing 100039, China

12

†

These authors contributed equally to this study

13

*To whom correspondence should be addressed. E-mail: [email protected].

14

Phone & fax: +86-411-84379720.

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ACS Paragon Plus Environment


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1

Abstract

2

Plasma membrane proteome analysis is of significance for screening candidate

3

biomarkers and drug targets. However, due to their low abundance and lack of

4

specific groups that can enable their capture, the plasma membrane proteins (PMPs)

5

are under-represented. Based on the fact that PMPs are embedded in or anchored to

6

the phospholipid bilayer of the plasma membrane, and the glycan moieties of proteins

7

and lipids located on the plasma membrane are exposed outside of cell surface, we

8

proposed a strategy to capture PMPs, termed as glycan moieties-directed PMPs

9

enrichment (GMDPE). With the glycan moieties exposed outside of the cells as bait to

10

ensure the selectivity, and the phospholipid bilayer as raft to provide the sensitivity,

11

we applied this strategy into the plasma membrane proteome analysis of HeLa cells,

12

and in total 772 PMPs were identified, increased by 4.5 times compared to those

13

identified by reported cell surface biotinylation method. Notably, among them 86 CD

14

antigens and 16 ion channel proteins were confidently identified. All these results

15

demonstrated that our proposed approach has great potential in the large scale plasma

16

membrane proteome profiling.

17

2


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1

Introduction

2

Plasma membrane proteins (PMPs) have many critical functions in signal

3

transduction, molecule transport, cell-cell/cell-matrix communication and so forth.1-3

4

PMP deficiency or impairment is associated with diseases, such as cancer,4 Alzheimer

5

disease5 and polio;6 more than 60% of all known drug targets are PMPs.7 Therefore,

6

the plasma membrane proteome profiling can provide insight into disease mechanisms

7

and reveal new therapeutic targets.8-10 However, PMPs pose analytical challenges and

8

remain under-represented in proteomic studies owing to their low abundance and lack

9

of specific groups that are amenable to capture.11,12

10

Nowadays, various methods have been developed for PMPs enrichment.

11

Density-gradient centrifugation was used to study the plasma membrane proteome of

12

human embryonic stem cells and human embryonal carcinoma cells, with 237 and

13

219 PMPs were identified, respectively;13 However, this accounted for only ~ 20% of

14

all identified proteins, which might be affected by the interference of subcellular

15

organelles with similar buoyant density, such as mitochondria and endoplasmic

16

reticulum.14,15

17

One study reported an improved strategy for isolating PMPs, involving the

18

attachment of the negatively charged plasma membrane to cationic colloidal silica

19

nanoparticles (CCSNs), followed by cross-linking with an anionic polymer. This

20

increased the density of the plasma membrane, thereby improving the separation

21

efficiency of PMPs from other lysed cellular components by centrifugation.16 In a

22

subsequent study, contamination from precipitate was reduced by attaching the

23

plasma membrane to magnetic CCSNs; with nanocomposites isolated from other

24

cellular components with a magnetic field, up to 422 proteins were found to be

25

associated with plasma membrane (36% of all identified proteins).17 However,

26

CCSNs may also be rapidly internalized by cells, leading to the co-capture of

27

intracellular components.18 Additionally, owing to the weak electrostatic force

28

between CCSNs and plasma membrane, some captured PMPs may be lost during

29

washing with high-salt and high-pH buffers.19,20

30

Another strategy for PMPs enrichment involves the chemical labeling of cell

31

surface proteins.11,21-24 In Kasvandik’s work, a biotinylating reagent containing an

32

N-hydroxysuccinimide (NHS) group has been used to label the amino groups of

33

PMPs, followed by differential centrifugation to achieve the efficient separation of

34

sperm surface proteins;24 owing to the combination of two independent enrichment 3



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1

methods, a high yield as 40.8% of PMPs was obtained. However, PMPs with

2

post-translationally modified amino acid residues, such as N-terminal acetylation25

3

and cysteine oxidation,26 cannot be labeled with biotinylation reagent, leading to the

4

underestimation of PMPs.

5

In addition, as most cell surface proteins are glycosylated,11 and it was found that

6

the terminal sialic acid of glycan moieties could be selective oxidized in mild

7

oxidization buffer,27-29 this modification were exploited for specific PMPs enrichment

8

by using chemical capture. Mostly, the glycosylated PMPs were first selectively

9

captured by biotinylated reagents, followed by affinity enrichment. This method was

10

with high selectivity for the cell surface glycoproteome analysis. However, to our best

11

knowledge, there is few reported work using this strategy to enrich the whole PMPs

12

and further perform the plasma membrane proteome analysis. In the present study, we

13

developed a glycan moiety-directed PMPs enrichment (GMDPE) strategy. Based on

14

the fact that most PMPs are glycosylated, hydrazide-functionalized magnetic

15

microspheres were used to capture the oxidized glycan moieties of proteins and lipids

16

exposed outer surface of the cell by covalent binding.28,30,31 After cell lysis, both

17

glycosylated and non-glycosylated PMPs could be enriched by the raft binding effect

18

of the phospholipid bilayer of plasma membrane under suspension in detergent.

19

Compared with cell surface biotinylation method, the identified PMPs number from

20

HeLa cell was significantly improved, indicating that GMDPE might be of great

21

promising for the large-scale PMPs profiling.

22 23

Experimental Section

24

Reagents and Materials

25 26

The details are listed in the Supporting Information. Sample Preparation with Cell Surface Biotinylation Method

27

Cell surface biotinylation was performed according to the manufacturer's

28

instructions for PMPs enrichment,32 and the details are shown in the Supporting

29

Information.

30

Afterward, the collected proteins were digested with trypsin according to the filter

31

aided sample preparation (FASP) method.33 Briefly, the enriched PMPs sample

32

containing UA buffer (8 M urea dissolved in 50 mM NH4HCO3, pH 8.5) was added to

33

the 10k filtration device, followed by centrifugation at 14,000 × g at 20 °C for 15 min.

34

All subsequent centrifugation steps were performed under the same condition. 4


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The sample was diluted with UA buffer and the device was centrifuged. After

2

incubation in the dark with 50 mM IAA dissolved in UA buffer for 20 min, the device

3

was successively washed three times with UA buffer and 50 mM NH4HCO3. Trypsin

4

was added to the protein at a mass ratio of 1:25, and the sample was incubated

5

overnight at 37 °C before the digested peptides were collected by centrifugation.

6

Sample Preparation with GMDPE Method

7

The protocol for GMDPE was shown in Figure 1. After washing twice with the

8

coupling buffer composed of 100 mM NaAc and 150 mM NaCl (pH 5.5), HeLa cells

9

(4×107 HeLa cells) were oxidized with 5 mM NaIO4 in the dark at 37 °C for 1 h.

10

Oxidized cells were washed twice with coupling buffer to remove residual oxidation

11

reagent and deplete broken cells or fragments. The cell pellet was resuspended in 3

12

mL coupling buffer before adding 150 mg SiMAG-Hydrazide magnetic microspheres.

13

The coupling reaction between the aldehyde group of oxidized cells and hydrazide

14

group was allowed to proceed overnight at room temperature with end-over-end

15

rotation. After incubation of magnetic microspheres-coated cells in 100 mM Tris

16

buffer (pH 8) for 30 min, captured cells were lysed in Tris buffer containing 80 µg

17

trypsin for 0.5 h at room temperature. Thereafter, the magnetic microspheres-

18

conjugated cell membrane fragments were sequentially washed with 2 M NaCl, 0.1 M

19

Na2CO3 (pH 11.5), 4 M urea and 50 mM NH4HCO3, then resuspended in 1 mL of 4%

20

SDS buffer containing 20 mM DTT, and maintained at 56 °C for 2 h for thermal

21

denaturation and PMPs extraction. The supernatant and magnetic microspheres were

22

simultaneously transferred to the 10k filtration device. PMPs were digested by FASP

23

method, and non-glycopeptides were collected by centrifugation. Magnetic

24

microspheres were sequentially washed with 0.1 M Na2CO3, H2O, 50% ACN, H2O, 8

25

M urea, and 50 mM NH4HCO3 buffer, and then incubated with PNGase F to obtain

26

the information of glycosylation sites.

27

Scanning Electron Microscopy (SEM) Analysis

28

HeLa cells were fixed with 3% (v/v) glutharaldehyde in 0.1 M phosphate-buffer

29

saline (PBS, pH 7.3), washed three times with PBS, and dehydrated in a graded series

30

of t-butanol (50%, 70%, 80%, 90%, and 100%, v/v), before critical point drying, gold

31

staining, and visualization with an S-570 Hitachi Scanning Electron Microscope

32

(Tokyo, Japan).

33

Nano-RPLC-ESI-MS/MS Analysis

34

Obtained peptides were analyzed by nano-RPLC-electrospray ionization tandem 5



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1

mass spectrometry (RPLC-ESI-MS/MS). The details are shown in the Supporting

2

Information.

3

Database Searching

4

All nano-RPLC-ESI-MS/MS raw files were searched with the MaxQuant v.1.3.0.5

5

search engine34 against the Human International Protein Index IPI v.3.87 (91,464

6

entries). The details are shown in the Supporting Information.

7

Bioinformatic Analysis

8

Protein cellular localization was determined based on Gene Ontology (GO) and

9

UniProt databases (http://www.uniprot.org). The TMHMM (http://www.cbs.dtu.dk/

10

services/TMHMM/) algorithm was used to predict transmembrane domains (TMDs)

11

of identified proteins. UniProt and PANTHER (Protein ANalysis THrough

12

Evolutionary Relationships) (http://www.pantherdb.org/) were used to determine

13

protein function. The isoelectric points (pIs) and molecular weights (Mws) of

14

identified proteins were calculated with the ExPASy Server tool (http://web.

15

expasy.org/compute_pi/). The grand average of hydropathicity (GRAVY) values of

16

identified peptides were calculated with the ProtParam program (http://web.expasy.

17

org/protparam/), and proteins with signal peptides were identified with TargetP

18

(http://www.cbs.dtu.dk/services/TargetP/).35

19 20

Results and Discussion

21

Principle of GMDPE

22

As illustrated in Figure 1, the first step for GMDPE strategy consisted of the

23

oxidation of glycan moieties of proteins and lipids located on the outer surface of the

24

cell; the specificity and efficiency of this step has great effects on the number of

25

PMPs which are selectively enriched. After optimization, the favorite concentration of

26

NaIO4 for the oxidation of glycan moieties was 5 mM (data not shown). SEM images

27

revealed that the cells were intact after oxidation (Figure 2A, D and Figure 2B, E),

28

critical for avoiding contamination by proteins from the cytosol and subcellular

29

organelles.

30

With the hydroxyl groups on the adjacent carbon atoms of glycan moieties oxidized

31

to aldehyde groups, the glycan moieties of glycoproteins and glycolipids located on

32

the outside of cell surface were further coupled specifically to hydrazide-

33

functionalized magnetic microspheres. As shown in Figure 2C and 2F, the cells were

34

intensively coated with magnetic microspheres, indicating that the glycan moieties 6


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1

located on the cell surface could be effectively oxidized and captured by hydrazide

2

microspheres.

3

To prevent reaction between magnetic microspheres and the aldehyde groups of

4

non-PMPs, cells were incubated in Tris buffer (pH 8) to block the aldehyde groups of

5

the intracellular proteins before cells lysis under mild condition.36 After lysis, as the

6

phospholipid bilayer were intact, which was confirmed by Blackler et al. using TEM

7

tomography,37 the plasma membrane fragments with PMPs were separated from other

8

subcellular organelles under a magnetic field using glycan moieties captured on the

9

outer surface as bait, which prevented the protein coprecipitation that occurred in the

10

conventional centrifugation method. To disrupt the noncovalent protein-protein

11

interaction and remove the proteins those were nonspecifically adsorbed to magnetic

12

microspheres, high-pH buffer (0.1 M Na2CO3, pH 11), high-salt buffer (2 M NaCl)

13

and 8 M urea were used for washing.38 Since the oxidized glycan moieties were

14

coupled to hydrazide-functionalized magnetic microspheres by covalent bonding,

15

even under such harsh washing conditions, the captured PMPs could not be lost.

16

Finally, the enriched PMPs were solubilized with SDS and subjected to tryptic

17

digestion. Glycopeptides conjugated to the microspheres were released by treatment

18

with PNGase F, which preserved the information of glycosylation sites.

19

Analysis of PMPs from HeLa cells by GMDPE Method

20

The efficiency of PMPs enrichment by GMDPE was assessed by HeLa cell plasma

21

membrane proteome analysis. Peptides released by trypsin and PNGase F treatment

22

were analyzed by nano-RPLC-ESI-MS/MS. A total of 2158 protein groups were

23

identified, of which 772 were annotated as PMPs, 388 (50.3%) were glycosylated and

24

384 (49.7%) were non-glycosylated (Supporting Information, Table S1),

25

confirming that with the raft-binding effect of the phospholipid bilayer, not only the

26

glycosylated

27

simultaneously.

PMPs,

but

the

non-glycosylated

PMPs

could

be

enriched

28

With GO molecular locating applied to the identified non-PMPs, it was found that

29

these proteins were mainly located in nucleus, mitochondrion, endoplasmic

30

reticulum-Golgi, and extracellular region (shown as Supporting Information, Figure

31

S1). The identification of these contaminant proteins might be attributed to the

32

unspecific adsorption to the silica surface of the used commercial magnetic

33

microspheres, as well as the protein-protein interaction between the PMPs and

34

non-PMPs. Given that extremely harsh washing might cause serious PMPs loss 7



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1

during the non-PMPs removal, the hydrophilic modification of the magnetic

2

microspheres and in combination with the other enrichment strategies like density

3

centrifugation could be alternative methods to further improve the selectivity of PMPs

4

isolation.

5

The dynamic range of PMPs identified by GMDPE was determined based on a

6

previously published report.39 With the PMPs obtained by our method covered the

7

complete dynamic range of this dataset (102-108) (Supporting Information, Figure

8

S2), it was indicated that our method can be applied to whole PMPs analysis, without

9

any bias in protein abundance. With the pI values of the identified PMPs ranged from

10

3.79 to 12.24 and Mws ranged from 8.0 to 1519.1 kDa, which were similar to those in

11

the Human International Protein Index (IPI) database v.3.87 (Supporting

12

Information, Figure S3), it was demonstrated that the PMPs with variable physical

13

characteristics could be enriched by our strategy.

14

Comparison with Other Method for PMPs Enrichment

15

According to the literature,11 the strategies based on CCSNs and cell surface

16

biotinylation were most commonly used for PMPs enrichment due to their high

17

selectivity, achieving 24%-36%17,40 and 27-31%,41 respectively. Therefore, a

18

comparative percentage of PMPs was identified with our protocol (36%), indicating

19

good selectivity of our developed protocol for PMPs analysis.

20

Furthermore, to evaluate the identification performance of our method for PMPs

21

analysis, a commercial kit that employs the cell surface biotinylation method of PMPs

22

enrichment was adopted for comparison. The kit uses sulfo-NHS-SS-biotin with a

23

cleavable -SS- unit as the labeling agent instead of the conventional sulfo-NHS-long

24

chain-biotin, which confers higher selectivity.11,24,42 With the same starting sample

25

amount of HeLa cells (4×107 cells), the two methods were respectively performed for

26

PMPs enrichment. In biotinylation method, a total of 332 protein groups were

27

identified and 140 proteins were annotated as PMPs in the GO and Uniprot databases.

28

The selectivity of PMPs enrichment was similar to that obtained by our method (42%

29

vs. 36%), but the number of identified PMPs was much lower (140 vs 772). The low

30

recovery by the biotinylation method may be attributed to steric hindrance in the

31

reaction between small amino groups and sulfo-NHS-SS-biotin, as well as the low

32

rate of PMP release from the NeutrAvidin column. Additionally, 70% (98/140) of

33

these PMPs were also identified by our method, providing evidence for the greater

34

efficiency of our method. 8


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1

Furthermore, the distributions of GRAVY values of peptides from PMPs identified

2

by the two methods were analyzed. The results showed that compared to the

3

biotinylation method, more peptides and a broader range of GRAVY values were

4

obtained with our method (Figure 3A); it is worth noting that the hydrophobic

5

peptides with positive GRAVY values increased more significantly than those

6

hydrophilic peptides with negative GRAVY values (8.5-fold vs 7.0-fold). Moreover,

7

the transmembrane domains of identified PMPs were analyzed with TMHMM

8

software. The number of integral PMPs was 6.8 times higher by the GMDPE than by

9

the biotinylation method (420 vs. 54) (Figure 3B). These results demonstrated the

10

advantage of using our method for identifying hydrophobic PMPs, which might owe

11

to the low sample loss contributed by our high enrichment efficiency. To demonstrate

12

this hypothesis, we analyzed the spectral counting and sequence coverage of the 98

13

PMPs identified by both GMDPE and biotinylation methods. As shown in Figure 4,

14

for most proteins, more spectral counting and higher sequence coverage could be

15

obtained with GMDPE method, indicating that PMPs were efficiently enriched and

16

identified with high confidence.

17

We further compared with the study of whole cell proteome analysis of Hela.39 In

18

Nagaraj’s work, with the obtained proteins fractionated into 72 fractions and

19

measured with 288 h, a total of 10255 proteins were identified. With the complexity

20

of the sample reduced by the fractionation step, 2206 proteins were annotated as

21

PMPs according to GO and Uniprot software, including those with low abundance.

22

However, with this method, the selectivity of PMPs was not high (22%) and the

23

method was labor and time consuming. In our method, the whole separation time for

24

LC-MS/MS was performed without MudPIT strategy in 10 h, and 772 (selectivity of

25

36%) PMPs were identified. In addition, in comparison with Nagaraj’s work, a total

26

of 211 PMPs were exclusively identified by GMDPE method, 58% of which

27

contained transmembrane domains, indicating that highly hydrophobic PMPs can be

28

identified by our GMDPE method.

29

The high performance of GMDPE method for PMP analysis can be explained as

30

follows. Firstly, glycan moieties of proteins and lipids are exposed to the outside of

31

the cell, making them accessible to oxidation and reaction with hydrazide, and the

32

covalent binding between PMPs and hydrazide microspheres allows nonspecifically

33

absorbed proteins to be removed by washing with high-pH and high-salt buffers

34

without the loss of PMPs; Secondly, the free aldehyde groups of non-plasma 9



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1

membrane proteins are blocked by incubation in Tris buffer, preventing their reaction

2

with hydrazide-functionalized magnetic microspheres after cell lysis; Thirdly, using

3

glycan moieties located on the plasma membrane as bait to ensure the selectivity, and

4

exploiting the raft-binding effect of the phospholipid bilayer to provide the sensitivity,

5

both glycosylated and non-glycosylated PMPs could be effectively enriched.

6

Function Characterization of Identified PMPs

7

The molecular functions of the identified PMPs by GMDPE were classified

8

according to the UniProt database and PANTHER software, with a large number of

9

these proteins were found to be involved in cell adhesion, signal transduction, and

10

other physiological processes.

11

Cluster of differentiation (CD) is a cell surface antigen that is used for cell

12

immunophenotyping; the number of CD molecules that are captured indicates the

13

degree of PMPs enrichment.43 Herein, a total of 79 CD antigens (corresponding to 86

14

PMPs) were identified with the GMDPE method, accounting for 22% of the total

15

number of CDs in human (364, as of November 2014), most of which are essential for

16

signaling, molecule transport and cell adhesion (Supporting Information, Table S2).

17

This is larger than the number of CD antigens identified in previous studies,28,44

18

demonstrating the high efficiency of our method for PMPs enrichment. Moreover,

19

307 and 36 of the identified PMPs were predicted to have signal peptides and

20

mitochondrial targeting peptides, respectively (Supporting Information, Table S1).

21

There is increasing evidence that signal peptides of tumor-associated antigens can

22

enhance the ability of autoantibodies to recognize their cognate immunogen;45,46

23

hence, our method has potential applications in cancer diagnosis and prognosis.

24

Among the 722 identified PMPs, 75 were implicated in cell-cell interaction and

25

signaling, including G-protein coupled receptor, and tumor necrosis factor and

26

epidermal growth factor receptor (Supporting Information, Table S3); and 16 were

27

annotated as ion channel (Supporting Information, Table S4) that maintain the

28

resting membrane potential, control the flow of ions across secretory and epithelial

29

cell membranes, and serve as drug targets in various diseases.47 Additionally, receptor

30

tyrosine-protein kinase erbB-2 (a marker for predicting the effiacy of Herceptin)48 and

31

Niemann-Pick-C1-like protein 1 (a direct molecular target of ezetimibe)49 were also

32

among the identified proteins.

33

Furthermore, we analyzed the molecular functions of the 211 exclusively identified

34

PMPs, which were identified by GMDPE method while not identified in the 10


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1

comprehensive dataset from HeLa cell line.39 These were primarily receptors,

2

followed by transporters, hydrolases and cell adhesion molecules involved in

3

integerin, Alzheimer’s disease-presenilin, Wnt signaling and other important

4

physiological pathways (Supporting Information, Figure S4). Our results provide

5

evidence that GMDPE can be a powerful tool for identifying potential drug targets.

6 7

Conclusions

8

In summary, to facilitate PMPs enrichment, we developed a method involving the

9

capture of plasma membrane fragments via covalent binding of oxidized glycan

10

moieties on the cell surface, followed by glycosylated and non-glycosylated PMPs

11

enrichment aided by the raft-binding effect of the phospholipid bilayer. This method

12

has the advantages of high efficiency and selectivity in identifying PMPs, especially

13

for those with high hydrophobicity as compared to previously reported methods.

14

These findings indicate that GMDPE can be used for the comprehensive analysis of

15

the plasma membrane proteome and discovery of novel drug targets.

16 17

Acknowledgements

18

We gratefully acknowledge funding from the National Basic Research Program of

19

China (2012CB910604), the National Natural Science Foundation (21375126 and

20

21505136), and the Creative Research Group Project by NSFC (21321064).

21 22 23

Notes The authors declare no competing financial interest.

24 25 26

Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org.

11



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1

Figure 1. Schematic diagram of GMDPE method for PMPs enrichment. 1) Glycan

2

moieties on the cell surface are oxidized and captured by hydrazide-functionalized

3

magnetic microspheres. 2) Aldehyde groups of non-PMPs are blocked by incubation

4

in Tris buffer. 3) Cell lysis and magnetic separation. 4) Unbound and nonspecifically

5

adsorbed proteins are removed with harsh washing. 5) PMPs are extracted. 6)

6

Non-glycopeptides and glycopeptides of PMPs are released.

7 8

Figure 2. SEM images of (A) unprocessed HeLa cells, (B) oxidized cells, and (C)

9

oxidized cells with bound hydrazide-functionalized magnetic microspheres. (D-F)

10

Enlarged images of the sections in red boxes are shown in the lower panels. Scale bars:

11

100 µm (A-B), 50 µm (C), 10 µm (D, E) and 5 µm (F).

12 13

Figure 3. Distribution of (A) GRAVY values of PMP peptides and (B) TMDs of PMPs

14

identified by GMDPE and cell surface biotinylation method.

15 16

Figure 4. Comparison of (A) spectral counting and (B) sequence coverage of the

17

PMPs identified by both GMDPE and cell surface biotinylation methods.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Glycan Moieties as Bait to Fish Plasma Membrane Proteins

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