Strategy Based on Deglycosylation, Multiprotease, and Hydrophilic

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A novel strategy based on deglycosylation, multi-protease and hydrophilic interaction chromatography for large-scale profiling of protein methylation Min Ma, Xinyuan Zhao, Shuo Chen, Yingyi Zhao, Lu Yang, Yu Feng, Weijie Qin, Lingjun Li, and Chenxi Jia Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 01 Nov 2017 Downloaded from http://pubs.acs.org on November 1, 2017

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Analytical Chemistry

1

Research Article

2 3

A novel strategy based on deglycosylation, multi-protease

4

and

5

large-scale profiling of protein methylation

hydrophilic

interaction

chromatography

for

6 7

Min Ma1, 2, 6, Xinyuan Zhao2, 6, Shuo Chen2, Yingyi Zhao2, Lu yang4，Yu Feng5, Weijie

8

Qin2, Lingjun Li1, 3* and Chenxi Jia2*

9

1

School of Life Sciences, Tianjin University, Tianjin 300072, China

10

2

National Center for Protein Sciences-Beijing, Beijing Proteome Research Center, State Key

11

Laboratory of Proteomics, Beijing Institute of Radiation Medicine, Beijing 102206, China

12

3

13

School of Pharmacy and Department of Chemistry, University of Wisconsin–Madison, WI 53705, USA

14

4

Department of Blood Transfusion, Chinese PLA General Hospital, Beijing, China

15

5

Beijing Hua LiShi Scientific Co. Ltd., Beijing 101300, China

16

6

17

Corresponding Authors:

18

E-mail: [email protected] (C.J.)

19

E-mail: [email protected] (L.L.)

These authors contribute equally to this work.

20 21 1

ACS Paragon Plus Environment


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ABSTRACT

2

Reversible methylation of proteins regulates the majority of cellular processes,

3

including signal transduction, mRNA splicing, transcriptional control, DNA repair, and

4

protein translocation. A fundamental understanding of these biological processes at the

5

molecular level requires comprehensive characterization of the methylated proteins.

6

Methylation is often substoichiometric, and only a very limited number of methylated

7

proteins and sites have been confidently identified to date. Although the intrinsically

8

basic/hydrophilic methylated peptides can be enriched by the hydrophilic interaction

9

liquid chromatography (HILIC), other hydrophilic peptides can co-elute during the

10

enrichment process and suppress the detection of methylated peptides. In addition, the

11

modified Arg and Lys residues cannot be efficiently cleaved by trypsin, the most

12

commonly used enzyme in shotgun proteomics. To overcome these caveats, we develop a

13

novel De-glycO-assisted MethylAtion site IdeNtification (DOMAIN) strategy which

14

enables straightforward, fast, and reproducible analysis of protein methylation in a

15

proteome-wide manner. Combining multi-dimensional fractionation and multi-protease

16

digestion, our method enabled the identification of 573 methylated forms in 270 proteins,

17

including 311 new methylation forms, in A549 cells. Combining this technique with

18

stable isotope labeling quantitative proteomics and RNA interference, we determined the

19

differential regulation of several putative methylated sites that are related to protein

20

arginine N-methyltransferase 3 (PRMT3). Collectively, our integrated proteomics

21

workflow for comprehensive mapping of methylation sites enables a better understanding 2


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of protein methylation, while providing a rapid and effective approach for global protein

2

methylation analysis in biomedical research.

3 4

Key words: Protein methylation, Deglycosylation, HILIC, Mass spectrometry, Proteomics

5 6

3



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INTRODUCTION

2

Protein methylation, occurring predominantly on arginine and lysine residues, is an

3

important modification that has been implicated in signal transduction, mRNA splicing,

4

transcriptional control, DNA repair, and protein translocation, among others.1 Arginine

5

methylation is catalyzed by protein arginine methyl transferases (PRMTs), and there are at

6

least nine distinct enzymes encoded in the human genome.2 PRMTs catalyze the transfer of a

7

methyl group from S-adenosylmethionine (SAM) to the guanidine nitrogen atoms of arginine,

8

resulting in three main forms of methylarginines: ω-NG-mono- methylarginine (MMA), ω

9

-NG,NG-asymmetric

dimethylarginine

(aDMA),

and

ω -NG,N’G-symmetric

10

dimethylarginine (sDMA).3 The methylation states of lysine are catalyzed by the protein

11

lysine

12

ε-N-dimethyllysine, or ε-N-trimethyllysine.4,5 Dysregulation of protein methylation is often

13

associated with various disease states, including cancer,6,7 cardiovascular and pulmonary

14

disease,8 neurodegenerative disease,9,10 and others.11-13 Thus, knowledge related to the

15

abnormally methylated proteins has the potential to provide some promising therapeutic

16

targets.14,15

methyltransferases

(PKMTs)

to

form

three

types:

ε-N-monomethyllysine,

17

Taking into account the importance of methylated proteins in biological processes and its

18

involvement in many human diseases, there is an urgent need for effective methodologies to

19

comprehensively characterize protein methylation. However, protein methylation is often

20

substoichiometric and many methylated molecules are expressed at low abundance, which

21

renders additional challenges for the analysis of methylated proteins. To date, various 4


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1

techniques have been developed to enrich the methylated proteins or peptides to solve these

2

problems. Techniques such as methylated-peptide immunoaffinity purification,16-19

3

strong-cation exchange chromatography (SCX),20,21 chemical proteomics approach22 and the

4

methylation-binding MBT domain,23 and combinations of various approaches.20,24,25 Among

5

them, methylated-peptide immunoaffinity purification has been the more successful approach,

6

and over 8000 arginine mono-methylation sites were identified in a single study.20 However,

7

this technique is limited to peptide sequences and methylation types corresponding to the

8

epitope recognized by the antibody. Chemically modifying methylated residues through

9

propionylation of mono-methylated lysine and immunoaffinity enrichment enabled

10

identification of the 446 lysine mono-methylation sites. However, this strategy is limited to

11

mono-methylated lysine and relies on antibodies.26 In addition, other researchers took

12

advantage of stable isotope labeling of amino acid in cell culture to label methyl groups using

13

[13CD3] S-adenosyl methionine in the cell culture to increase the confidence of identification

14

of methylation sites.16,21 Despite this, the time-consuming procedure of metabolic labeling

15

makes it less effective for clinical related research. Therefore, it is still a daunting challenge

16

to develop a method to enable highly efficient affinity enrichment of methylated proteins or

17

peptides at a global level.

18

Proteomics identifications of proteins and post-translational modifications (PTMs)

19

require proteolytic digestion of proteins into peptides. Trypsin is the most commonly used

20

enzyme in proteolytic digestion. However, if Arg or Lys carries PTMs, such as methylation,

21

trypsin cannot efficiently cleave at its usual Arg or Lys sites.21 Recent development of a new 5



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protease, LysargiNase,27 has presented a new possibility for a comprehensive study of

2

methylation. LysargiNase mirrors trypsin in specificity and overcomes the limitations of

3

LysN28 in controlled N-terminal placement of basic residues, with the added advantages of

4

cleaving N-terminal to methylated forms of arginine and lysine.27 In addition to generating

5

methylated tryptic peptides, a reliable enrichment method is critical for the efficient

6

identification of protein methylation sites. Arginine methylation occurs predominantly in

7

conserved motif sequence, such as glycine/arginine rich sequence. Therefore, methylated

8

peptides inherently contain hydrophilic residues29 . Besides, previous studies showed that

9

more than 90% of tryptic methylated peptides displayed high basic isoelectric points (pIs)

10

and hydrophilicity.30 Furthermore, three different enrichment methods were compared,

11

including SCX, isoelectric focusing (IEF), and hydrophilic interaction chromatography

12

(HILIC).31 The comparison indicated that HILIC had an excellent capacity to enrich

13

methylated peptides.30 Nevertheless, the identification of 215 methylated sites in HeLa cells

14

suggested limitation in comparison to those reports for other PTMs. It is clear from this study

15

that the HILIC method alone is not sufficient to enrich all methylated peptides and proteins,

16

and additional technical advancements are needed to enable more comprehensive

17

characterization of protein and peptide methylation.

18

To overcome these limitations, we developed a novel antibody-free strategy to enable

19

specific enrichment of

methylated peptides, called DOMAIN

(De-glycO-assisted

20

MethylAtion site IdeNtification, Figure 1), which contains three main steps: (1) digestion of a

21

complex sample by complementary use of trypsin and LysargiNase; (2) affinity enrichment 6


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of the methyl peptides using HILIC-tip; (3) fractionation with high-pH small reversed phase

2

tip (sRP-tip) followed by LC-MS/MS analysis. The utility and robustness of this strategy

3

were demonstrated by large-scale screening of methylated sites in A549 cells, and 573

4

methylated forms and 270 methylated proteins were identified, including 312 new

5

methylation forms.

6

EXPERIMENTAL PROCEDURES

7

Cell Culture and SILAC labeling

8 9

SGC7901 (Human gastric cancer) and A549 (Human lung cancer) cells were cultured in DMEM

(Hyclone)

supplemented

with

10%

fetal

bovine

serum

(Gibco),

1%

10

penicillin/streptomycin (Sigma-Aldrich) and incubated at 37 Co in a humidified chamber with

11

5% CO2. For the SILAC labeling, HeLa (Human cervical cancer) cells were grown in SILAC

12

DMEM (Thermo Scientific) supplemented with 10% dialyzed FBS (Gibco), 1%

13

penicillin/streptomycin

14

L-lysine-U-13C6-15N2 (lys8) and L-arginine-U-13C6-15N4 (Arg10) (Cambridge Isotope

15

Laboratory, Inc.) as described previously.32 Dimethylated modified synthetic peptide R1

16

(WGGYSR(di)GGYGGW) was obtained from GenScript.

17

Sample Preparation

(Sigma-Aldrich),

and

either

L-lysine

and

L-arginine,

or

18

Cells were harvested by washing with PBS buffer (Gibco, pH 7.4), and lysed in

19

denaturing buffer consisting of 9 M urea, 20 mM HEPES (pH 8.0), 1 mM sodium

20

orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM β -glycerophosphate, 1% (v/v)

21

protease inhibitor cocktail (Roche). Lysates were sonicated three times at 15% power for 15 7



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1

secs each with intermittent cooling on ice, followed by centrifugation at 20,000 g for 15 min

2

at room temperature. The supernatant was transferred into a new tube and measured

3

concentration using the BCA Protein Assay kit (Solarbio). Subsequently, proteins were

4

digested with multi-enzyme using the FASP protocol.33 Briefly, proteins of 1 mg were

5

reduced with 5 mM DTT at 55 Co for 30 min and alkylated with 10 mM iodoacetamide in the

6

dark at RT for 30 min. The solution was transferred to a 10-kDa-MWCO filter (Millipore)

7

and washed three times with 20 mM HEPES (pH 8.0). Next, add either 0.1 mL of 50 mM

8

HEPES, 10 mM CaCl2 (pH 7.5) containing LysargiNase in an enzyme-to-protein ratio of 1:50

9

(w/w), as previously described

27

or 20 mM HEPES (pH 8.0) containing Trypsin Gold

10

(Promega) in an enzyme-to-protein ratio of 1:100 (w/w) to the filter and incubated at 37 Co

11

for 4 h. At this point, the digestion was split into two parts, and one half was quenched with

12

trifluoroacetic acid (Sigma-Aldrich) at a final concentration of 1% (v/v), while another half

13

was continued to be digested with PNGase F (500 units/µl, New England Biolabs) at 37 Co

14

for 2 h.

15

Methylated-peptide enrichment by HILIC-tip and fractionation by sRP-tip

16

Approximately, 100 µg of digested peptides was dissolved in binding buffer (80% ACN,

17

0.5% FA) and incubated at room temperature with 2 mg of ZIC-HILIC media (Merck,

18

particle size 10 µm), which was pre-washed three times with 100 µl of binding buffer. Then,

19

ZIC-HILIC media was loaded into the gel-loader-tip that was pre-filled with a C8 disk

20

(EMpore). The HILIC-tip was washed five times with 100 µl of binding buffer and then the

21

bound peptides were eluted three times with elution buffer containing 0.5% of FA. The elute 8


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1

was dried and then fractionated on a C18 sRP-tip with different percentage of acetonitrile

2

(ACN) as 6%, 12%, 15%, 18%, 25% and 35%. The six fractions were combined to three ones

3

and dried in vacuum. Peptides were stored at -80 oC until re-dissolved for MS analysis.

4

MALDI-MS and LC-MS/MS Analysis

5

MALDI-MS was used to characterize the synthetic methylated peptides. In our study,

6

MALDI-MS analysis was performed on an ultrafleXtreme time-of-flight mass spectrometer

7

(Bruker). The HILIC-enriched peptides and DHB matrix (10 mg/mL in 50% ACN containing

8

0.1% TFA) was sequentially deposited on the plate and allowed to dry. All the mass spectra

9

(500 laser shots for every spectrum) were obtained in positive reflectron mode in the mass

10

range of (1000-4500 Da). The acquired data was analyzed using flexAnalysis software.

11

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed with

12

Orbitrap Q-Exactive HF mass spectrometer (Thermo) equipped with an online Easy-nLC

13

1200 nano-HPLC system (Thermo) to analyze more complex samples.34,35 More detailed

14

LC-MS/MS parameters can be found in Supporting Information.

15

Methylproteomics and Quantitative Proteomics Data Analyses

16

All raw MS files were processed with MaxQuant software suite version 1.5.2.8 supported

17

by the Andromeda search engine.36,37 For global methylproteomics, three technical replicates

18

were searched individually against the Uniprot human proteome database (downloaded on

19

March 7, 2017, encompassing 20,180 protein sequence entries). Further detailed search

20

engine parameter setting can be found in Supporting Information. For stable isotope labeling

21

by amino acids (SILAC) quantification, two biological replicates were searched against the 9



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same database. Using the same MaxQuant parameters as supporting information, except:

2

multiplicity was set to 2 (heavy/light) with Arg10 and Lys8 selected, “re-quantify” was

3

unchecked, and a minimum of two ratio-counts were required. For methylation site analysis,

4

statistical analysis and hierarchical clustering were performed using Perseus software

5

package38. Detailed workflow of the process with Perseus can be found in Supporting

6

Information. Significantly enriched Gene Ontology terms were determined using the

7

Functional Annotation Tool of the DAVID Bio-informatics database.

8

analyses were performed using the Motif-X 40 and iceLogo.41

9

RNA interference and Western Blot

10

39

Sequence motif

Details are included in the Supporting Information.

11 12

RESULTS AND DISCUSSION

13

Development of the DOMAIN strategy.

14

Due to the particularly low abundance of the methylated peptides, developing strategies

15

to specifically enrich them from complex samples is a prerequisite. This can be problematic if

16

one uses HILIC enrichment alone because HILIC has become a powerful analytical tool for

17

glycopeptide enrichment.42-44 The modified N/O-linked glycopeptides are hydrophilic

18

competing with methylated peptides during enrichment. Therefore, for the proposed

19

procedure for methyl-proteomic screening to be efficient, the elimination of glycopeptides

20

should be as complete as possible. Herein, we proposed that deglycosylation before HILIC

10


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1

enrichment can eliminate the interference from glycopeptides, thus enhancing the selectivity

2

of methylated peptides recovery from non-methylated peptides.

3

To carry out proof of principle studies, the PNGase F was chosen for deglycosylation,

4

since it is an amidase that cleaves between the innermost GlcNAc and asparagine residues of

5

high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins.

6

Glycoprotein fetuin that contains three asparagine-linked carbohydrate chains is widely used

7

to simulate the real glycopeptides environment in methylated peptides enrichment.45 By using

8

the mixture of the synthetic di-methylated peptide (WGGYSR(di)GGYGGW) and fetuin

9

digested peptides as the test sample, we investigated the impact of PNGase F treatment on the

10

enhancement of detection of the methylated peptides using MALDI MS. Three experiments

11

were performed: (1) HILIC enrichment without PNGase F treatment; (2) HILIC enrichment

12

prior to PNGase F treatment; (3) HILIC enrichment after PNGase F treatment. As shown in

13

the upper spectrum of Figure 2A “HILIC enrichment without PNGase F treatment”, the

14

dimethylated peptide standard and some other tryptic peptides were detected simultaneously.

15

To verify if the tryptic peptides in the range of m/z above 3300 were glycopeptides, the same

16

enrichment product was treated with PNGase F and analyzed by MALDI-MS (middle

17

spectrum of Figure 2A “HILIC enrichment prior to PNGase F treatment”). The intensive

18

signals from deglycosylated tryptic peptides from fetuin were clearly observed at m/z 1741,

19

3018 and 3673, which are identical to previously reported glycopeptide data of fetuin.45 On

20

the contrary, the peaks with m/z of above 3300 mostly disappeared upon PNGase F treatment

21

and HILIC enrichment. This result indicates that the HILIC-tip approach shows effective 11



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Page 12 of 28

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affinity to enrich the fetuin glycopeptides as well as the di-methylated peptide. This

2

observation confirmed our previous speculation that the presence of abundant glycopeptides

3

could be co-eluted with methylated peptides and hinder their effective recovery using

4

HILIC-based enrichment strategy. To overcome this issue, the PNGase F treatment was

5

performed to eliminate the interference of hydrophilic glycopeptides to HILIC enrichment.

6

As shown in the bottom spectrum of Figure 2A “HILIC enrichment after PNGase F

7

treatment”, the di-methylated peptide standard was effectively enriched while other peaks

8

exhibited much weaker signals, especially the peaks above m/z 3300. Based on these results,

9

we conclude that the HILIC enrichment after PNGase F treatment can help to enhance the

10

detection of methylated peptide through eliminating the interference of hydrophilic

11

glycopeptides.

12

In order to validate our approach for other proteomic studies, we prepared peptides from

13

digests of SGC7901 cells. A total of 200 µg FASP digested peptides were divided into two

14

aliquots. One of them was treated with PNGase F to remove the glycopeptides. Then the two

15

aliquots were enriched with HILIC-tip (the details were described in the Experimental

16

methods section). In HCD fragmentation mode, five typical glycan fragments were detected

17

as an indicator of a glycopeptide spectra.46 To ensure the accuracy of glycopeptide

18

determination, the criteria were set that only spectra containing all of the five glycan

19

fragment ions were recognized as potential glycopeptides. With the in-house code to extract

20

the glycan fragments ions (m/z 126, 138, 168, 186, 204), we obtained the sum of intensities of

21

all the five glycan fragment ions in each spectrum and the number of MS/MS scans of 12


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1

potential glycopeptides. We found a significant decrease of the total glycopeptide PSM

2

intensity after treatment with PNGase F as expected. At the same time, the percentage of the

3

glycopeptide scan number against the total peptide scan number were reduced from 31.4% to

4

8.4% (inner pie chart in Figure 2B). As PNGase F cannot cleave certain types of N-glycans

5

and numerous O-GalNAc modified peptides are in existence, there are still some spectra

6

containing glycan fragments after the PNGase F treatment. Although the interference of

7

glycopeptides could not be completely removed, substantial enhancement of methyl peptide

8

enrichment was achieved. As shown in Figure 2C, the intensity of methyl-peptides increased

9

dramatically, especially the di-methylated peptides (shown as green trace in Figure 2C). This

10

result further supports that deglycosylation before HILIC enrichment can help to not only

11

increase the number of identified methylation forms but also the intensity of methylation

12

forms, demonstrating that our strategy is highly efficient.

13 14

Extending the methylation site identifications by complementary use of LysargiNase.

15

To investigate the applicability of LysargiNase in methyl-proteomics applications, we

16

compared LysargiNase and trypsin digests of SGC7901 cells followed by the HILIC

17

enrichment. In short, we performed two replicates of HILIC-tip enrichments on the

18

LysargiNase and tryptic SGC7901 cell digests to enrich methyl-peptides, using just 100 µg of

19

peptides per enrichment. Despite the shared amino acid preference of the two enzymes and

20

the fact that the technical reproducibility (overlap in identifications of methylated sites) of the

21

mass spectrometric runs was on average more than 70% at site-specific level (Figure S-1A,

22

Figure S-1B). After removal of the N- or C- terminal cleavage sites, there was only a 17% 13



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Page 14 of 28

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overlap in the identifications of methylated modified peptides between the two enzymes

2

(Figure S-1C). Furthermore, the data from two replicates revealed that the combined use of

3

two proteases can increase the methylated sites coverage by about 80% (Figure 3A).

4

Although the number of methylated sites did not differ substantially between the two

5

enzymes, the number of modified sites at the peptide termini for LysargiNase（N-termini）and

6

trypsin（C-termini）are distinct (Figure 3B). The number of terminal methylation-containing

7

peptides resulting from trypsin are sequentially decreased on mono, di and tri-methylated

8

residues, respectively. One possible reason is that the trypsin shows sequentially decreased

9

cleavage efficiency on the three types of methylated residues due to increased steric

10

hindrance. It is consistent with previous report that methylation on Arg and Lys prohibits the

11

cleavage by trypsin, while the mono-methylated peptides are partially cleaved.21 In contrast

12

with trypsin digestion, the results from LysargiNase indicate significantly enhanced cleavage

13

on di- and tri- methylated residues. Moreover, after HCD fragmentation, the fragment-ion

14

sequences retaining the basic residue were different when generated with LysargiNase versus

15

trypsin.27 The spectra of LysargiNase-cleaved methylated-peptides had a strong series of

16

b-type ions due to N-terminal cleavage, whereas the spectra corresponding to tryptic

17

methylated peptides were dominated by y-type ions due to C-terminal cleavage (Figure 3C).

18

Overall, the complementary use of both proteases can increase methyl-proteome coverage.

19

Landscape of methylated sites of A549 cells.

20

To further demonstrate the enrichment capability of the combined deglycosylation and

21

HILIC enrichment strategy, A549 cell (lung carcinomas) protein sample was used. Although 14


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1

several studies on methylated proteins of lung carcinomas have been conducted,47 the global

2

characterization of methylation modification in lung carcinomas has not reported. In our

3

study, we performed three technical replicates on tryptic and LysargiNase A549 cell digests

4

to enrich methyl-peptides, using just 1 mg of peptide material per enrichment. Three

5

technical replicate analyses demonstrated that strong reproducibility (the average R2 value

6

greater than 0.9) using our established methodology, furthered by high reproducibility and

7

high pH sRP (Figure 4 A). However, for the technical replicates of two different proteases,

8

the average R2 value was only 0.5 (Figure 4A). Moreover, we find that 70% of identified sites

9

from one replicate were also identified in the other (Figure S-2A, Figure S-2B), and the

10

overlap between consecutive sRP-tip fraction were quite low (Figure S-2C, Figure S-2D),

11

indicating that overall the stability of one protease group was much higher than that of

12

different proteases group and that there was good separation between fractions. Collectively,

13

by applying strict quality control (the FDR at the peptide and sites level to be 40)48, we obtained

15

573 methylation forms on 516 sites from 270 proteins, combining the results from three

16

technical replicates (Table S1). Among them, 184 forms were on the lysine residues and 389

17

forms were on the arginine residues (Figure S-2E). Compared to publicly available database49,

18

more than 54% of identified methylation forms constitute novel modification forms (Figure

19

S-2F). At the same time, nearly half (46%) of the identified sites were reported previously.

20

For example, as annotated in the Uniprot database, the MS/MS fragmentation spectrum of the

21

identified peptide (*KSAPATGGVKPHR) of H3K2750 using our method with all three 15



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1

methylation (K) forms (Figure S3) matched very well, further validating the methylation site

2

identification and assignment with our approach. Previous reports suggested that H3K27

3

methylation is linked to several types of cancers, such as lung cancers48. Therefore, by

4

comparing our results with previous studies, we confirm that not only our data are reliable,

5

but also our new approach enables a more comprehensive and deeper understanding of the

6

cellular extent of methylproteome.

7

To this end, we first assessed the distribution of methylated sites and found that 31.1% of

8

identified proteins harbor more than one methylation site (Figure S-2G). Especially the

9

TATA-binding associated factor (TAF15) protein, which is an important protein responsible 51

10

for transcription initiation at distinct promoters

and 27 methylated sites were identified,

11

while in the UniProt database this protein was only annotated with 8 sites, all of which were

12

included in our data; Additionally, HNRNPU52 is involved in a variety of biological process,

13

like mRNA splicing, circadian regulation of gene expression, CRD-mediated mRNA

14

stabilization53 and 13 methylated sites were identified in our data.

15

Next, we assessed the potential contribution of the LysargiNase methylproteome data set.

16

As show in Figure 4B, only 107 methyl sites can be identified in both proteases, and about

17

45.6% methylated forms are unique to the LysargiNase treatment. In addition, it was found

18

that the LysargiNase significantly increased the identification of di-methylation forms and the

19

modified sites at the peptide termini for LysargiNase (25.6% sites at the N-termini) was 1.7

20

times that of trypsin (14.8% sites at the C-termini) (Figure S-2H). After HCD orbitrap

21

fragmentation, mass spectra of LysargiNase-cleaved peptides produced a strong series of 16


Page 17 of 28

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1

b-ions, and the corresponding spectra of tryptic peptides identified with the identical

2

cleavage-site were dominated by y-type fragment ions (Figure S-2I). Annotated MS/MS

3

spectra of di-methylated R407 of FUS protein (RNA-binding protein FUS) are shown in

4

Figure S-5; the first is trypsin digestion results and the second is a spectrum of LysargiNase

5

digest peptide. The fragmentation of the same composition peptide under different digestion

6

conditions were different, with trypsin-cleaved peptide displaying a strong y-ion series

7

whereas LysargiNase digested peptide exhibiting strong b-type fragment ions. Furthermore,

8

the methyl forms identified using both protease treatments would be highly confident due to

9

more fragments being detected and the cross validation of the two methods, which was

10

consistent with our previous experiment. In summary, compared to the results obtained by

11

the conventional HILIC approach where only 249 sites were identified from twelve HILIC

12

fractions,30 the DOMAIN approach reported in this study enabled more comprehensive

13

mapping of methyl-proteome with less time and cost for the analysis.

14 15

Bioinformatics Analysis of Identified Methylation Forms.

16

Since this is the first global identification of methylated forms in lung carcinoma A549

17

cells, Gene Ontology annotation analysis using the bioinformatics tool DAVID39 was

18

conducted to gain a better functional understanding of the methylated proteins. In terms of

19

the category of biological processes, we found that methylated proteins are mainly involved

20

in RNA binding, nucleotide binding and mRNA splicing process (Figure S-4A), regardless

21

of the treatment by LysargiNase or trypsin. To analyze the sequence specificity of the 573 17



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Page 18 of 28

1

identified methylation forms, the motif-x40 was used. Compared with tryptic methylated

2

peptides, LysargiNase generated methylation peptides showed higher representation of RG,

3

and RGG motif (Figure S-4B). One possible reason is that, as with trypsin, basic residues in

4

missed LysargiNase cleavage sites were frequently preceded or followed by prolines.27 Of the

5

total 389 methylation forms on Arginine, 60% occurred at RG and RGG of

6

glycine-arginine-rich sequences. However, no significant motifs were found on lysine

7

methyl-sites (Figure 4C), implying that different amino acids may have different

8

regulation mechanisms. In addition, methylation has previously been reported to participate

9

in crosstalk with phosphorylation.54

To investigate colocalization between methylation and

10

phosphorylation, we used the identified methylation sites to match the known

11

phosphorylation sites (from phosphosite.org). The colocalization of methylation and

12

phosphorylation was observed to be very close as expected (Figure 4D). Of all the identified

13

methylated sites, 59.8% were observed close to known phosphorylation sites within ±6

14

amino acids window. It reveals that methylation and phosphorylation may have the same

15

target substrate, and the two PTMs may function synergistically in some biological processes.

16

Furthermore, the results of our experiments showed that the majority of methylated

17

peptides were highly hydrophilic with a median GRAVY score55 of -1.3 and more than 90%

18

modified peptides displayed a PI ≥9, which was consistent with previous studies (Figure

19

S-4C and D).30

20 21

Quantitative analysis of putative methylation sites by cellular knockdown of PRMT3 18


Page 19 of 28

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1


enzymes.

2

PRMT3 is unique among PRMTs for its zinc-finger at its N terminus, which confers

3

substrate specificity.56 Therefore, the discovery of putative target methylated sites is

4

conducive to further study of its mechanism. To identify cellular PRMT3 putative

5

methylation sites and obtain regulatory information of arginine methylation, we next

6

combined our established proteomic workflow with SILAC quantification and RNAi of

7

PRMT3 (Figure 5A). Heavy isotope-labeled cells were left untreated, while the light

8

isotope-labeled cells were treated with RNAi. After efficient knockdown effect in HeLa

9

cells were obtained (Figure S-5A), heavy and light cells were harvested and mixed 1:1

10

based on total protein content and digested with trypsin or LysargiNase. 1mg methylated

11

peptides were enriched using DOMAIN method, followed with LC-MS/MS for

12

methylation site identification.

13

revealed strong Pearson correlations in measured SILAC ratios (Figure S-5B),

14

demonstrating high reproducibility in our experimental setup. Using the quantitative

15

information obtained in the SILAC experiment, we found 69 methylation sites decreased

16

or increased in abundance by more than 1.5 fold, as compared with the untreated cells

17

(Figure 5B, Table S2). Among them nearly half of these sites have been reported to be

18

related to methylation57, such as, the down-regulation of the methylation sites of Histone

19

H3.3 (H3F3A-28R),58 Scaffold attachment factor B2(SAFB2-903R), and 78 KDa

20

glucose-regulated protein (HSPA5-591K,585K), which further provides solid support for

21

our study. Besides, A-kinase anchor protein 3(AKAP3), one of the A-kinase anchoring

Two biological replicates RNAi analyses of PRMT3

19



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Page 20 of 28

1

proteins (AKAPs), has two new methylation sites (400,410) up-regulated (Figure 5B);

2

Heterogeneous

3

pre-mRNA-binding proteins, also contains two newly identified up-regulated methylation

4

sites. These newly identified sites may uncover novel regulatory mechanism of the

5

PRMT3. Other identified differential methylation sites are presented in Table S2,

6

providing additional targets for future biological studies. In addition, a heat map based on

7

the ratios of down/up regulation of PRMTs revealed that replicate experiments (for

8

trypsin or LysargiNase, separately) were clustered together (Figure 5C). This regulation

9

pattern suggests that both trypsin and LysargiNase treatments provided overall similar

10

trends for methylation sites down/up-regulation, validating the SILAC results.

11

Furthermore, the same protease treatment provided very similar quantitative results,

12

further highlighting the excellent reproducibility of our methodology.

13

CONCLUSIONS

nuclear

ribonucleoprotein

K

(HNRNPK),

one

of

the

major

14

In this study, a novel strategy combining selective enzymatic deglycosylation and

15

HILIC-tip enrichment was developed for the global analysis of methylated peptides by

16

mass spectrometry. Selective enzymatic deglycosylation was introduced before HILIC

17

enrichment to remove majority of the N-linked glycans that would interfere detection of

18

methylated peptides. This strategy leads to the first efficient enrichment and large-scale

19

identification of methylated peptides from lung cancer cells, which could provide new

20

insights into the study of methylation related human physiological and pathological

21

changes for biomarker discovery. Like many other enrichment methods, our approach also 20


Page 21 of 28

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1

has some limitations.

For example, there are still some methylated peptides that are less

2

hydrophilic and cannot be enriched with the HILIC-based methodology, which could

3

reduce the coverage of methylation sites. However, considering its simplicity and

4

robustness, this strategy shows significant promise to become a powerful tool for

5

systematically analyzing protein methylation in various biological samples, such as

6

tissues, cells and biofluids, enabling accelerated discovery and in-depth characterization

7

of methylproteome in a variety of biological processes and complex biological systems.

8 9

ACKNOWLEGEMENTS

10

This work is supported in part by the National Key R&D Program of China (No.

11

2016YFA0501302 and No. 2017YFA0505702 to CJ), the National Science Foundation of

12

China (No. 21675006 to CJ) and Changjiang Scholars Program sponsored by the Chinese

13

Ministry of Education (to LL). We are grateful to Professor Wantao Ying for helpful

14

discussions and insightful suggestions on this project. We thank Yue Zhu, Quan Zhou,

15

Kai Li from mass spectrometry facility of the National Center for Protein Science-Beijing

16

(Phoenix Center) for expert technical assistance.

17

Supporting Information

18

The supporting information is available and noted in the text. The material is available free of

19

charge via the Internet at http://pubs.acs.org.

20 21

Reference:

22

(1) Afjehi-Sadat, L.; Garcia, B. A. Curr Opin Chem Biol 2013, 17, 12-19. 21



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Figure 1. The DOMAIN strategy for comprehensive analysis of protein methylation. Proteins from cell lysates were digested with trypsin or LysargiNase followed with PNGase F treatment. The HILIC-tip enrichment and the high pH sRP-tip fractionation were then processed for large-scale identification of protein methylation by LC-MS/MS followed by data analysis and bioinformatics analysis.

24


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1

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Figure 2. Proof of principle study of the proposed DOMAIN strategy. (A). MALDI-TOF/TOF analysis of a glycoprotein fetuin digest and methylpeptide standard under different conditions. The upper spectrum shows the result of HILIC enrichment without PNGase F treatment; the middle spectrum is resulting from HILIC enrichment followed by PNGase F treatment; and the bottom spectrum displays the result of PNGase F treatment followed by HILIC enrichment. Methylpeptide, WGGYSR(di)GGYGGW. Deglycosylated

peptides,

m/z

1741,

LCPDCPLLAPLN(156)DSR;

m/z

3018,VVHAVEVALATFNAESN(176)GSYLQLV EISR; m/z 3673, RPTGEVYDIEIDTLETTCHVLDPTPLAN(99)CSVR. (B). Distribution of spectral counts which contain glycan fragments before and after PNGase F treatment. The blue histogram represents PNGase F treatment and the white one is no treatment. The inner pie chart demonstrates that the percentage of glycan-containing spectra in total spectral counts is reduced from 31.4% to 8.4% due to PNGase F treatment. (C). Accumulative intensity of the identified methylated peptides in three different methylation forms with and without PNGase F treatment.

25



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Page 26 of 28

1 2

3 4 5 6 7 8

Figure 3. Evaluation of the complementary use of trypsin and LysargiNase for methylation protein identification. (A) Venn diagram comparison of the identified methylated sites using two different enzymes. (B) Distribution of the percentage of the identified methylpeptides with the methylated Arg or Lys at the termini of peptide chains. (C). Boxplot of the b fragments and y fragments resulting from the use of two different enzymes.

26


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1 2 3 4 5 6 7


Figure 4. Global view of mehylproteome of A549 cells. (A) Pearson correlation coefficiency of identification results from complementary use of trypsin and LysargiNase. Each experiment contains three technical replicates. (B) Venn diagram showing the identified methylated sites. (C) Sequence logo of identified Arg and Lys methylated sites within ±6 amino acids distance. (D) Methylation colocalization with phosphorylation within ±6 amino acids distance.

27



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Page 28 of 28

1 2 3

4 5 6 7 8 9 10

Figure 5. Quantitative analysis of the cellular effects of siPRMT3. (A) Workflow of the SILAC-based quantification and RNAi of PRMT3. The experiments using trypsin have two replicates, and the same as LysargiNase. Cells were labeled with light or heavy stable isotope amino acids and combined in a protein ratio of 1:1. (B) The site-speciﬁc changes (log2(L/H)) of all identified methylation sites. (C) Hierarchical clustering of methylated sites under siPRMT3 treatment. High reproducibility was observed in replicates with the same enzyme.

28


Strategy Based on Deglycosylation, Multiprotease, and Hydrophilic

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