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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
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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
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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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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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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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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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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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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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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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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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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
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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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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
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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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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
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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:
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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.
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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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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.
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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.
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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.
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