Chem. Res. Chin. Univ., 2015, 31(1), 21―24
doi: 10.1007/s40242-014-4317-2
Mass Spectrometric Analysis Revealing Phosphorylation Modifications of Periostin ZHAO Yueming1,3, SONG Lina1, LI Lingxia1, LI Xiao’ou3, SHI Qinghong1, HONG Xin1, GUO Jia1, FANG Ling1, HE Chengyan1*, LI Hongjun1 and ZHAO Haifeng2 1. China-Japan Union Hospital of Jilin University, Changchun 130033, P. R. China; 2. The First Affiliated Hospital of Jiamusi University, Jiamusi 154002, P. R. China; 3. Tumor Hospital of Jilin Province, Changchun 130012, P. R. China Abstract Human periostin was over-expressed in HEK293T cells, which was enriched by nickel ion affinity resin, and further purified by gel electrophoresis. Phosphopeptides contained in the tryptic digestion of the purified periostin were enriched by TiO2 affinity chromatography, and then analyzed by liquid chromatography-tandem mass spectrometry(LC-MS/MS). LC-MS/MS analysis reveals three phosphorylation modification sites of periostin at IKVIEGpSLQPIIK(682―694), pSLHEKLKQDK(498―507) and p[TT]VLYEC*C*PGYM*R(73―85). The established method could be a great help to profiling the phosphorylation status of periostin under different physiological environments, such as inflammatory and tumor micro-environments. Keywords Mass spectrometry; Periostin; Phosphorylation
1
Introduction
Periostin was originally identified as osteoblast-specific factor in osteoblasts, regulating the formation and maintenance of bone and tooth[1]. This protein was named because of its preferential location in the periosteum. Despite its important role in physiology in bone, a large body of evidence has shown that periostin has various biological functions in the fields, such as cardiovascular physiology[2―4] or oncology[5―7]. Periostin is rich in glutamate residues[8] and contains several N-glycosylation sites[9]. So far, there have been few studies to decipher the types and locations of post-translational modification of periostin. For example, the phosphorylation status of periostin remains unclear. As a kind of widely studied post-translational modifications, phosphorylation plays vital roles in a variety of physiological and pathological processes in life[10―12]. Therefore, identification of the status of phosphorylation of periostin could prove beneficial to deep understanding its biological functions. During the past decade, mass spectrometry has been developed into one of the most powerful tools in both the chemical and the biological sciences[13,14], especially in analyzing the post-translational modifications including phosphorylation[15,16]. When coupled with other chromatographic techniques, such as high performance liquid chromatography(HPLC), mass spec-
trometry has the potential to characterize samples with extreme complexity in a single run. Owing to its low abundance and transient feature of periostin, phosphorylation modification usually cannot be easily characterized. Efforts have been made to develop strategies to specifically enrich and purify the phosphorylated peptides. Enrichment of phosphorylated peptides by some kinds of metal oxides, such as Fe3O4, ZrO2 and TiO2 has been well established and widely used in proteomics studies. Tips embedded with TiO2 were utilized in the present study to enrich the phosphorylated peptides from in-gel tryptic digestion of periostin. TiO2 enrichment is superior to other methods, such as immobilized metal ion affinity chromatography(IMAC) and it possesses less non-specific bindings during the enrichment process. Additionally, enrichment with TiO2 is easy to handle, which can greatly reduce the artifacts during sample preparation. In this study, HEK293T cells in which periostin with a six-histidine tag was over-expressed were prepared. The over-expressed periostin was then enriched by nickel ion affinity resin and further purified by gel electrophoresis. After in-gel digestion, the phosphorylated peptides in the peptide digestion mixture were enriched by TiO2 chromatography, followed by mass spectrometry analysis. Three phosphorylation sites of periostin were identified.
——————————— *Corresponding author. E-mail:
[email protected] Received August 25, 2014; accepted October 20, 2014. Supported by the National Natural Science Foundation of China(Nos.81472454, 31100930), the Natural Science Foundation of Jilin Provincial Science and Technology Department, China(Nos.2011713, 20090461, 20110739, 20130413017GH), the Natural Science Foundation of Heilongjiang Province, China(Nos.201000742, D201075, Gc09c317), the Natural Science Foundation of Jilin Provincial Development and Reform Commission, China(No.2013c026-5) and the Natural Science Foundation of Changchun Science and Technology Bureau, China(No.2011125). © Jilin University, The Editorial Department of Chemical Research in Chinese Universities and Springer-Verlag GmbH
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2 2.1
Chem. Res. Chin. Univ.
Materials and Methods Chemicals and Materials
Sequencing-grade TPCK-modified trypsin was obtained from Promega(Madison, WI, USA). Ni affinity resin, as well as TiO2 tips, was obtained from Pierce Biotechnology(Rockford, IL, USA). Bradford protein assay kit was from Bio-Rad (Hercules, CA, USA). All other chemicals were obtained from Sigma-Aldrich(St. Louis, MO, USA). His-periostin expression plasmid was kindly provided by Prof. LIU Ning from Jilin University, China. Ultra-pure water was produced on a MilliQ water purification system.
2.2 Expression, Enrichment and Purification of Periostin by Ni Affinity Resin and SDS-PAGE Expression of periostin was performed by transfecting His-periostin expression plasmid into HEK293T cells. After 28 h, the transfected cells were lyzed and the lysates were cleared by centrifugation. The expressed full-length periostin, attached with six-histidine tag, was then enriched and purified by Ni-NTA(NTA=nitrilotriacetic acid) agarose resin(Qiagen). The bound periostin was released by boiling the Ni-NTA agarose resin in sample loading buffer containing 2% sodium dodecyl sulfate(SDS), 20% glycerol, 20 mmol/L Tris-HCl(pH=6.8), 6 mmol/L β-mercaptoethanol and trace bromoxylenol blue. The released periostin was further purified by SDS-polyacrylamide gel electrophoresis(PAGE) separation.
2.3 Tryptic Digestion of Protein Sample and Enrichment of Phosphorylated Peptides The protein band of interest was cut from the gel, and then destained. After the destained gel band was reduced and alkylated, it was completely dried with ACN and then incubated with 50 mmol/L ammonium bicarbonate aqueous buffer containing sequencing-grade N-tosyl-L-phenylalanine chloromethyl ketone(TPCK)-modified trypsin at 37 °C for 16 h. One microliter of 10% formic acid was used to quench the enzymatic reaction. The tryptic peptides from gel bands were extracted with a buffer containing 10% ACN and 25 mmol/L ammonium bicarbonate. A small aliquot of the extraction was pipetted out and transferred into a new vial submitted to MS analysis for protein identification purpose. The rest of extraction sample was lyophilized and re-dissolved in 0.1% formic acid and then loaded onto the Supel-Tips Ti Pipette Tips that were well pre-equilibrated with a buffer containing 50% ACN and 0.1% formic acid. The TiO2 tips were extensively washed with a buffer containing 0.1% formic acid, 50% ACN and 100 mmol/L KCl. Elution of the bound peptide was performed with 1% aqueous ammonia. The eluted peptides were then lyophilized and stored at –20 °C.
2.4
LC-MS/MS Analyses and MS Data Processing
The lyophilized tryptic peptides were dissolved in a buffer containing 0.1% formic acid and 1% ACN, and then loaded
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onto a C18 reverse capillary column(100 mm×0.17 mm). Elution of the peptides was performed with a buffer containing 0.1% formic acid and 99% ACN, on which a gradient of 5%―45% was applied at 0.30 μL/min for 180 min. An LTQ mass spectrometer(Thermo Fisher Scientific) was used to detect the eluted peptides. For tandem mass spectroscopy(MS/MS) analyses, data-dependent acquisition was employed and the normalized collision energy was set at 35%. The collected mass data were analyzed by Thermo Proteome Discoverer 1.1 software. Briefly, mass data were searched against SwissProt protein database by the built-in SEQUEST. Parameters in database searching included as following: up to two missed cleavage during trypsin digestion; a peptide tolerance of 15 ppm; an MS/MS tolerance of 0.8 Da; fixed modification as carbamidomethylation of cysteine; variable modifications as phosphorylations of serine, thronine and tyrosine, as well as oxidation of methionine. Peptide Confidence as High was chosen to filter the results from database searching.
3
Results and Discussion
3.1 Enrichment and Purification of His-tagged Periostin Plasmid with full length periostin gene that is attached a six-His tag sequence was transfected into HEK293T cells. The cells were maintained in Dulbecco’s modified Eagle medium(DMEM) containing 10% fetal bovine serum for 28 h at 37 °C, then collected with a scrap into a clean vial. The collected cell pellets were washed in cold phosphate buffered saline(PBS) three times, which were then lyzed. The expressed periostin with a six-His tag was enriched by Ni-NTA agarose resin, and further purified by SDS-PAGE with high purity as indicated in Fig.1.
Fig.1
3.2 tin
SDS-PAGE of periostin purified by Ni-NTA agarose resin
Mass Spectrometric Identification of Perios-
The gel band of interest was cut off and subjected to in-gel digestion. An aliquot of the obtained tryptic peptide mixture was analyzed by liquid chromatography-tandem mass spectroscopy(LC-MS/MS). LC-MS/MS analyses were performed in triplicate. Database searching identified a total of 16 specific tryptic peptides from perisotin. The details of the peptides in
No.1
ZHAO Yueming et al.
the identification of perisotin are indicated in Table 1. Of note, even though Ser, Thr and Tyr phosphorylations were considered as viable modifications during the database searching, no positive identification of phosphopeptide within periostin was obtained. Table 1 Summary of proteolytic peptides identified in periostin over-expressed in HEK293 cells m/z(MH+)
Residue
VIEGSLQPIIK TTVLYECCPGYMR
Charge status 2 2
1197.2256 1650.5649
684―694 73―85
T3
IMGDKVASEALMK
2
1393.3987
290―302
T4
AAAITSDILEALGR
2
1401.6148
252―265
T5
EVNDTLLVNELK
2
1381.4639
597―608
Peak
Peptide sequence
T1 T2
T6
YSDASKLR
2
939.9941
116―123
T7
EIPVTVYTTK
2
1151.3217
663―672
T8
LLQGDTPVR
2
999.1057
807―815
T9
NGAIHIFR
2
917.8214
482―489
T10
QVIELAGK
2
857.9634
372―379
T11
LILQNHILK
2
1092.3377
427―435
T12
TAVCIENSCMEK
2
1442.3198
464―475
T13
TEGPTLTK
2
847.2174
695―702
T14
LLQEEVTK
2
959.9649
779―786
T15
VKIEGEPEFR
2
1204.4398
703―712
T16
YFSTCKNWYK
2
1397.4663
56―65
3.3 Identification of Phosphorylation Sites of Periostin The phosphorylated peptides often cannot be easily detected by mass spectrometry in positive-ion mode due to their low ionization efficiency and low abundance. Therefore, efforts have been made to develop methods to analyze phosphorylated peptides by mass spectrometry. One of the most well established and widely applied methods is the enrichment of phosphorylated peptides prior to mass spectrometry analysis. The enrichment of phosphorylated peptides is often achieved by immobilized metal ion affinity chromatography(IMAC) or metal oxide particles(ZrO2, TiO2, and Fe3O4) adsorption. In this study, TiO2 was used to selectively enrich and purify the phosphorylated peptides contained in the tryptic digest of periostin over-expressed in HEK293T cells. Fig.2 shows the MS/MS spectrum of a doubly-charged molecular ion peak at m/z 759.83. Database searching identified the corresponding peptide as IKVIEGpSLQPIIK (682―694), in which Serine 688 in periostin was phosphorylated. A series of both b and y types of fragment ions, such as b3, b4, b5, b6, b7, b9, b10 and y2―y11 was detected. It should
23
be noted that there was a missing cleavage at Lysine 683 by trypsin digestion. Fig.3 shows the MS/MS spectrum of a doubly-charged molecular ion peak at m/z 653.74. The peptide was identified as pSLHEKLKQDK(498―507), in which Serine 498 was phosphorylated. Most of the y series of fragment ions were detected with high sensitivity. Many fragment ions of b series could be assigned. But the abundance was low. Similar to the sequence IKVIEGpSLQPIIK(682―694) identified in Fig.2, there were two missing cleavages at Lysine 502 and 504 by trypsin digestion in pSLHEKLKQDK(498―507).
Fig.3
MS/MS spectrum of a doubly-charged peak molecular ion at m/z 653.74 identified as pSLHEKLKQDK(498―507)
Phosphorylation modification at Serine 498 in periostin was identified.
Besides the two above phosphorylated peptides identified, a third peptide sequence p[TT]VLYEC*C*PGYM*R(73―85) was identified as illustrated in Fig.4. Fig.4 shows the MS/MS spectrum of a doubly-charged molecular ion peak at m/z 873.63. Most of the fragment ions of y series were easily assigned. The detection of b2 and b3 ions suggested that either Thronine 73 or Thronine 74 was phosphorylated. Because b1 ion is not stable in the collision-induced dissociation process, it cannot be easily observed by tandem mass spectrometry. Therefore, it is impossible to determine the exact phosphorylated position for this peptide sequence by examining the MS/MS spectrum of this peptide. Additionally, the Methionine 84 was found to be oxidazed. It could be easy to see that there was no missing cleavage site in the identified peptide sequence.
Fig.4
MS/MS spectrum of a doubly-charged molecular ion peak at m/z 873.63 identified as p[TT]VLYEC*C*PGYM*R(73―85)
Phosphorylation modification at Thronine 74 in periostin was identified, whereas Methionine 84 was found to be oxidized. C* denotes the carbarmidomethylation of the Cysteine residue.
4
Fig.2
MS/MS spectrum of a doubly-charged molecular ion peak at m/z 759.83 identified as IKVIEGpSLQPIIK(682―694)
Phosphorylation modification at Serine 688 in periostin was identified.
Conclusions
In this study, human periostin was over-expressed in HEK293T cells, which was enriched by nickel ion affinity resin and further purified by gel electrophoresis. Phosphopeptides contained in the tryptic digestion of the purified periostin were enriched by TiO2 affinity chromatography, and then analyzed by LC-MS/MS. Three phosphorylation sites within periostin
24
Chem. Res. Chin. Univ.
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were identified. The established method can be a great help to profiling the phosphorylation status of periostin in different physiological environments, such as inflammatory and tumor micro-environments.
[10] Yang G. Y., Zhao H. F., Xie F., Li Y., Sun L., Shi Q. H., He C. Y., Liu
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