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(1.Department of Biochemistry and Molecular Biology, Gannan Medical University, Ganzhou, Jiangxi 341000；2.Department of Pharmaceutical Sciences, UNT System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, TX 76107)

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Identification of albumin precursor protein from the mitochodria by the diagonal gel electrophoresis*

LUOXiao-ting1,2,YANLiang-jun2

(1.DepartmentofBiochemistryandMolecularBiology,GannanMedicalUniversity,Ganzhou,Jiangxi341000；2.DepartmentofPharmaceuticalSciences,UNTSystemCollegeofPharmacy,UniversityofNorthTexasHealthScienceCenter,FortWorth,TX76107)

Abstract:Objective: to separate and indentify the albumin precursor protein from the mitochodria. Methods: the mitochodria were separated from rat liver or kidney by centrifugation; the albumin precursor protein was separated from the mitochodria by the diagonal gel electrophoresis with the first non-reducing SDS-PAGE and the second reducing SDS-PAGE. Protein was identified further by mass spectrometric peptide sequencing. Results: there was a distinct spot that ran above the diagonal line in the gel after the diagonal gel electrophoresis; the albumin precursor proteins were identified in this spot by the mass spectrometric peptide sequencing. Conclusion: the albumin precursor protein from the mitochodria can be identified by the diagonal gel electrophoresis method with the mass spectrometric peptide sequencing.

Key words:albumin precursor protein;diagonal gel electrophoresis;disulfides;mitochondria;redox modifications

0Introduction

Albumin precursor protein was first found in the cytoplasmic particles of chicken liver in 1957[1]. It is initially synthesized and is subsequently transformed into serum albumin[2].Albumin precursor proteins exist in the liver[3-4],kidney[5-6],brain[7],skin[8],ovary[9],amnion[10],vascular smooth muscle cell[11],plasma[12],tear[13-17],aqueous humor[18],cerebrospinal fluid[19-21],and so on. The expression of albumin precursor protein was regulated in various diseases[3-21].In addition,the endogenous peptide conserved and generated from the albumin precursor by pH-regulated proteases, antagonizes CXCL 12-induced tumor cell migration,mobilizes stem cells, and suppresses inflammatory responses in mice[22].

Albumin precursor protein has 35 cysteine residues. Cysteine residues in proteins and enzymes often fulfill rather important roles, particularly in the context of cellular signaling, protein-protein interactions, substrate and metal binding, and catalysis. At the same time, some of the most active cysteine residues are also quite sensitive toward (oxidative) modification. S-Thiolation,S-nitrosation,and disulfide bond and sulfenic acid formation are processes which occur frequently inside the cell and regulate the function and activity of many proteins and enzymes[23].Many proteins are involved in disulfide formation. Diagonal electrophoresis is a relatively simple technique to analyze the formation of protein disulfides by sequential non-reducing/reducing electrophoresis. In this paper, we explored whether albumin precursor proteins could be identified by the diagonal electrophoresis method with the mass spectrometric peptide sequencing.

1Materials and methods

1.1Chemicalsβ-mercaptoethanol was purchased from Sigma. Ammonium persulfate, bis-acrylamide, acrylamide and Coomassie brilliant blue (CBB) G-250 were from Bio-Rad laboratories (Richmond, CA). Bis-Tris was from Calbiochem (La Jolla, CA). SDS-PAGE markers were from Invitrogen (Carlsbad, CA).

1.2Isolation of mitochondrial Rat liver or kidney mitochondria were prepared as previously described[24].Briefly, one gram of liver was homogenized in 10 mL mitochondria isolation buffer containing 70 mM sucrose, 230 mM mannitol, 15 mM MOPS (pH 7.2), and 1 mM EDTA. The homogenate was centrifuged at 800 g for 10 min and the resulting supernatant was collected for further centrifugation at 8,000 g for another 10 min. The resulting pellet containing mitochondria was washed once with the homogenization buffer and centrifuged once again at 8,000 g for 10 min. All the centrifugation steps were performed at 4 ℃. The isolated mitochondria were either used immediately or stored at -80 ℃until analysis.

1.3Diagonal gel electrophoresis The diagonal gel electrophoresis using a Bio-Rad Mini-PROTEAN III electrophoresis cell involves two dimensional SDS-PAGE.Samples for the first dimension were usually non-reducing and that for the second dimension are usually reducing (non-reducing/reducing).The isolated mitochondria were resuspended in nonreducing sample buffer (4×) to give a final protein concentration of 1 mL·mg-1. Prepare a 10% polyacrylamide resolving gel with 4% stacking gel. Add 30 μL non-reducing sample buffer containing 30 μg of mitochondrial protein to a well.The first dimensional electrophoresis was carried out at 150V until dye reaches the end of the gel. The entire lane was excised using a sharp fine scalpel and incubated in a reducing buffer that contained the reductant 2% β-mercaptoethanol at room temperature on a rocker for 30 min. The lane was then over-laid horizontally onto a second dimensional SDS-PAGE. The second dimensional electrophoresiswas carried out at 150V until dye reaches the end of the gel. The gel was stained by coomassie brilliant blue G-250 on a rocker for 1 h and then destained by the decoloring agent containing 10% glacial acetic acid and 10% methanol on a rocker for 4 h. However, two controls were devised:samples for the first dimension were non-reducing and that for the second dimension are non-reducing (non-reducing/non-reducing); samples for the first dimension were reducing and that for the second dimension are reducing (reducing/reducing).

1.4Mass spectrometric identification of proteins Protein identification was carried out at ProtTech (Norristown, PA) using the NanoLC-MS/MS peptide sequencing technology. Briefly, a given gel spot was destained, cleaned, and in-gel digested with sequencing grade trypsin. The resulting peptide mixture was analyzed by an LC-MS/MS system, in which a high pressure liquid chromatography with a reverse phase C18 column (inner diameter: 75 μm) was coupled on-line with an ion trap mass spectrometer. The collected mass spectrometric data were used to search the most recent non-redundant protein database using ProtTech’s proprietary software suite.

2Results

Liver mitochondria were isolated from rats. Mitochondrial samples underwent 3 different process modes (control groups: non-reducing/non-reducing and reducing/reducing, experimental group: non-reducing/reducing ). As shown in Fig. 1, the two control experiments did not yield any protein spots that deviate from the diagonal line. Instead, a perfect linear line under both conditions could be visualized (Fig. 1).

Shown are (A): Non-reducing/non-reducing; (B) Reducing/reducing. In A, protein samples were not treated by β-mercaptoehanol before gel loading, nor the resulting gel bands before applying onto the second dimensional gel. In B, samples for both dimensions were treated by β-mercaptoehano.

Fig 1Diagonal gel analysis of kidney mitochondrial proteins

As shown in Fig. 2A, there is a distinct spot that ran above the diagonal line in the experimental group. This spot was then excised and subjected to mass spectrometric peptide sequencing, a total of 17 peptides were obtained that matched to albumin precursor protein (Table 1, left side). To further confirm that albumin precursor protein indeed exists in mitochondria, we then analyzed rat kidney mitochondria under the same experimental conditions. As shown in Fig. 2B, there was also a spot above the line, which was also sequenced. As a result, a total of 15 peptides were obtained that also matched to albumin precursor protein (Table 1, right side).

Fig.2Identification of albumin precursor protein in both liver (A) and kidney (B) mitochondria

Table 1Peptides obtained from gel spots indicated in

Fig. 2, panels A and B, respectively

Livermitochondrialspot(17peptides)Kidneymitochondrialspot(15peptides)KYEATLEKLSQKFPKYEATLEKDLGEEHFKFKDLGEQHFKFKDLGEEHFKAPQVSTPTLVEAARHLVDEPQNLIKLVQEVTDFAKLKHLVDEPQNLIKAPQVSTPTLVEAARKVPQVSTPTLVEVSRSIHTLFGDKLVNELTEFAKQTALAELVKRHPEYAVSVLLRFPNAEFAEITKRHPEYAVSVLLRRHPDYSVSLLLRKQTALVELLKLGEYGFQNAILVRQTALVELLKRHPYFYAPEL-LYYAEKLGEYGFQNALIVRRHPYFYAPEL-LYYAEKRHPYFYAPELLYYANKRHPYFYAPEL-LYYAEKHPYFYAPELLYYANKHPYFYAPELLYYAEKDAFLGSFLYEYSRDVFLGTFLYEYSRGLVLIAFSQYLQK

The arrow indicates the spot excised for mass spectrometric identification. For liver mitochondria, the spot yielded 17 peptides that matched to albumin precursor protein; while for kidney mitochondria, the spot produced 15 peptides that matched to albumin precursor protein.

3Discussion

From these results, albumin precursor was identified sucessfully from the mitochondria of liver or kidney. These are in good agreement with previous studies that albumin precursor can be localized to mitochondria[25]. In many literatures, albumin precursor proteins were identified by proteomic profiling using two-dimensional electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry[5-21].Nevertheless, we have, for the first time, shown that mitochondrial albumin precursor protein has endogenous disulfide bonds that can be analyzed by the diagonal gel electrophoresis method with the mass spectrometric peptide sequencing.Based on the nature of disulfides that are formed, the diagonal gel can elegantly characterize whether the disulfides are intra- or inter-proteins: after staining, proteins that do not contain disulfide bonds congregate along a diagonal since they electrophorese identically in both reducing and non-reducing conditions; proteins involved in inter-chain disulfide bonds will fall below the diagonal as they have a lower apparent molecular mass in reducing conditions; proteins that contain intra-chain disulfide bonds will run above the diagonal as they have a larger apparent molecular mass[26].Albumin precursor protein ran above the diagonal line in the experimental group, so albumin precursor protein contain intra-chain disulfide. However, it should be noted that albumin precursor has 35 cysteine residues, 34 of which are in the form of disulfides. It’s likely that the second dimensional reducing gel analysis broke more than one disulfide bridges; nonetheless, it appears that we could only observe one spot above the diagonal line under our experimental conditions. There were other proteins present in each spot, but the abundance of which was much less than that of albumin precursor protein based on spectral count number that reflects the number of peptides sequenced[24,27].It should also be noted that albumin precursor protein has a single free cysteine residue that is highly redox sensitive[11].Excess levels of reactive oxygen species (ROS) can lead to reversible redox proteomic responses on protein thiols that can include, inter-/intra-disulfide bond formation, glutathionylation, nitrosylation, and sulfenic acid formation. This cysteine residue theoretically can form a disulfide bond with the same cysteine residue from another albumin precursor molecule, but we could not detect this type of disulfide in our gel analysis.

Protein disulfides are an integral feature of a given proteome[23，28-29].They are not only an inherent part of protein structure and function, but also involved in redox signaling and protein-protein interactions[30-31].While endogenous disulfides are indispensible for numerous redox-sensitive proteins and enzymes[23-32], those formed upon oxidative stress are usually involved in stress response and redox signaling[32-34].Protein cysteine residues are constantly undergoing redox modifications, and many of them are involved in disulfide formation. In this article,we first overviewed a diagonal gel analysis method for identification of protein disulfides for identification of mitochondrial albumin precursor proteins that form disulfide bonds upon oxidative stress. Nevertheless, it’s our intention to follow the identified proteins in our future studies, especially in the context of aging and aging-related diseases as well as in ischemic tolerance[35].

Acknowledgements:ThisworkwassupportedinpartbyNationalInstitutesofHealth(R01NS079792toL.J.Y.).TheauthorsthankDr.DrakeZhangatProtTechInc.formassspectrometricpeptidesequencing.XiaotingLuowassupportedbyVisiting-scholarspecificfundofyoungandmiddle-agedteachers’developmentprogrammeofgeneraluniversityinJiangxiProvince,China.

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斜線電泳分離線粒體白蛋白前體蛋白

羅曉婷1，2，晏良軍2

(1.贛南醫(yī)學(xué)院，江西贛州341000；2.美國北德州大學(xué),美國76107)

摘要：目的：分離并鑒定線粒體內(nèi)的白蛋白前體蛋白。方法：采用離心法分離大鼠肝及腎線粒體，采用斜線電泳法(第一相非還原SDS-PAGE，第二相還原SDS-PAGE)分離線粒體白蛋白前體蛋白，采用質(zhì)譜肽測序法鑒定蛋白質(zhì)。結(jié)果：斜線電泳膠上斜線上方出現(xiàn)一個點，該點經(jīng)質(zhì)譜肽測序進一步鑒定為白蛋白前體蛋白。結(jié)論：斜線電泳聯(lián)合質(zhì)譜肽測序能鑒定出線粒體中的白蛋白前體蛋白。

關(guān)鍵詞：白蛋白前體蛋白；斜線電泳；二硫化物；線粒體；氧化還原修飾

CLC Number：Q503

Document code：AArticle ID：1001-5779(2016)02-0167-05

DOI:10.3969/j.issn.1001-5779.2016.02.001

(收稿日期：2015-10-24)(責任編輯：敖慧斌)

*Founding support:This work was supported in part by National Institutes of Health (R01NS079792 to L.J.Y.) and visiting-scholar specific fund of young and middle-aged teachers’ development programme of general university in Jiangxi Province, China.Corresponding author:YAN Liang-Jun，male,associate professor,Ph.D.Research on protein oxidative modification.E-mail:liang-jun.yan@unthsc.edu