Chiang Mai J. Sci. 2008; 35(2) 311

ABSTRACTDeglycosylation with anhydrous hydrogen fluoride (HF) is an alternative method for

removing oligosaccharides from glycoproteins which can be extremely useful for identificationof proteins and the biological roles of post-translational modifications. In this study, aglycosylated proteome of human serum was treated by anhydrous HF to deglycosylate thenumber of oligosaccharides from glycoproteins which facilitated protein identification usingproteomic analysis. In the preliminary result, the high performance liquid chromatography(HPLC) and liquid chromatography mass spectrometry (LC-MS) showed that there was nocleavage of disulfide bonds in HF-treated insulin as a negative control. The effect of HFdeglycosylation on electrophoresis pattern was studied by resolving on one-dimensional (1-D)and two-dimensional (2-D) gels. Deglycosylation of glycoproteins in human serum resulted indifferent protein patterns on 1-DE and 2-DE gels with the clearer protein patterns and lowamount of complexity. The deglycosylated serum proteins could be enriched and identifiedby MS analysis. Using this approach, it indicated that the proteins in human serum have someglycosylation that affected to the protein analysis and might possess the diverse biologicalfunctions. Therefore, this deglycosylation technique is an effective method to solve the problemof oligosaccharide interference in proteome analysis and be able to use for further glycananalysis.

Keywords: glycoprotein, deglycosylation, anhydrous hydrogen fluoride, human serum,two-dimensional electrophoresis.

Proteomic Analysis of Deglycosylated Proteins inNormal Human Serum Using Anhydrous HydrogenFluoride TreatmentSupawadee Sriyam [a], Supachok Sinchaikul [b], Payungsak Tantipaiboonwong [c],Suree Phutrakul* [a] and Shui-Tein Chen** [b, d][a] Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand.[b] Institute of Biological Chemistry and Genomic Research Center, Academia Sinica, Taipei, 11529, Taiwan.[c] School of Cosmetic Science, Mae Fah Luang University, Chiang Rai, 57100, Thailand.[d] Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, 10617, Taiwan.Author for correspondence; *e-mail: scchi007@chiangmai.ac.th; **e-mail: bcchen@gate.sinca.edu.tw

Received: 3 August 2007Accepted: 16 January 2008.

1. INTRODUCTIONMany proteins in eukaryotes are glyco-

sylated proteins that are occurred inside thecells, both in cytoplasm and subcellularorganelles. Protein expression is important in

many disease states, but the post-translationalmodification (PTMs) of proteins also play amajor role in the biological system and mostlyoccurred during disease development and

Chiang Mai J. Sci. 2008; 35(2) : 311-323www.science.cmu.ac.th/journal-science/josci.htmlContributed Paper

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progression [1,2]. Glycosylation is one of amajority of PTMs in eukaryotic cells andconsists of mainly two types of proteinglycosylation: N-glycosylation, the structureof the linkage region between carbohydrateand protein is β-glycosidically attached viaN-acetylglucosamine (GlcNAc) to the amidegroup of asparagine residues; and O-glyco-sylation, the glycans attached at a serine,theronine, hydroxylysine, or hydroxylproline[3,4]. However, several methods have beendeveloped for the analysis of glycoproteinsby proteomic approaches. Gel electrophoresisis one of the most widely employedbiochemical techniques for detection of glyco-protein from various biological materials[5-7]. Although glycoproteins can be analyzedand separated by protein separation techniquesincluding two-dimensional gel electrophoresis(2-DE), which separated the proteinsaccording to their molecular weight and pIvalues, the number of oligosaccharidescomposed in glycoproteins often resulted inmany glycoforms of glycoprotein spots and/or bands, board protein bands and even smearbands or streaks on 2-D gel due to theheterogeneous glycosylation pattern [8,9].

Deglycosylation technique has beenused in attempts to remove the glycan chainsattached to proteins in order to reduce thecarbohydrate interactions and proteomecomplexity. Therefore, deglycosylationtechnique may become an efficient way toremove the oligosaccharides resulted inmultiple forms of glycoproteins and to obtaina single protein. It may also offer the key toobtain high quality of protein pattern for thenext step of protein identification. In general,the carbohydrate chains that covalently linkedto protein or peptide backbone can bereleased by either enzymatic or chemicalmethod that has been used to identify theglycosylation site in cellular organelles orsurface membrane. Many reports have been

presented the removal of glycan groups fromglycoprotein by chemical methods to effectthe intact polysaccharide according to theanalysis by molecular mass determinationand amino acid sequencing [10-12]. Amongthese methods, the deglycosylation withchemical reagent is one of the most effectivemethods that can be completely removedall carbohydrate group both N-linked andO-linked glycosylation in only one step.

In the present study, we focused on theremoval of all oligosaccharides that attachedto proteins by chemical deglycosylationmethod using anhydrous HF reagent treatedto samples prior to analysis. The effects ofHF treatment on the cleavage of oligosac-charides and the change of molecular weightand/or pI value of proteins in samples weredetermined using proteomic approach. Aftertreatment, the protein pattern in human serumsample could be visible on 1-DE and 2-DEgel patterns with the unique protein spots and/or bands. Therefore, this strategy can be usedin sample preparation that would enable torelease of carbohydrate chains from differentbiological materials.

2. MATERIALS AND METHODS2.1 Chemicals and Materials

Standard glycoprotein alpha-1-acidglycoprotein (Orosomucoid, 99% purity) andPNGase F from Elizabethkingia (Chryseobac-terium/Flavobacterium) meningosepticum werepurchased from Sigma-Aldrich (St. Louis,MO, USA). Anhydrous hydrogen fluoride waspurchased from Matheson Gas Products Inc.(Ottawa, ON, USA). HF cleavage apparatus(model FC2002S) was obtained from TOHOCo. (Chigasaki, Japan). Human serum samples(healthy donors) were provided fromLampang Regional Cancer Center, Lampang,Thailand. A commercial protein assay kit waspurchased from Bio-Rad (Hercules, CA, USA).Gel electrophoresis running system and

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reagents were obtained from GE Healthcare(Uppsala, Sweden). Ruby was purchasedfrom Molecular Probes (Eugene, OR, USA).

2.2 Deglycosylation of Glycoprotein2.2.1 Anhydrous Hydrogen Fluoride (HF)Treatment

Deglycosylation of sample using anhy-drous HF treatment was performed in a HFcleavage apparatus [13]. Briefly, the glyco-protein sample was added with anhydrous HFand incubated at 0oC for 1 h, with consistentgentle agitation. The reaction was quenchedby freezing in liquid N2 and the remained HFwas removed by vacuum pump. The deglyco-sylated protein was dissolved in distilled waterand lyophilized. Since anhydrous HF isextremely hazardous and toxic, the wholeapparatus was kept in a fume hood. The proteinconcentration was determined using acommercial protein assay kit with bovinealbumin (BSA) as a standard.

2.2.2 Removal of N-Linked Oligosac-charides by PNGase F Treatment

PNGase F was used to release the N-linked oligosaccharides from glycoprotein.Briefly, 100 μg of lyophilized glycoproteinsample was dissolved in 50 μl of 50 mMphosphate buffer, pH 7.5. The sample wasadded with the denaturing buffer containing0.1% SDS and 2% β-mercaptoethanol andheated at 95oC for 5 min. For deglycosylation,the addition of 5 μl of 7.5% NP-40 and 5Units of PNGase F was performed. Thereaction mixture was incubated overnight at37oC. The free glycan was removed fromsample by precipitation with ice-cold ethanoland centrifugation at 10,000 rpm for 15 min[14]. The deglycoslyated protein was desaltedby using PD10 desalting column and thenlyophilized. The protein concentration wasdetermined using a commercial protein assaykit with bovine albumin (BSA) as a standard.

2.3 Protein Analysis Methods2.3.1 Reverse-Phase High PerformanceLiquid Chromatography (RP-HPLC)

Analysis of deglycosylated sample wasperformed using a reverse-phase HPLCcolumn (4.6x250 mm, Nucleosil 7C18)attached to a L-7400 UV-VIS detector and aL-7100 pump (Hitachi model, Tokyo, Japan)with a 20 μL sample injection loop. Twomobile phases, A (0.1% v/v trifluoroaceticacid (TFA) and 5% v/v acetronitrile (ACN))and B (0.1% v/v TFA and 95% v/v ACN),were used for all samples. All mobile phaseswere filtered through a 45-μm Millipore filterand degas before use. Each sample wasresuspended in mobile phase A , filtered andinjected into the HPLC column with a finalvolume of 10 μL. A linear gradient of 5%ACN to 95% ACN (0-100% mobile phase B)was used for 30 min running time at a flowrate of 1.0 ml/min. Protein was monitoredby measuring absorbance at 280 nm.

2.3.2 Liquid Chromatography Mass Spec-trometry (LC-MS)

The active compound/protein wasanalyzed by using a high-resolution ESI-TOFmass spectrometer (BioTOF III; BrukerBruker Daltonics, Inc.; Billerica, MA, USA).

2.3.3 SDS-PAGEThe lyophilized protein sample was

resuspended in 10 mM Tris-HCl, pH 7.5 anddesalted with dialysis bag overnight at roomtemperature, followed to PD-10 desaltingcolumn. Samples were separated underdenaturing conditions in 15% polyacrylamidegel using a Laemmli buffer system [15]. Thesamples were dissolved in sample buffercontaining 50 mM Tris-HCl, pH 6.8, 0.1 MDTT, 10% glycerol, 2% SDS and 0.1%bromophenol blue at a concentration of1 mg/ml and heated at 95oC for 5 min. Eachsample solution (10 μg/well) was separately

314 Chiang Mai J. Sci. 2008; 35(2)

loaded into gel wells. The SDS-PAGE gel wasrun in a BioRad Mini-Protean II apparatus at20 mA per gel. After completion of electro-phoresis, the protein bands in the gel werevisualized by Ruby staining andscanned by using a Typhoon 9200 imagescanner (GE Healthcare). The Low-rangemolecular weight calibration kit (GE Healthcare)was used as standard molecular weight proteinmarkers.

2.3.4 2-DEThree hundred and fifty microgram of

proteins was firstly treated with SDS solutionbuffer containing 0.2% SDS and 2.5 mMdithioerythreitol (DTE) and heated in aheating block at 95oC for 5 min [16]. Aftertreatment, the protein sample was solubilizedin 350 μl of lysis buffer containing 7 M urea,2 M thiourea, 4% CHAPS, 4 mM Tris base,65 mM DTE, 5 mM tributylphosphine (TBP)and 0.5% IPG buffer (pH 4-7 or pH 3-10NL)and followed to incubate for 1 h at roomtemperature. The sample was centrifuged at12000 rpm for 20 min and then loaded intoan 18 cm IPG strip (pH 4-7 or pH 3-10NL,GE Healthcare). Gel rehydration was carriedout for 14 h at 50 V according to theprogrammed setting: (1) 100 V, 100 Vh; (2)250 V, 250 Vh; (4) 500 V, 500 Vh; (5) 1000,1000 Vh; (6) 3000, 3000 Vh; and (7) 8000 V,60000 Vh. Following IEF, the IPG strips werereduced and alkylated in equilibration buffer(50 mM Tris-HCl, pH 8.8, 6M urea, 30% v/vglycerol, 2% w/v DTE and a trace ofbromophenol blue), and then subsequentlyalkylated in the same buffer that replaced DTEwith 2.5% w/v iodoacetamide (IAA) for 15min. Each equilibrated IPG strip wastransferred onto vertical 10-18% lineargradient polyacrylamide gel (18x18x1.5 cm)and covered with 0.5% agarose. The second-dimensional separation was carried out at45 mA per gel for approximately 5 h at 15oC

until the bromophenol blue dye front reachedthe bottom of the gel. After electrophoresis,the 2-D gels were stained with Rubyand scanned using a Typhoon 9200 imagescanner at 200 nm resolution. Image analysiswas carried out using the ImageMaster2D Platinum software version 5.0 (GEHealthcare).

2.3.5 Tryptic DigestionThe interested proteins were excised and

transferred into a cleaned 0.5 ml siliconizedmicrocentrifuge tube. The gel pieces wereextensively washed twice with 200 μl of 50%ACN/25 mM ammonium bicarbonatebuffer, pH 8.5, for 15 min each. Then, the gelpieces were washed once with 200 μl of 100%ACN and dried using a Speed-Vacuumconcentrator. Reduction and alkylation wereaccomplished with 50 mM DTE and 100mM IAA. Dried gel pieces were swollen in10 μl of 25 mM ammonium bicarbonatebuffer, pH 8.5, containing 0.0225 μg trypsin,crushed with siliconized blue stick andincubated at 37oC for at least 16 h. Peptideswere subsequently extracted twice with 50 μlof 50% ACN/1% TFA, then the extractedsolutions were combined and dried using aSpeed-Vacuum concentrator. The peptides orpellets were resuspended in 5 μl of 50%ACN/0.1% TFA.

2.3.6 MALDI-TOF MS and MS/MSThe samples were premixed in a ratio

of 1:1 with matrix solution (5 mg/ml CHCAin 50% ACN, 0.1% v/v TFA and 2% w/vammonium citrate) and spotted onto the 96-wells formatted MALDI sample stage. Datawas directed acquisition on the Q-TOFUltimaTM MALDI instrument (M@LDITM;Micromass, Manchester, UK) was fullyautomated with predefined probe motionpattern and the peak intensity threshold forswitching over from MS survey scan to MS/

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MS, from one MS/MS to another. Within eachwell, as many parent ions meeting thepredefined criteria (any peak within the m/z800-3000 range with intensity above 10 count± include/exclude list) will be selected forCID MS/MS using argon as collision gas anda mass dependent ±5V rolling collision energyuntil the end of the probe pattern was reached(all details are available at http://proteome.sinica.edu.tw) Proteins were identified fromthe peptide mass maps using the MASCOTonline database (http://www.matrixscience.com) to search the non-redundant proteindatabase.

3. RESULTS AND DISCUSSION3.1 Effect of HF Deglycosylation on ProteinStability

Chemical deglycosylation method is non-specific for the removal of either N-linkedor O-linked glycans. A variety of reagents andtreatment conditions are very important fordeglycosylation and lead to cause denaturationof proteins [17,18]. In this study, anhydrousHF is a deglycosylated reagent and used tostudy the effect of chemical deglycosylationon protein stability, especially disulfidebond that can be occurred both inter- and

intramolecular polypeptide chain of proteins.Since the structure of insulin is a small proteinscontaining fifty one amino acid residues andcomposed of two polypeptide chains thatheld together by two interchain disulfidebonds [19], it was therefore used as a negativecontrol or standard protein to determine thestability of peptide bonds under the HF-deglycosylation treatment. After treatment, thestability of disulfide bonds in HF-treatedinsulin was examined by RP-HPLC and themolecular weight of HF-treated insulin wasanalyzed by LC-MS. The RP-HPLC resultshowed single peak of HF-treated insulin withthe same retention time as standard insulin(Figure 1). It indicated that anhydrous HFreagent could not cleave the disulfide bondsin HF-treated insulin sample. In addition, therewas no change of its molecular weight afterHF treatment with a constant molecularweight (MW) of approximately 5735 Da(Figure 2). These results indicated thatdeglycosylation method using HF treatmenthad no effect on the stability of peptide bondsin standard protein used. Therefore, thisdeglycosylation technique would beparticularly useful for identifying andcharacterizing the deglycosylated proteome.

Figure 1. HPLC chromatograms of (A) a standard insulin and (B) HF-treated insulin thatanalyzed using RP-HPLC for investigation of the disulfide bond digestion, showing a majorpeak of insulin treated with anhydrous HF and non-treated appearing at the same peak ofretention time approximately 18 min, is indicated by the arrow.

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Figure 2. LC-MS profile obtained from the standard insulin treated with anhydrous HF,shown in (A) and (B) shows the signal from the HF-treated insulin which was observed atm/z 5735 Da.

Figure 3. SDS-PAGE results represent the deglycosylated samples by two deglycosylationmethods, PNGase F and HF treatment. Two standard glycoproteins of AGP1 and AHSGwere used in these treatments and separated on 15% polyacrylamide gel, then stained withCoomassie blue. (A) Deglycosylation of AGP1. Lane 1, standard AGP1; lane 2, AGP1deglycosylated with PNGase F. (B) Deglycosylation of AHSG with PNGase F. Lane 1, standardAHSG; lane 2, AHSG deglycosylated with PNGase F. (C) Deglycosylation of AGP1 usinganhydrous HF treatment. Lane 1, standard AGP1; lane 2, HF-deglycosylated AGP1; M, proteinmarkers.

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3.2 Determination of DeglycosylatedStandard Glycoprotein

Comparison of deglycosylation methodsbetween enzymatic deglycosylation (PNGaseF) and chemical deglycosylation (HFtreatment) was carried out by using twostandard glycoproteins, alpha-1-acidglycoprotein (AGP1) containing N-linkedglycans and alpha-2-HS glycoprotein (AHSG)containing both N- and O-linked glycans[20,21].The deglycosylated AGP1 and AHSGsamples under both treatments were resolvedon SDS-PAGE and the change of protein’smolecular weight was examined. AfterPNGase F treatment, the protein band ofAGP1 generally located at 45 kDa shifted to20 kDa (Figure 3A) whereas the protein bandof AHSG generally located at 57 kDa shiftedto 45 kDa (Figure 3B). In addition to HFtreatment, the protein band of AGP1 showed

a few of deglycosylated protein bands withthe major protein band or completeddeglycosylated protein band at 20 kDa (Figure3C). It indicated that the deglycosylation withPNGase F provided the completeddeglycosylation of oligosaccharides from bothAGP1 and AHSG whereas the deglycosylationwith HF treatment provided uncompleteddeglycosylation reaction of AGP1. In orderto confirm the uncompleted deglycosylationby HF treatment, the upper protein bandsover 45 kDa were excised from SDS-PAGEgel and subsequently identified the proteinsby MALDI-TOF MS (Figure 4). The HF-deglycosylated AGP1 upper proteins were stillidentified as AGP1. We suggested that thedeglycosylation of AGP1 by HF treatmentmight not sufficiently remove the glycans fromglycoprotein and the HF treatment conditionshould be optimized to get the high efficiency

Figure 4. Protein identification result of a HF deglycosylated AGP1. (A) MALDI-TOF-MSpeptide mapping analyses of the deglycosylated AGP1; (B) MS mowse score and (C) summaryof protein identification based on NCBI database search.

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of deglycosylation. Although the enzymatictreatment has a high specificity of anappropriate enzyme that allowed to cleavageof a definite saccharide depending onglycosylation sites [22] and gave the completelydeglycosylation result rather than HFtreatment, the enzymatic treatment still had alimitation of individual enzyme and necessaryto use multiple enzymes to remove severaltypes of glycan chains attached on glycopro-teins [23,24]. In contrast, deglycosylation byHF treatment has the advantage in removalof all glycans in glycoprotein regardless ofstructure or glycosylation site. However, thechemical deglycosylation technique using HFreagent can be used as an alternative methodfor further study of deglycosylated proteomein human serum samples.

3.3 Effect of HF Deglycosylation onGlycoproteins in Human Serum

Human serum is a complex biologicalsample containing a large number of proteinsand glycoproteins. Many proteins in serumpresent complex combinations of post-translational modifications (PTMs) , such asglycosylation that can be discriminated by 2-DE [25-27]. Due to the complexity of humanserum, a number of variables need to beconsidered including sample preparation priorto analyisis. Deglycosylation technique is thusan alternative method for enriching theamount of proteins and determining thestructure of glycoproteins in humanproteome. In the present study, we usedanhydrous HF reagent to deglycosylate theglycoproteins in normal human serum andexamined the deglycosylated proteomepatterns. The pattern of proteins betweenuntreated serum sample (control) and HF-deglycosylated serum sample were inves-tigated by proteomic approaches. Thedeglycosylated proteins in human serumsamples resulted in different protein patterns

on SDS-PAGE (Figure 5). Besides the use of2-DE, it is another way to separate and analyzethe isoforms of deglycosylated proteins withthe high-resolution of separation. Using2-DE, both 2-DE gel patterns of HF-treatedsample in a wide pH range of 3-10 NL and anarrow pH range of 4-7 were distinctlydifferent from untreated sample, in which the

Figure 5. Analysis of normal human serumsample before and after deglycosylation withanhydrous HF by a 12.5% SDS–PAGE.Following electrophoresis, the gel was staineddirectly by Ruby staining. Labels; M,protein markers; Lane 1, normal humanserum; lane 2, HF-treated human serumsample.

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complexity of protein patterns was reducedand the neat or unique proteins could beenriched (Figure 6). Figure 7 shows a represen-tative example of the protein spots on a narrowrange 2-D gel image that were matchedbetween untreated and treated with anhydrousHF in normal human serum using ImageMaster software. After matching analysis,

there was significant change in the total numberof detectable protein spots by 2-DE analysis.Approximately 46 total match of proteinspots were detected whereas the controlsample exhibits the reduction of total proteinspots were observed from 364 to 222 proteinspots in HF deglycosylated serum sample.

The appearance of broad albumin band

Figure 6. 2-DE image of normal human serum as control and HF-deglycosylated serumsample. The samples were applied to a pH 3-10 NL IPG strip (A, B) and a narrow range pH4–7 IPG strip (C, D) for the first dimension and the 10-18% SDS–PAGE for the seconddimension and stained with SYPRO Ruby gel staining.

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in both treated and untreated serum sampleswas still revealed on 2-DE gel image with thehigh molecular weight at 65 kDa because thealbumin is the highest abundant protein inhuman serum and the deglycosylated albuminstill contained the major protein that locatedat the same position and seemed to be notdifferent. In contrast to the high abundanthaptoglobin at the MW approximately 45 kDaas to be haptoglobin β-chain, the 2-DE gelimages showed the clear protein band,indicating the successfully deglycosylation ofhaptoglobin and enriching the proteins at thatarea. In addition, some deglycosylated proteinsin HF treated samples had some modificationswith MW and pI shifts. It indicated thatHF could remove the oligosaccharides inglycoproteins and provided the deglycosylatedproteins with different pI and MW. In additionto these modifications, it may be caused bythe strongly acidification of proteins duringHF treatment. Unfortunately, the chemical

Figure 7. Spot detection using ImageMaster software for comparison of the narrow range2-DE gel image between (A) normal human serum and (B) HF-treated human serum sample.The marked spots in green color were proteins that showed the match protein spots.Additionally, the differential expressed spots in HF treatment sample were indicated byred color.

deglycosylation method for deglycosylatingglycoproteins in human serum resulted in theincompletely removal of carbohydrate andextensive degradation of peptide core, whichis a limitation of this technique. The 2-DE gelpatterns also showed a vertically smearedprotein bands due to the remaining anhydrousHF in samples. Although the 2-DE results ofHF treated serum showed a slightly poorresolution of protein separation, the HFtreatment could deglycosylate the oligosac-charides of glycoproteins and enriched thelow abundant proteins. Moreover, thedeglycosylated proteins in human serumshowing in neat proteins may be served as apotential indicators or a wealthy biologicalsource for discovery of biomarker [28, 29].Therefore, deglycosylation using HF may bean alternative method for deglycosylation ofglycoproteins, reducing the sample complexityand enriching the neat proteins. However, thecondition of this method still needs to be

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optimized for improving the deglycosylationefficiency and then able to use in the first stepin the human serum proteome analysis.

4. CONCLUSIONDeglycosylation is an alternatively

challenging method for analyzing the size ofthe protein and the biological role of post-translational modification. After HF deglyco-sylation, there was no effect of HF on thecleavage of disulfide bonds in the insulin asstandard protein. The protein patterns of HF-treated samples showed the high reductionof complexity, in which the contributionof N- and O-linked glycosylations waseliminated, and the enrichment of lowabundant proteins or neat proteins. Therefore,this method may be alternative used in thesample preparation of various humanmaterials such as serum and urine that havethe high complexity of protein samples. Inaddition, the anhydrous HF reagent may beused as a valuable chemical reagent fordeglycosylation of glycoprotein to determineunknown structures of glycoproteins and toreduce all glycosylation variants. However, ourresults are the preliminary study of HFdeglycosylation in proteome analysis and thismethod will be further optimized forimproving the deglycosylation of glycoproteinprior to protein analysis.

ACKNOWLEDGEMENTSThis research is part of the Royal Golden

Jubilee Ph.D. project of Ms. SupawadeeSriyam supported by the Thailand ResearchFund (TRF), Thailand. This work is alsosupported by the Graduate School, ChiangMai University, Chiang Mai, Thailand. Wegratefully acknowledge the System BiologyLaboratory (SBL), Institute of BiologicalChemistry, Academia Sinica, Taiwan, for thepart of proteomics experiments. Finally, wegratefully thank the Lampang Regional Cancer

Center, Lampang, Thailand for providing theserum samples.

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