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A mass spectrometry approach for the study of deglycosylated proteins
Microchemical Journal 99 (2011) 309–311
Contents lists available at ScienceDirect
Microchemical Journal j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i c r o c
A mass spectrometry approach for the study of deglycosylated proteins Lancia N.F. Darville a, Mark E. Merchant b, Kermit K. Murray a,⁎ a b
Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, United States Department of Chemistry, McNeese State University, Lake Charles, LA 70609, United States
a r t i c l e
i n f o
Article history: Received 22 May 2011 Accepted 26 May 2011 Available online 1 June 2011 Keywords: Proteomics Mass spectrometry Deglycosylation
a b s t r a c t Mass spectrometry (MS) analysis, after enzymatic or chemical deglycosylation, requires preparatory steps to remove salts and buffers. In this work, the glycosylated protein fetuin and a lectin protein isolated from the serum of Alligator mississippiensis were used to evaluate methods for desalting samples after an enzymatic or chemical deglycosylation. Precipitation and dialysis were used to prepare the deglycosylated samples for MS analysis. Both the precipitation and dialysis methods were suitable for sample preparation prior to analysis by matrix assisted laser desorption ionization (MALDI) MS. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Proteomics is the study of the structure and functions of proteins in an organism or tissue, and mass spectrometry is now a routinely used method for protein identification in complex mixtures [1]. Prior to a typical mass spectrometry analysis, separation techniques such as two-dimensional gel electrophoresis and high performance liquid chromatography (HPLC) are used to reduce the complexity of protein mixtures [1,2]. Some proteins are post-translationally modified to help regulate gene expression, protein turnover and cellular structure [3]. Glycosylation is a common post-translational modification that is involved in biological functions such as immune recognition and inflammation [4]. There are two major types of protein carbohydrate linkages: N- and Olinked glycosylation. During N-glycosylation, the glycan is attached to an asparginine residue followed by any amino acid other than proline, which is linked to a serine or threonine residue. During O-glycosylation, the glycan is attached to a serine or threonine residue. Glycoproteins have been characterized by mass spectrometry, but this is challenging due to the heterogeneity of the carbohydrate moieties and the complexity of the resulting mass spectra [5]. Glycosylation of the protein may also limit the extent of proteolytic digestion. To avoid this, the N- and/or O-linked carbohydrates can be removed prior to further analysis [6,7]. A direct approach using enzymatic or chemical cleavage of the glycan can be used to enable the analysis of the protein without the oligosaccharides. An enzymatic approach using peptide: N-glycosidase (PNGase F) [8] and a chemical approach using trifluoromethanesulfonic ⁎ Corresponding author at: Louisiana State University, 337 Choppin Hall, Baton Rouge, LA 70803, United States. Tel.: + 1 225 578 3417; fax: + 1 225 578 3458. E-mail address: [email protected] (L.N.F. Darville). 0026-265X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2011.05.020
acid (TFMS) have been used to remove the oligosaccharides [9,10]. PNGase F is an enzyme that removes N-linked oligosaccharides, leaving both the protein and oligosaccharides intact for further analysis. To date, there have been no enzymatic techniques reported that cleave O-linked oligosaccharides. TFMS is a non-specific deglycosylating agent that cleaves both N-linked and O-linked oligosaccharides to leave the intact protein [11]. The proteins used in this study were enzymatically or chemically deglycosylated, followed by matrix assisted laser desorption ionization (MALDI) MS. The steps for both chemical and enzymatic deglycosylation approaches are fully established and has been reported previously [9,12,13], but the deglycosylated product requires cleanup prior to MALDI mass spectrometry. We have highlighted important sample desalting procedures that follow deglycosylation prior to mass spectrometry analysis, which are not clearly outlined in many procedures. This study focuses on the treatment of deglycosylated proteins for mass spectrometry analysis.
2. Experimental 2.1. Materials HPLC grade acetonitrile, formic acid, and trifluoroacetic acid (TFA) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. Fetuin, a plasma glycoprotein produced by the liver, was obtained from Sigma-Aldrich and the lectin protein was isolated from the blood plasma of Alligator mississippiensis. Blood from seven American alligators was collected; anticoagulated with heparin, and the blood was allowed to clot overnight at ambient temperature. The serum was collected and the lectin was isolated using mannan-agarose affinity chromatography.
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L.N.F. Darville et al. / Microchemical Journal 99 (2011) 309–311
2.2. Enzymatic deglycosylation Proteins containing oligosaccharides attached through N-linked glycosylation were deglycosylated using a previously developed protocol for enzymatic deglycosylation from New England BioLabs (Ipswich, MA) [8]. Approximately 100 μg of protein was dissolved in 18 μl of nanopure water. Denaturing buffer (2 μl), containing 0.6 mg/mL SDS and 0.5 mg/mL DTT, was added and heated at 100 °C for 10 min to increase the rate of deglycosylation. The sample was incubated on ice at 0 °C for five minutes and centrifuged for 10 s. To the denatured sample, a non-ionic detergent (NP-40) was added to prevent loss of enzyme activity. The PNGaseF enzyme was added to the sample and it was incubated at 37 °C for four hours [8,14]. The intact protein was isolated from the mixture via protein precipitation, which removed the buffers that can interfere with the MALDI signal. Acetone or trichloroacetic acid (TCA) was used to precipitate the protein [15].
was changed and dialysis was continued for an additional two hours. After a final buffer change, the sample was dialyzed overnight at room temperature. 2.7. Mass spectrometry Glycoproteins and deglycosylated proteins were analyzed in linear MS mode with a MALDI-mass spectrometer (Ultraflextreme, Bruker Daltonics, Billerica, MA, USA). The glycoproteins and the deglycosylated proteins (after dialysis or precipitation) were mixed on a steel target
2.3. TCA precipitation TCA precipitation was accomplished by the addition of one volume of 60% TCA. The sample was gently vortexed and incubated on ice overnight. The sample mixture was centrifuged at 10,000 g for 30 min and the supernatant was removed. Chilled acetone was added to wash the precipitate and it was centrifuged again at 10,000 g [16]. This procedure was repeated twice. The precipitate was air-dried and suspended in aqueous 0.1% trifluoroacetic acid (TFA) or 0.1% formic acid (FA) for MALDI analysis. 2.4. Acetone precipitation Acetone precipitation was achieved by the addition of four volumes of acetone to the protein sample, followed by an overnight incubation on ice. The sample was centrifuged at 10,000 g for 30 min at 4 °C. The supernatant was removed and the precipitate was air dried for 30 min, then suspended in the same buffer as used above in the TCA precipitation. 2.5. Chemical deglycosylation In-solution deglycosylation was performed using a deglycosylation kit containing trifluoromethanesulphonic acid (TFMS) (ProZyme, Hayward, CA, USA). TFMS removes most glycan types from glycoproteins and leaves the protein intact [9]. Briefly, approximately 0.3 mg of protein in a vial was dried overnight using a lyophilizer. The dried protein was capped with a septum and placed on a dry ice-ethanol bath for 20 s to reduce the temperature of the reaction. A mixture of TFMS and toluene solution was prepared and 50 μl was slowly added using a gas-tight syringe. Precautions were taken to prevent the dried protein from being exposed to moisture due to the potential for hydrolysis of the peptide bonds [9,17]. The vials containing the protein with TFMS mixture were incubated at − 20 °C for five minutes then shaken briefly to help dissolve the protein. The solvation process was repeated and followed by a four-hour incubation at −20 °C. 2.6. Dialysis Dialysis was performed prior to mass spectrometry analysis. After the four hour incubation, the vials containing the samples were placed in a dry ice-ethanol bath for 20 s, and then 150 μl of pyridine solution was added to each vial. The vials were incubated on dry ice for five minutes, and then in a dry ice-ethanol bath for 15 min. A 400 μl quantity of 0.5% ammonium bicarbonate was added to each vial to neutralize the sample. Following deglycosylation, the protein was isolated using dialysis. Each sample was added to a dialysis cassette (Thermo Scientific, Rockford, IL, USA) and dialyzed against 10 mM ammonium formate for two hours at room temperature. The buffer
Fig. 1. (a) MALDI spectrum of intact fetuin. (b) MALDI spectra comparing glycosylated and deglycosylated fetuin after acetone precipitation. (c) Fetuin after TFMS deglycosylation followed with dialysis.
L.N.F. Darville et al. / Microchemical Journal 99 (2011) 309–311
using a 1:1 ratio of sample and saturated matrix solution. The matrices used were 2-hydroxy-5-methoxybenzoic acid (sDHB) and 2,4dimethoxy-3-hydroxycinnamic acid (sinapininc acid, SA). The samples were analyzed in positive ion mode and 500 shots averages were collected using a mass range from 30 to 100 kDa. Masses were determined using FlexAnalysis software (Version 2.0, Bruker Daltonics).
3. Results and discussion Two methods were used for deglycosylation: PNGaseF and trifluoromethanesulfonic acid. The N- and O-linked glycoprotein protein, fetuin, was enzymatically deglycosylated to remove N-linked oligosaccharides and chemical deglycosylation was used to remove both N- and O-linked oligosaccharides. Fetuin is a glycoprotein with 359 amino acids containing N-linked glycosylation sites at Asn 99, 156 and 176, and numerous O-linked glycosylation sites [18]. As seen in Fig. 1A a mass of 45.3 kDa was measured for fetuin before deglycosylation which is consistent with previous studies [19]. The deglycosylated fetuin was analyzed with MALDI after deglycosylation with PNGaseF and no further cleanup. No signal was observed possibly due to buffers used during the deglycosylation. Using acetone and TCA precipitation independently, the protein was separated from the PNGaseF released oligosaccharides. The deglycosylated protein pellet of fetuin measured 39.5 kDa and was compared with the glycosylated fetuin using MALDI and a mass shift of 5.8 kDa was observed (Fig. 1B). Both acetone and TCA precipitation yielded sufficient protein recovery for MALDI analysis and the MALDI results were comparable. The chemical deglycosylation method using trifluoromethanesulfonic acid was also used to treat the fetuin protein. Following the deglycosylation, a MALDI analysis showed no signal in the protein mass range. However, following dialysis, which was used to desalt and concentrate the deglycosylated protein, a signal was observed. Compared to the precipitation methods, dialysis was more timeconsuming and the protein signal observed was lower (Fig. 1C). However, it removed interfering salts and produced a MALDI signal, although not as intense as the signal obtained from the precipitated deglycosylated protein. This approach was applied to an alligator lectin protein. The nonspecific deglycosylation method using TFMS as described above was used to remove oligosaccharides from the protein. The intact protein mass was measured at 34.6 kDa before deglycosylation. After deglycosylation and dialysis a mass of 32.2 kDa was observed in the MALDI mass spectrum (Fig. 2). The mass shift suggests the loss of
Fig. 2. MALDI mass spectrometry of alligator lectin.
311
oligosaccharides and the less intense signal indicates lower protein recovery from the dialysis procedure. 4. Conclusions Appropriate sample preparation is necessary to obtain mass spectrometry data after deglycosylation. To demonstrate the necessity for sample cleanup prior to mass spectrometry, we have used a common glycosylated protein, fetuin, and a lectin protein isolated from Alligator mississippiensis serum. Protein precipitation with acetone and TCA was used following deglycosylation to concentrate and desalt the proteins. Alternatively, dialysis was used to purify the protein from the other unwanted substances, as demonstrated with the chemical deglycosylation procedure. The precipitation and dialysis methods resulted in efficient desalting and protein concentration. However, the precipitation methods produced a higher protein signal as compared to dialysis. Acknowledgements This research was supported by the Louisiana Board of Regents Fund (LEQSF(2007-10)-RD-A-25). References [1] R. Aebersold, M. Mann, Mass spectrometry-based proteomics, Nature 422 (6928) (2003) 198–207. [2] J.R. Yates, Mass spectral analysis in proteomics, Annu. Rev. Biophys. Biomol. Struct. 33 (2004) 297–316. [3] N. Sundararajan, et al., Ultrasensitive detection and characterization of posttranslational modifications using surface-enhanced Raman spectroscopy, Anal. Chem. 78 (11) (2006) 3543–3550. [4] R.A. Dwek, Glycobiology: toward understanding the function of sugars, Chem. Rev. 96 (2) (1996) 683–720. [5] I.M. Lazar, et al., Recent advances in the MS analysis of glycoproteins: theoretical considerations, Electrophoresis 32 (1) (2011) 3–13. [6] K.F. Medzihradszky, In-solution digestion of proteins for mass spectrometry, Mass spectrometry: modified proteins and glycoconjugates, 2005, pp. 50–65. [7] K.L.S.a.K.R. Williams, The Protein Protocols Handbook. Enzymatic Digestion of Proteins in Solu, in: J.M. Walker (Ed.), Vol. Enzymatic Digstion of Proteins in Solution and in SDS Polyacrylamide Gels, Totowa: Humana Press Inc. (1996). [8] T.H. Plummer, A.L. Tarentino, Purification of the oligosaccharide-cleaving enzymes of Flavobacterium meningosepticum, Glycobiology 1 (3) (1991) 257–263. [9] A.S.B. Edge, Deglycosylation of glycoproteins with trifluoromethanesulphonic acid: elucidation of molecular structure and function, Biochem. J. 376 (2003) 339–350. [10] A.S.B. Edge, et al., Deglycosylation of glycoproteins by trifluoromethanesulfonic acid, Anal. Biochem. 118 (1) (1981) 131–137. [11] A.L. Tarentino, T.H. Plummer, Enzymatic deglycosylation of asparagine-linked glycans — purification, properties, and specificity of oligosaccharide-cleaving enzymes from flavobacterium–meningosepticum, Guide to Techniques in Glycobiology, 1994, pp. 44–57. [12] T. Patel, et al., Use of hydrazine to release in intact and unreduced form both N- and O-linked oligosaccharides from glycoproteins, Biochemistry 32 (2) (1993) 679–693. [13] K.O. Lloyd, et al., Comparison of O-linked carbohydrate chains in MUC-1 mucin from normal breast epithelial cell lines and breast carcinoma cell lines — demonstration of simpler and fewer glycan chains in tumor cells, J. Biol. Chem. 271 (52) (1996) 33325–33334. [14] D. Koutsioulis, D. Landry, E.P. Guthrie, Novel endo-alpha-N-acetylgalactosaminidases with broader substrate specificity, Glycobiology 18 (10) (2008) 799–805. [15] E. Fic, et al., Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis, Electrophoresis 31 (21) (2010) 3573–3579. [16] L. Jiang, L. He, M. Fountoulakis, Comparison of protein precipitation methods for sample preparation prior to proteomic analysis, J. Chromatogr. A 1023 (2) (2004) 317–320. [17] B.G. Fryksdale, et al., Impact of deglycosylation methods on two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry for proteomic analysis, Electrophoresis 23 (14) (2002) 2184–2193. [18] K.M. Dziegielewska, et al., The complete cDNA and amino acid sequence of bovine fetuin. Its homology with alpha 2HS glycoprotein and relation to other members of the cystatin superfamily, J. Biol. Chem. 265 (8) (1990) 4354–4357. [19] M. Gey, K. Unger, A strategy for chromatographic and structural analysis of monosaccharide species from glycoproteins, Fresn. J. Anal. Chem. 356 (8) (1996) 488–494.
Contents lists available at ScienceDirect
Microchemical Journal j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m i c r o c
A mass spectrometry approach for the study of deglycosylated proteins Lancia N.F. Darville a, Mark E. Merchant b, Kermit K. Murray a,⁎ a b
Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, United States Department of Chemistry, McNeese State University, Lake Charles, LA 70609, United States
a r t i c l e
i n f o
Article history: Received 22 May 2011 Accepted 26 May 2011 Available online 1 June 2011 Keywords: Proteomics Mass spectrometry Deglycosylation
a b s t r a c t Mass spectrometry (MS) analysis, after enzymatic or chemical deglycosylation, requires preparatory steps to remove salts and buffers. In this work, the glycosylated protein fetuin and a lectin protein isolated from the serum of Alligator mississippiensis were used to evaluate methods for desalting samples after an enzymatic or chemical deglycosylation. Precipitation and dialysis were used to prepare the deglycosylated samples for MS analysis. Both the precipitation and dialysis methods were suitable for sample preparation prior to analysis by matrix assisted laser desorption ionization (MALDI) MS. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Proteomics is the study of the structure and functions of proteins in an organism or tissue, and mass spectrometry is now a routinely used method for protein identification in complex mixtures [1]. Prior to a typical mass spectrometry analysis, separation techniques such as two-dimensional gel electrophoresis and high performance liquid chromatography (HPLC) are used to reduce the complexity of protein mixtures [1,2]. Some proteins are post-translationally modified to help regulate gene expression, protein turnover and cellular structure [3]. Glycosylation is a common post-translational modification that is involved in biological functions such as immune recognition and inflammation [4]. There are two major types of protein carbohydrate linkages: N- and Olinked glycosylation. During N-glycosylation, the glycan is attached to an asparginine residue followed by any amino acid other than proline, which is linked to a serine or threonine residue. During O-glycosylation, the glycan is attached to a serine or threonine residue. Glycoproteins have been characterized by mass spectrometry, but this is challenging due to the heterogeneity of the carbohydrate moieties and the complexity of the resulting mass spectra [5]. Glycosylation of the protein may also limit the extent of proteolytic digestion. To avoid this, the N- and/or O-linked carbohydrates can be removed prior to further analysis [6,7]. A direct approach using enzymatic or chemical cleavage of the glycan can be used to enable the analysis of the protein without the oligosaccharides. An enzymatic approach using peptide: N-glycosidase (PNGase F) [8] and a chemical approach using trifluoromethanesulfonic ⁎ Corresponding author at: Louisiana State University, 337 Choppin Hall, Baton Rouge, LA 70803, United States. Tel.: + 1 225 578 3417; fax: + 1 225 578 3458. E-mail address: [email protected] (L.N.F. Darville). 0026-265X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.microc.2011.05.020
acid (TFMS) have been used to remove the oligosaccharides [9,10]. PNGase F is an enzyme that removes N-linked oligosaccharides, leaving both the protein and oligosaccharides intact for further analysis. To date, there have been no enzymatic techniques reported that cleave O-linked oligosaccharides. TFMS is a non-specific deglycosylating agent that cleaves both N-linked and O-linked oligosaccharides to leave the intact protein [11]. The proteins used in this study were enzymatically or chemically deglycosylated, followed by matrix assisted laser desorption ionization (MALDI) MS. The steps for both chemical and enzymatic deglycosylation approaches are fully established and has been reported previously [9,12,13], but the deglycosylated product requires cleanup prior to MALDI mass spectrometry. We have highlighted important sample desalting procedures that follow deglycosylation prior to mass spectrometry analysis, which are not clearly outlined in many procedures. This study focuses on the treatment of deglycosylated proteins for mass spectrometry analysis.
2. Experimental 2.1. Materials HPLC grade acetonitrile, formic acid, and trifluoroacetic acid (TFA) were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. Fetuin, a plasma glycoprotein produced by the liver, was obtained from Sigma-Aldrich and the lectin protein was isolated from the blood plasma of Alligator mississippiensis. Blood from seven American alligators was collected; anticoagulated with heparin, and the blood was allowed to clot overnight at ambient temperature. The serum was collected and the lectin was isolated using mannan-agarose affinity chromatography.
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L.N.F. Darville et al. / Microchemical Journal 99 (2011) 309–311
2.2. Enzymatic deglycosylation Proteins containing oligosaccharides attached through N-linked glycosylation were deglycosylated using a previously developed protocol for enzymatic deglycosylation from New England BioLabs (Ipswich, MA) [8]. Approximately 100 μg of protein was dissolved in 18 μl of nanopure water. Denaturing buffer (2 μl), containing 0.6 mg/mL SDS and 0.5 mg/mL DTT, was added and heated at 100 °C for 10 min to increase the rate of deglycosylation. The sample was incubated on ice at 0 °C for five minutes and centrifuged for 10 s. To the denatured sample, a non-ionic detergent (NP-40) was added to prevent loss of enzyme activity. The PNGaseF enzyme was added to the sample and it was incubated at 37 °C for four hours [8,14]. The intact protein was isolated from the mixture via protein precipitation, which removed the buffers that can interfere with the MALDI signal. Acetone or trichloroacetic acid (TCA) was used to precipitate the protein [15].
was changed and dialysis was continued for an additional two hours. After a final buffer change, the sample was dialyzed overnight at room temperature. 2.7. Mass spectrometry Glycoproteins and deglycosylated proteins were analyzed in linear MS mode with a MALDI-mass spectrometer (Ultraflextreme, Bruker Daltonics, Billerica, MA, USA). The glycoproteins and the deglycosylated proteins (after dialysis or precipitation) were mixed on a steel target
2.3. TCA precipitation TCA precipitation was accomplished by the addition of one volume of 60% TCA. The sample was gently vortexed and incubated on ice overnight. The sample mixture was centrifuged at 10,000 g for 30 min and the supernatant was removed. Chilled acetone was added to wash the precipitate and it was centrifuged again at 10,000 g [16]. This procedure was repeated twice. The precipitate was air-dried and suspended in aqueous 0.1% trifluoroacetic acid (TFA) or 0.1% formic acid (FA) for MALDI analysis. 2.4. Acetone precipitation Acetone precipitation was achieved by the addition of four volumes of acetone to the protein sample, followed by an overnight incubation on ice. The sample was centrifuged at 10,000 g for 30 min at 4 °C. The supernatant was removed and the precipitate was air dried for 30 min, then suspended in the same buffer as used above in the TCA precipitation. 2.5. Chemical deglycosylation In-solution deglycosylation was performed using a deglycosylation kit containing trifluoromethanesulphonic acid (TFMS) (ProZyme, Hayward, CA, USA). TFMS removes most glycan types from glycoproteins and leaves the protein intact [9]. Briefly, approximately 0.3 mg of protein in a vial was dried overnight using a lyophilizer. The dried protein was capped with a septum and placed on a dry ice-ethanol bath for 20 s to reduce the temperature of the reaction. A mixture of TFMS and toluene solution was prepared and 50 μl was slowly added using a gas-tight syringe. Precautions were taken to prevent the dried protein from being exposed to moisture due to the potential for hydrolysis of the peptide bonds [9,17]. The vials containing the protein with TFMS mixture were incubated at − 20 °C for five minutes then shaken briefly to help dissolve the protein. The solvation process was repeated and followed by a four-hour incubation at −20 °C. 2.6. Dialysis Dialysis was performed prior to mass spectrometry analysis. After the four hour incubation, the vials containing the samples were placed in a dry ice-ethanol bath for 20 s, and then 150 μl of pyridine solution was added to each vial. The vials were incubated on dry ice for five minutes, and then in a dry ice-ethanol bath for 15 min. A 400 μl quantity of 0.5% ammonium bicarbonate was added to each vial to neutralize the sample. Following deglycosylation, the protein was isolated using dialysis. Each sample was added to a dialysis cassette (Thermo Scientific, Rockford, IL, USA) and dialyzed against 10 mM ammonium formate for two hours at room temperature. The buffer
Fig. 1. (a) MALDI spectrum of intact fetuin. (b) MALDI spectra comparing glycosylated and deglycosylated fetuin after acetone precipitation. (c) Fetuin after TFMS deglycosylation followed with dialysis.
L.N.F. Darville et al. / Microchemical Journal 99 (2011) 309–311
using a 1:1 ratio of sample and saturated matrix solution. The matrices used were 2-hydroxy-5-methoxybenzoic acid (sDHB) and 2,4dimethoxy-3-hydroxycinnamic acid (sinapininc acid, SA). The samples were analyzed in positive ion mode and 500 shots averages were collected using a mass range from 30 to 100 kDa. Masses were determined using FlexAnalysis software (Version 2.0, Bruker Daltonics).
3. Results and discussion Two methods were used for deglycosylation: PNGaseF and trifluoromethanesulfonic acid. The N- and O-linked glycoprotein protein, fetuin, was enzymatically deglycosylated to remove N-linked oligosaccharides and chemical deglycosylation was used to remove both N- and O-linked oligosaccharides. Fetuin is a glycoprotein with 359 amino acids containing N-linked glycosylation sites at Asn 99, 156 and 176, and numerous O-linked glycosylation sites [18]. As seen in Fig. 1A a mass of 45.3 kDa was measured for fetuin before deglycosylation which is consistent with previous studies [19]. The deglycosylated fetuin was analyzed with MALDI after deglycosylation with PNGaseF and no further cleanup. No signal was observed possibly due to buffers used during the deglycosylation. Using acetone and TCA precipitation independently, the protein was separated from the PNGaseF released oligosaccharides. The deglycosylated protein pellet of fetuin measured 39.5 kDa and was compared with the glycosylated fetuin using MALDI and a mass shift of 5.8 kDa was observed (Fig. 1B). Both acetone and TCA precipitation yielded sufficient protein recovery for MALDI analysis and the MALDI results were comparable. The chemical deglycosylation method using trifluoromethanesulfonic acid was also used to treat the fetuin protein. Following the deglycosylation, a MALDI analysis showed no signal in the protein mass range. However, following dialysis, which was used to desalt and concentrate the deglycosylated protein, a signal was observed. Compared to the precipitation methods, dialysis was more timeconsuming and the protein signal observed was lower (Fig. 1C). However, it removed interfering salts and produced a MALDI signal, although not as intense as the signal obtained from the precipitated deglycosylated protein. This approach was applied to an alligator lectin protein. The nonspecific deglycosylation method using TFMS as described above was used to remove oligosaccharides from the protein. The intact protein mass was measured at 34.6 kDa before deglycosylation. After deglycosylation and dialysis a mass of 32.2 kDa was observed in the MALDI mass spectrum (Fig. 2). The mass shift suggests the loss of
Fig. 2. MALDI mass spectrometry of alligator lectin.
311
oligosaccharides and the less intense signal indicates lower protein recovery from the dialysis procedure. 4. Conclusions Appropriate sample preparation is necessary to obtain mass spectrometry data after deglycosylation. To demonstrate the necessity for sample cleanup prior to mass spectrometry, we have used a common glycosylated protein, fetuin, and a lectin protein isolated from Alligator mississippiensis serum. Protein precipitation with acetone and TCA was used following deglycosylation to concentrate and desalt the proteins. Alternatively, dialysis was used to purify the protein from the other unwanted substances, as demonstrated with the chemical deglycosylation procedure. The precipitation and dialysis methods resulted in efficient desalting and protein concentration. However, the precipitation methods produced a higher protein signal as compared to dialysis. Acknowledgements This research was supported by the Louisiana Board of Regents Fund (LEQSF(2007-10)-RD-A-25). References [1] R. Aebersold, M. Mann, Mass spectrometry-based proteomics, Nature 422 (6928) (2003) 198–207. [2] J.R. Yates, Mass spectral analysis in proteomics, Annu. Rev. Biophys. Biomol. Struct. 33 (2004) 297–316. [3] N. Sundararajan, et al., Ultrasensitive detection and characterization of posttranslational modifications using surface-enhanced Raman spectroscopy, Anal. Chem. 78 (11) (2006) 3543–3550. [4] R.A. Dwek, Glycobiology: toward understanding the function of sugars, Chem. Rev. 96 (2) (1996) 683–720. [5] I.M. Lazar, et al., Recent advances in the MS analysis of glycoproteins: theoretical considerations, Electrophoresis 32 (1) (2011) 3–13. [6] K.F. Medzihradszky, In-solution digestion of proteins for mass spectrometry, Mass spectrometry: modified proteins and glycoconjugates, 2005, pp. 50–65. [7] K.L.S.a.K.R. Williams, The Protein Protocols Handbook. Enzymatic Digestion of Proteins in Solu, in: J.M. Walker (Ed.), Vol. Enzymatic Digstion of Proteins in Solution and in SDS Polyacrylamide Gels, Totowa: Humana Press Inc. (1996). [8] T.H. Plummer, A.L. Tarentino, Purification of the oligosaccharide-cleaving enzymes of Flavobacterium meningosepticum, Glycobiology 1 (3) (1991) 257–263. [9] A.S.B. Edge, Deglycosylation of glycoproteins with trifluoromethanesulphonic acid: elucidation of molecular structure and function, Biochem. J. 376 (2003) 339–350. [10] A.S.B. Edge, et al., Deglycosylation of glycoproteins by trifluoromethanesulfonic acid, Anal. Biochem. 118 (1) (1981) 131–137. [11] A.L. Tarentino, T.H. Plummer, Enzymatic deglycosylation of asparagine-linked glycans — purification, properties, and specificity of oligosaccharide-cleaving enzymes from flavobacterium–meningosepticum, Guide to Techniques in Glycobiology, 1994, pp. 44–57. [12] T. Patel, et al., Use of hydrazine to release in intact and unreduced form both N- and O-linked oligosaccharides from glycoproteins, Biochemistry 32 (2) (1993) 679–693. [13] K.O. Lloyd, et al., Comparison of O-linked carbohydrate chains in MUC-1 mucin from normal breast epithelial cell lines and breast carcinoma cell lines — demonstration of simpler and fewer glycan chains in tumor cells, J. Biol. Chem. 271 (52) (1996) 33325–33334. [14] D. Koutsioulis, D. Landry, E.P. Guthrie, Novel endo-alpha-N-acetylgalactosaminidases with broader substrate specificity, Glycobiology 18 (10) (2008) 799–805. [15] E. Fic, et al., Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis, Electrophoresis 31 (21) (2010) 3573–3579. [16] L. Jiang, L. He, M. Fountoulakis, Comparison of protein precipitation methods for sample preparation prior to proteomic analysis, J. Chromatogr. A 1023 (2) (2004) 317–320. [17] B.G. Fryksdale, et al., Impact of deglycosylation methods on two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry for proteomic analysis, Electrophoresis 23 (14) (2002) 2184–2193. [18] K.M. Dziegielewska, et al., The complete cDNA and amino acid sequence of bovine fetuin. Its homology with alpha 2HS glycoprotein and relation to other members of the cystatin superfamily, J. Biol. Chem. 265 (8) (1990) 4354–4357. [19] M. Gey, K. Unger, A strategy for chromatographic and structural analysis of monosaccharide species from glycoproteins, Fresn. J. Anal. Chem. 356 (8) (1996) 488–494.