Glycopeptide Analysis, Recent Developments and Applications
2013; Elsevier BV; Volume: 12; Issue: 4 Linguagem: Inglês
10.1074/mcp.r112.026567
ISSN1535-9484
Autores Tópico(s)Mass Spectrometry Techniques and Applications
ResumoGlycopeptide-based analysis is used to inform researchers about the glycans on one or more proteins. The method's key attractive feature is its ability to link glycosylation information to exact locations (glycosylation sites) on proteins. Numerous applications for glycopeptide analysis are known, and several examples are described herein. The techniques used to characterize glycopeptides are still emerging, and recently, research focused on facilitating aspects of glycopeptide analysis has advanced significantly in the areas of sample preparation, MS fragmentation, and automation of data analysis. These recent developments, described herein, provide the foundation for the growth of glycopeptide analysis as a blossoming field. Glycopeptide-based analysis is used to inform researchers about the glycans on one or more proteins. The method's key attractive feature is its ability to link glycosylation information to exact locations (glycosylation sites) on proteins. Numerous applications for glycopeptide analysis are known, and several examples are described herein. The techniques used to characterize glycopeptides are still emerging, and recently, research focused on facilitating aspects of glycopeptide analysis has advanced significantly in the areas of sample preparation, MS fragmentation, and automation of data analysis. These recent developments, described herein, provide the foundation for the growth of glycopeptide analysis as a blossoming field. The reasons for analyzing glycopeptides are nearly as diverse as the glycopeptides themselves. In some cases, new and interesting molecules are being discovered and characterized, such as glycosylated snake venom (1Quinton L. Gilles N. Smargiasso N. Kiehne A. De Pauw E. An unusual family of glycosylated peptides isolated from Dendroaspis angusticeps venom and characterized by combination of collision-induced and electron transfer dissociation.J. Am. Soc. Mass Spectrom. 2011; 22: 1891-1897Crossref PubMed Scopus (14) Google Scholar). In other cases, comparative glycopeptide analysis is done on endogenous compounds to differentiate between a disease state and a healthy state (2Wang D. Hincapie M. Rejtar T. Karger B.L. Ultrasensitive characterization of site-specific glycosylation of affinity-purified haptoglobin from lung cancer patient plasma using 10-μm i.d. porous layer open tubular liquid chromatography-linear ion trap collision-induced dissociation/electron transfer dissociation mass spectrometry.Anal. Chem. 2011; 83: 2029-2037PubMed Google Scholar, 3Li Y. Tian Y. Rezai T. Prakash A. Lopez M.F. Chan D.W. Zhang H. Simultaneous analysis of glycosylated and sialylated prostate-specific antigen revealing differential distribution of glycosylated prostate-specific antigen isoforms in prostate cancer tissues.Anal. Chem. 2011; 83: 240-245Crossref PubMed Scopus (73) Google Scholar). These types of analyses can be useful both in diagnosing or treating disease and also in developing a better understanding of disease progression and pathology. Furthermore, comparative analyses can be done on endogenous glycoproteins that are isolated from various species (4Dalpathado D.S. Irungu J. Go E.P. Butnev V.Y. Norton K. Bousfield G.R. Desaire H. Comparative glycomics of the glycoprotein follicle-stimulating hormone (FSH): glycopeptide analysis of isolates from two mammalian species.Biochemistry. 2006; 45: 8665-8673Crossref PubMed Scopus (56) Google Scholar), with the goal of increasing the understanding of how the same protein, from different species, can have slightly different properties, such as different circulation half-life, or receptor binding affinity, etc. Glycopeptides originating from recombinantly expressed glycoproteins are also frequent subjects of investigation. These analyses are completed for a variety of reasons. For example, human erythropoietin, an endogenous compound, can be expressed in mammalian cells and is consumed illicitly by athletes in an effort to enhance their performance (5Giménez E. Ramos-Hernan R. Benavente F. Barbosa J. Sanz-Nebot V. Analysis of recombinant human erythropoietin glycopeptides by capillary electrophoresis electrospray-time of flight mass spectrometry.Anal. Chim. Acta. 2012; 709: 81-90Crossref PubMed Scopus (41) Google Scholar). Because EPO 1The abbreviations used are:EPOerythropoietinUVPDUV-photodissociationHILIChydrophilic interaction liquid chromatographyMALDImatrix-assisted laser desorption ionizationESIelectrospray ionizationCIDcollision-induced dissociationETDelectronic transfer dissociationCEcapillary electrophoresisoaTOForthogonal acceleration time of flight. is classified as a banned substance, drug testers need to be able to distinguish between recombinant and endogenous forms, and the glycosylation on EPO provides quite a useful means of distinguishing the compounds. In other cases, recombinant proteins are expressed for human and animal consumption for quite legitimate reasons, such as to aid in fertility, as is the case with follicle-stimulating hormone (6Grass J. Pabst M. Chang M. Wozny M. Altmann F. Analysis of recombinant human follicle-stimulating hormone (FSH) by mass spectrometric approaches.Anal. Bioanal. Chem. 2011; 400: 2427-2438Crossref PubMed Scopus (36) Google Scholar). In these instances, characterizing glycosylation of recombinantly expressed proteins is one important step is assessing the overall drug product quality. Finally, glycopeptide analysis can even aid in the development of new products, such as in the development of an HIV vaccine (7Zhu X. Borchers C. Bienstock R.J. Tomer K.B. Mass spectrometric characterization of the glycosylation pattern of HIV-gp120 expressed in CHO cells.Biochemistry. 2000; 39: 11194-11204Crossref PubMed Scopus (207) Google Scholar, 8Go E.P. Irungu J. Zhang Y. Dalpathado D.S. Liao H.-X. Sutherland L.L. Alam S.M. Haynes B.F. Desaire H. Glycosylation site-specific analysis of HIV envelope proteins (JR-FL and CON-S) reveals major differences in glycosylation site occupancy, glycoform profiles, and antigenic epitopes' accessibility.J. Proteome Res. 2008; 7: 1660-1674Crossref PubMed Scopus (121) Google Scholar, 9Go E.P. Chang Q. Liao H.-X. Sutherland L.L. Alam S.M. Haynes B.F. Desaire H. Glycosylation site-specific analysis of clade C HIV-1 envelope proteins.J. Proteome Res. 2009; 8: 4231-4242Crossref PubMed Scopus (79) Google Scholar, 10Go E.P. Hewawasam G. Liao H.X. Chen H. Ping L.H. Anderson J.A. Hua D.C. Haynes B.F. Desaire H. Characterization of glycosylation profile of HIV-1 transmitted/founder envelopes by mass spectrometry.J. Virol. 2011; 85: 8270-8284Crossref PubMed Scopus (103) Google Scholar). Glycopeptide analysis has been used to extensively compare the properties of numerous HIV envelope proteins under investigation for their potential to elicit a strong immune response against the HIV-1 virus. The following section highlights, in more detail, the examples mentioned above. These highlights are by no means an exhaustive list of important problems that are being addressed with glycopeptide analysis. Rather, the examples give the reader a sense of scope of the types of samples that are currently being studied using this technique and the types of problems that can be addressed. erythropoietin UV-photodissociation hydrophilic interaction liquid chromatography matrix-assisted laser desorption ionization electrospray ionization collision-induced dissociation electronic transfer dissociation capillary electrophoresis orthogonal acceleration time of flight. One interesting example of the need for characterization of biologically relevant, isolated glycoproteins is the analysis of several glycopeptides from the venom of Dendroaspis angusticeps (1Quinton L. Gilles N. Smargiasso N. Kiehne A. De Pauw E. An unusual family of glycosylated peptides isolated from Dendroaspis angusticeps venom and characterized by combination of collision-induced and electron transfer dissociation.J. Am. Soc. Mass Spectrom. 2011; 22: 1891-1897Crossref PubMed Scopus (14) Google Scholar). These highly active polypeptide compounds were found to be both glycosylated and rich in proline; both features are unusual in snake venom compounds. Because the analytes of interest were generally under 4 kDa, no enzymatic digestion was required prior to analysis; and a top-down approach was used for assigning both the protein sequence, the glycosylation site occupancy, and the glycans themselves (1Quinton L. Gilles N. Smargiasso N. Kiehne A. De Pauw E. An unusual family of glycosylated peptides isolated from Dendroaspis angusticeps venom and characterized by combination of collision-induced and electron transfer dissociation.J. Am. Soc. Mass Spectrom. 2011; 22: 1891-1897Crossref PubMed Scopus (14) Google Scholar). The species contained small O-linked glycans. In this research, a variety of MS techniques were used, including MALDI and ESI for ionization and CID and ETD for fragmentation (1Quinton L. Gilles N. Smargiasso N. Kiehne A. De Pauw E. An unusual family of glycosylated peptides isolated from Dendroaspis angusticeps venom and characterized by combination of collision-induced and electron transfer dissociation.J. Am. Soc. Mass Spectrom. 2011; 22: 1891-1897Crossref PubMed Scopus (14) Google Scholar). This study represents the first step necessary for understanding the structure/function connection for this particular snake's venom, but it also serves as an illustrative example of the need for classifying glycosylation, as a first step, for achieving a better understanding of a glycoprotein of interest. Often, classifying the glycosylation of a protein in one species is a very useful start to understanding that protein's characteristics, but if the protein of interest is present in organisms from multiple species, which is almost always the case, a comparison of the glycosylation on the same protein from different species can provide important insights into the changes in protein properties that are observed in different animals. For example, the glycosylation on the protein's FSH was characterized from human and horse isolates, and between these two species more glycoforms were identified that were unique to one species or the other than those glycoforms that were common to both species (4Dalpathado D.S. Irungu J. Go E.P. Butnev V.Y. Norton K. Bousfield G.R. Desaire H. Comparative glycomics of the glycoprotein follicle-stimulating hormone (FSH): glycopeptide analysis of isolates from two mammalian species.Biochemistry. 2006; 45: 8665-8673Crossref PubMed Scopus (56) Google Scholar). The protein, with four different glycosylation sites, was characterized using a proteinase K digestion and analyzed using high resolution ESI-MS and CID data, and ∼30 glycoforms were detected for each species (4Dalpathado D.S. Irungu J. Go E.P. Butnev V.Y. Norton K. Bousfield G.R. Desaire H. Comparative glycomics of the glycoprotein follicle-stimulating hormone (FSH): glycopeptide analysis of isolates from two mammalian species.Biochemistry. 2006; 45: 8665-8673Crossref PubMed Scopus (56) Google Scholar). The key data are shown in Fig. 1. Because glycosylation is dependent on the local environment of the cell, this modification can even change when samples are obtained from the same species but from two different biological conditions (2Wang D. Hincapie M. Rejtar T. Karger B.L. Ultrasensitive characterization of site-specific glycosylation of affinity-purified haptoglobin from lung cancer patient plasma using 10-μm i.d. porous layer open tubular liquid chromatography-linear ion trap collision-induced dissociation/electron transfer dissociation mass spectrometry.Anal. Chem. 2011; 83: 2029-2037PubMed Google Scholar, 3Li Y. Tian Y. Rezai T. Prakash A. Lopez M.F. Chan D.W. Zhang H. Simultaneous analysis of glycosylated and sialylated prostate-specific antigen revealing differential distribution of glycosylated prostate-specific antigen isoforms in prostate cancer tissues.Anal. Chem. 2011; 83: 240-245Crossref PubMed Scopus (73) Google Scholar). About 10 years ago, the glycosylation state of haptoglobin was shown to be different between healthy cells and cancer cells (11Poon T.C. Mok T.S. Chan A.T. Chan C.M. Leong V. Tsui S.H. Leung T.W. Wong H.T. Ho S.K. Johnson P.J. Quantification and utility of monosialylated α-fetoprotein in the diagnosis of hepatocellular carcinoma with nondiagnostic serum total α-fetoprotein.Clin. Chem. 2002; 48: 1021-1027Crossref PubMed Scopus (52) Google Scholar), and this finding, along with other notable works showing that the glycosylation in certain proteins from individuals with cancer varies, compared with healthy controls (12Peracaula R. Tabarés G. Royle L. Harvey D.J. Dwek R.A. Rudd P.M. de Llorens R. Altered glycosylation pattern allows the distinction between prostate-specific antigen (PSA) from normal and tumor origins.Glycobiology. 2003; 13: 457-470Crossref PubMed Scopus (254) Google Scholar, 13Kuzmanov U. Jiang N. Smith C.R. Soosaipillai A. Diamandis E.P. Differential N-glycosylation of kallikrein 6 derived from ovarian cancer cells or the central nervous system.Mol. Cell. Proteomics. 2009; 8: 791-798Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), opens up the possibility for using the glycosylation state both as a marker for disease state and also as a potential window into understanding the biology of the disease state itself. Although such studies are tantalizing, the analytical techniques to support the work must be in place and capable of processing clinical samples. Both the ability to detect the glycoforms from the protein of interest, from a highly complex sample, and the ability to quantify those resulting glycoforms are current challenges that are being tackled by emerging methods (2Wang D. Hincapie M. Rejtar T. Karger B.L. Ultrasensitive characterization of site-specific glycosylation of affinity-purified haptoglobin from lung cancer patient plasma using 10-μm i.d. porous layer open tubular liquid chromatography-linear ion trap collision-induced dissociation/electron transfer dissociation mass spectrometry.Anal. Chem. 2011; 83: 2029-2037PubMed Google Scholar, 3Li Y. Tian Y. Rezai T. Prakash A. Lopez M.F. Chan D.W. Zhang H. Simultaneous analysis of glycosylated and sialylated prostate-specific antigen revealing differential distribution of glycosylated prostate-specific antigen isoforms in prostate cancer tissues.Anal. Chem. 2011; 83: 240-245Crossref PubMed Scopus (73) Google Scholar). In addition to the need to characterize endogenous analytes, equally important is the analysis of glycosylation from recombinantly expressed proteins. An interesting example highlighting this need is provided in Ref. 5Giménez E. Ramos-Hernan R. Benavente F. Barbosa J. Sanz-Nebot V. Analysis of recombinant human erythropoietin glycopeptides by capillary electrophoresis electrospray-time of flight mass spectrometry.Anal. Chim. Acta. 2012; 709: 81-90Crossref PubMed Scopus (41) Google Scholar, where the goal is to understand the glycosylation profile of recombinantly expressed EPO, a banned substance for professional athletes, so that this protein could be detected and differentiated from endogenous EPO. In this case, the recombinant protein was treated with trypsin and Glu-C, and the glycopeptide products were monitored by CE-MS using a TOF detector (5Giménez E. Ramos-Hernan R. Benavente F. Barbosa J. Sanz-Nebot V. Analysis of recombinant human erythropoietin glycopeptides by capillary electrophoresis electrospray-time of flight mass spectrometry.Anal. Chim. Acta. 2012; 709: 81-90Crossref PubMed Scopus (41) Google Scholar). Both O-linked and N-linked glycopeptides were detected, and the authors found several glycopeptides containing Neu5Gc in the recombinant form of the protein, which is significant because this glycoform is not native to humans and could identify the sample as being recombinantly expressed. This study is a more recent example of the successful glycopeptide analysis of recombinant EPO, which has also been accomplished previously. For examples, see Refs. 14Sasaki H. Ochi N. Dell A. Fukuda M. Site-specific glycosylation analysis of human recombinant erythropoietin. Analysis of glycopeptides or peptides at each glycosylation site by fast atom bombardment mass spectrometry.Biochemistry. 1988; 27: 8618-8626Crossref PubMed Scopus (116) Google Scholar, 15Rahbek-Nielsen H. Roepstorff P. Reischl H. Wozny M. Koll H. Haselbeck A. Glycopeptide profiling of human urinary erythropoietin by matrix-assisted laser desorption/ionization mass spectrometry.J. Mass Spectrom. 1997; 32: 948-958Crossref PubMed Scopus (53) Google Scholar, 16Takegawa Y. Ito H. Keira T. Deguchi K. Nakagawa H. Nishimura S. Profiling of N- and O-glycopeptides of erythropoietin by capillary zwitterionic type of hydrophilic interaction chromatography/electrospray ionization mass spectrometry.J. Sep. Sci. 2008; 31: 1585-1593Crossref PubMed Scopus (59) Google Scholar. Although glycopeptide analysis in the drug testing arena is a niche application, this analysis is much more widely practiced for the characterization of legally prescribed therapeutic glycoproteins. One report describes the glycopeptide analysis of recombinant FSH, a glycoprotein used to treat infertility (6Grass J. Pabst M. Chang M. Wozny M. Altmann F. Analysis of recombinant human follicle-stimulating hormone (FSH) by mass spectrometric approaches.Anal. Bioanal. Chem. 2011; 400: 2427-2438Crossref PubMed Scopus (36) Google Scholar). Characterizing the glycosylation on therapeutic glycoproteins is one important way that manufacturers can monitor the batch-to-batch consistency of their manufacturing process, and these analyses can also help to distinguish biosimilars, such as generic forms of therapeutic proteins. To obtain coverage of all four glycosylation sites on FSH, the investigators found that a chymotrypsin digestion was more useful than simply digesting with trypsin (6Grass J. Pabst M. Chang M. Wozny M. Altmann F. Analysis of recombinant human follicle-stimulating hormone (FSH) by mass spectrometric approaches.Anal. Bioanal. Chem. 2011; 400: 2427-2438Crossref PubMed Scopus (36) Google Scholar). Data were acquired on a qTOF mass spectrometer, and both high resolution MS and CID data were used to confirm the glycopeptide compositions (6Grass J. Pabst M. Chang M. Wozny M. Altmann F. Analysis of recombinant human follicle-stimulating hormone (FSH) by mass spectrometric approaches.Anal. Bioanal. Chem. 2011; 400: 2427-2438Crossref PubMed Scopus (36) Google Scholar). In addition to characterizing the glycosylation of glycoprotein pharmaceuticals that are already on the market or in late stages of development, another important aspect of characterizing recombinant glycoproteins is supporting drug discovery. For the last decade, glycopeptide analysis has served to aid HIV researchers who are interested in developing the recombinant protein gp120 into an effective vaccine for HIV-1 (7Zhu X. Borchers C. Bienstock R.J. Tomer K.B. Mass spectrometric characterization of the glycosylation pattern of HIV-gp120 expressed in CHO cells.Biochemistry. 2000; 39: 11194-11204Crossref PubMed Scopus (207) Google Scholar, 8Go E.P. Irungu J. Zhang Y. Dalpathado D.S. Liao H.-X. Sutherland L.L. Alam S.M. Haynes B.F. Desaire H. Glycosylation site-specific analysis of HIV envelope proteins (JR-FL and CON-S) reveals major differences in glycosylation site occupancy, glycoform profiles, and antigenic epitopes' accessibility.J. Proteome Res. 2008; 7: 1660-1674Crossref PubMed Scopus (121) Google Scholar, 9Go E.P. Chang Q. Liao H.-X. Sutherland L.L. Alam S.M. Haynes B.F. Desaire H. Glycosylation site-specific analysis of clade C HIV-1 envelope proteins.J. Proteome Res. 2009; 8: 4231-4242Crossref PubMed Scopus (79) Google Scholar, 10Go E.P. Hewawasam G. Liao H.X. Chen H. Ping L.H. Anderson J.A. Hua D.C. Haynes B.F. Desaire H. Characterization of glycosylation profile of HIV-1 transmitted/founder envelopes by mass spectrometry.J. Virol. 2011; 85: 8270-8284Crossref PubMed Scopus (103) Google Scholar). Although this protein is extensively glycosylated, with 20–30 N-linked glycosylation sites, depending on the exact sequence of the protein, glycopeptide analysis of this protein has been useful in developing a three-dimensional model of the full protein, including the glycoforms, which consume about half of the protein's mass (7Zhu X. Borchers C. Bienstock R.J. Tomer K.B. Mass spectrometric characterization of the glycosylation pattern of HIV-gp120 expressed in CHO cells.Biochemistry. 2000; 39: 11194-11204Crossref PubMed Scopus (207) Google Scholar). These analyses have also shown that the glycosylation on different HIV-1 variants changes (8–10). Furthermore, the glycoforms derived from recombinant gp120 proteins known to be on transmitted viruses are different from the glycoforms that are typically found on the recombinantly expressed version of the analogous protein found on the virus from chronically infected patients (10Go E.P. Hewawasam G. Liao H.X. Chen H. Ping L.H. Anderson J.A. Hua D.C. Haynes B.F. Desaire H. Characterization of glycosylation profile of HIV-1 transmitted/founder envelopes by mass spectrometry.J. Virol. 2011; 85: 8270-8284Crossref PubMed Scopus (103) Google Scholar). Researchers have shown that this particular protein's glycosylation is also highly diverse, with over 300 glycoforms typically characterized per recombinantly expressed protein (8Go E.P. Irungu J. Zhang Y. Dalpathado D.S. Liao H.-X. Sutherland L.L. Alam S.M. Haynes B.F. Desaire H. Glycosylation site-specific analysis of HIV envelope proteins (JR-FL and CON-S) reveals major differences in glycosylation site occupancy, glycoform profiles, and antigenic epitopes' accessibility.J. Proteome Res. 2008; 7: 1660-1674Crossref PubMed Scopus (121) Google Scholar, 9Go E.P. Chang Q. Liao H.-X. Sutherland L.L. Alam S.M. Haynes B.F. Desaire H. Glycosylation site-specific analysis of clade C HIV-1 envelope proteins.J. Proteome Res. 2009; 8: 4231-4242Crossref PubMed Scopus (79) Google Scholar, 10Go E.P. Hewawasam G. Liao H.X. Chen H. Ping L.H. Anderson J.A. Hua D.C. Haynes B.F. Desaire H. Characterization of glycosylation profile of HIV-1 transmitted/founder envelopes by mass spectrometry.J. Virol. 2011; 85: 8270-8284Crossref PubMed Scopus (103) Google Scholar). These studies were all done using a tryptic digestion and HPLC-MS and MS/MS on high resolution instruments, and in some instances the work was supported with off-line HPLC followed by MALDI-TOF/TOF analyses (7Zhu X. Borchers C. Bienstock R.J. Tomer K.B. Mass spectrometric characterization of the glycosylation pattern of HIV-gp120 expressed in CHO cells.Biochemistry. 2000; 39: 11194-11204Crossref PubMed Scopus (207) Google Scholar, 8Go E.P. Irungu J. Zhang Y. Dalpathado D.S. Liao H.-X. Sutherland L.L. Alam S.M. Haynes B.F. Desaire H. Glycosylation site-specific analysis of HIV envelope proteins (JR-FL and CON-S) reveals major differences in glycosylation site occupancy, glycoform profiles, and antigenic epitopes' accessibility.J. Proteome Res. 2008; 7: 1660-1674Crossref PubMed Scopus (121) Google Scholar, 9Go E.P. Chang Q. Liao H.-X. Sutherland L.L. Alam S.M. Haynes B.F. Desaire H. Glycosylation site-specific analysis of clade C HIV-1 envelope proteins.J. Proteome Res. 2009; 8: 4231-4242Crossref PubMed Scopus (79) Google Scholar, 10Go E.P. Hewawasam G. Liao H.X. Chen H. Ping L.H. Anderson J.A. Hua D.C. Haynes B.F. Desaire H. Characterization of glycosylation profile of HIV-1 transmitted/founder envelopes by mass spectrometry.J. Virol. 2011; 85: 8270-8284Crossref PubMed Scopus (103) Google Scholar). In summary, the types of applications warranting glycopeptide analysis are quite diverse, and the methods used to solve each problem at hand also vary. Frequently, a digestion strategy must be designed that takes into account the nature of the specific protein to be characterized. In the examples above, no digestion was necessary in one case (1Quinton L. Gilles N. Smargiasso N. Kiehne A. De Pauw E. An unusual family of glycosylated peptides isolated from Dendroaspis angusticeps venom and characterized by combination of collision-induced and electron transfer dissociation.J. Am. Soc. Mass Spectrom. 2011; 22: 1891-1897Crossref PubMed Scopus (14) Google Scholar), and in another case, chymotrypsin was shown to be the enzyme of choice (6Grass J. Pabst M. Chang M. Wozny M. Altmann F. Analysis of recombinant human follicle-stimulating hormone (FSH) by mass spectrometric approaches.Anal. Bioanal. Chem. 2011; 400: 2427-2438Crossref PubMed Scopus (36) Google Scholar). Proteinase K was also chosen in one of the cited references (4Dalpathado D.S. Irungu J. Go E.P. Butnev V.Y. Norton K. Bousfield G.R. Desaire H. Comparative glycomics of the glycoprotein follicle-stimulating hormone (FSH): glycopeptide analysis of isolates from two mammalian species.Biochemistry. 2006; 45: 8665-8673Crossref PubMed Scopus (56) Google Scholar). In most cases, trypsin was used as the sole enzyme; however, sometimes pairing trypsin with a second enzyme, such as Glu-C in the case of the EPO analysis, provides significant advantages over trypsin alone (5Giménez E. Ramos-Hernan R. Benavente F. Barbosa J. Sanz-Nebot V. Analysis of recombinant human erythropoietin glycopeptides by capillary electrophoresis electrospray-time of flight mass spectrometry.Anal. Chim. Acta. 2012; 709: 81-90Crossref PubMed Scopus (41) Google Scholar). Although the analysis of recombinant glycoproteins typically does not require the use of an up front protein purification method, this step is essential when characterizing glycoproteins from endogenous sources. Again, this step is typically dependent upon the exact protein to be isolated, but more research in enhancing the detection of glycopeptides in general, along with glycopeptides from a specific protein of interest, is a developing field that is expanded upon more below. Another interesting observation can be made in considering the above-described applications. No consensus exists in the field as to the best MS method to characterize glycoproteins. In the examples above, both MALDI and ESI data were used, along with several different types of mass spectrometers, including TOF/TOF, qTOF, oaTOF, ion traps, FT-ICR, and triple quadrupoles. Additionally, both ETD and CID data were employed, and in at least one case, no MS/MS data were used to support the assignments (5Giménez E. Ramos-Hernan R. Benavente F. Barbosa J. Sanz-Nebot V. Analysis of recombinant human erythropoietin glycopeptides by capillary electrophoresis electrospray-time of flight mass spectrometry.Anal. Chim. Acta. 2012; 709: 81-90Crossref PubMed Scopus (41) Google Scholar). When a diversity of MS methods is employed, a diversity of data analysis strategies naturally follows. In many of the examples above, no particular strategy for analyzing the data, other than manually matching the candidate mass to the mass of its assigned glycopeptide, was explicitly described. In other cases, CID data were acquired and manually interpreted to support the high resolution assignments. In still other cases, some tools designed to support glycopeptide analysis were cited as being important aids. Clearly, data interpretation aspects of glycopeptide analysis are an important area that must be further developed, if glycopeptide analysis is to become routinely and more widely implemented. One final important observation should be acknowledged regarding all the glycopeptide analysis applications described above. In each case, the analysis method provides composition information about the glycans but not full structural information, i.e. sequence, branching, linkage, and anomeric assignments. Often, investigators using glycopeptide data obtain some degree of glycan connectivity information, particularly when CID data are employed, but linkage and anomeric information is absent in typical MS/MS data for glycopeptides. It is common practice in the field to depict "best guess" assignments for the glycan structures, such as those shown in Fig. 1, using a combination of the fragmentation data and "biological precedence" or a basic understanding of the types of structures that are typically formed, based on the glycosylation processing enzymes available. If complete structural data for the glycans are required, glycopeptide analysis can be paired with glycan analysis. For more information about obtaining structural information for glycans, readers may explore Refs. 17Zaia J. Mass spectrometry and the emerging field of glycomics.Chem. Biol. 2008; 15: 881-892Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 18Harvey D.J. Proteomic analysis of glycosylation: structural determination of N- and O-linked glycans by mass spectrometry.Expert Rev. Proteomics. 2005; 2: 87-101Crossref PubMed Scopus (139) Google Scholar. The remaining portion of this review focuses on the emerging work in methods development supporting the field of glycopeptide analysis. Specifically, what sample enrichment and chromatographic methods are emerging? What new tools from the MS community could aid in data acquisition? What is the state of software development for supporting the MS analysis of glycopeptide data? Sample enrichment of glycoproteins and glycopeptides is a key step prior to acquiring useful data for glycopeptide analysis. Except for recombinantly expressed proteins, where the glycoprotein of interest is already available in high concentration and high purity, affinity enrichment is necessary for a successful analysis. Various methods of affinity enrichment have recently been reviewed elsewhere (19Rebecchi K.R. Desaire H. Recent mass spectrometric based methods in quantitative N-linked glycoproteomics.Curr. Proteomics. 2011; 8: 269-277Crossref Scopus (4) Google Scholar). Briefly, common strategies involve using an antibody that recognizes the protein
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