Elucidation of N-Glycosylation Sites on Human Platelet Proteins

Among known platelet proteins, a prominent and functionally important group is represented by glycoprotein isoforms. They account e.g. for secretory proteins and plasma membrane receptors including integrins and glycoprotein VI as well as intracellular components of cytosol and organelles including storage proteins (multimerin 1 etc.). Although many of those proteins have been studied for some time with regard to their function, little attention has been paid with respect to their glycosylation sites. Here we report the analysis of N-glycosylation sites of human platelet proteins. For the enrichment of glycopeptides, lectin affinity chromatography as well as chemical trapping of protein bound oligosaccharides was used. Therefore, concanavalin A was used for specific interaction with carbohydrate species along with periodic acid oxidation and hydrazide bead trapping of glycosylated proteins. Derivatization by peptide:N-glycosidase F yielded deglycosylated peptides, which provided the basis for the elucidation of proteins and their sites of modification. Using both methods in combination with nano-LC-ESI-MS/MS analysis 70 different glycosylation sites within 41 different proteins were identified. Comparison with the Swiss-Prot database established that the majority of these 70 sites have not been specifically determined by previous research projects. With this approach including hydrazide bead affinity trapping, the immunoglobulin receptor G6f, which is known to couple to the Ras-mitogen-activated protein kinase pathway in the immune system, was shown here for the first time to be present in human platelets. Among known platelet proteins, a prominent and functionally important group is represented by glycoprotein isoforms. They account e.g. for secretory proteins and plasma membrane receptors including integrins and glycoprotein VI as well as intracellular components of cytosol and organelles including storage proteins (multimerin 1 etc.). Although many of those proteins have been studied for some time with regard to their function, little attention has been paid with respect to their glycosylation sites. Here we report the analysis of N-glycosylation sites of human platelet proteins. For the enrichment of glycopeptides, lectin affinity chromatography as well as chemical trapping of protein bound oligosaccharides was used. Therefore, concanavalin A was used for specific interaction with carbohydrate species along with periodic acid oxidation and hydrazide bead trapping of glycosylated proteins. Derivatization by peptide:N-glycosidase F yielded deglycosylated peptides, which provided the basis for the elucidation of proteins and their sites of modification. Using both methods in combination with nano-LC-ESI-MS/MS analysis 70 different glycosylation sites within 41 different proteins were identified. Comparison with the Swiss-Prot database established that the majority of these 70 sites have not been specifically determined by previous research projects. With this approach including hydrazide bead affinity trapping, the immunoglobulin receptor G6f, which is known to couple to the Ras-mitogen-activated protein kinase pathway in the immune system, was shown here for the first time to be present in human platelets. During recent years substantial advances have been made within and by the field of proteomics. In addition to the common differential proteomic analyses focusing on the characterization of protein patterns in two different, well controlled states, e.g. healthy and diseased, the inventory of proteins from complete organisms, cell types (1Maguire P.B. Fitzgerald D.J. Platelet proteomics.J. Thromb. Haemostasis. 2003; 1: 1593-1601Google Scholar), or organelles (2Sickmann A. Reinders J. Wagner Y. Joppich C. Zahedi R. Meyer H.E. Schonfisch B. Perschil I. Chacinska A. Guiard B. Rehling P. Pfanner N. Meisinger C. The proteome of Saccharomyces cerevisiae mitochondria.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13207-13212Google Scholar) received considerable attention. However, most of these investigations paid little attention to the post-translational modifications of the proteins investigated, e.g. glycosylation and phosphorylation sites. Most data in this respect are still obtained by studies of individual proteins. Glycosylations are known as a most heterogenic group of modifications to proteins in general. Abundant types of O- and N-glycosylation comprise most commonly serine, threonine, and asparagine residues as well as glycosylphosphatidylinositol anchors. Furthermore, carbohydrate additions such as C-mannosylation and O-fucosylation are known to be present on proteins of interest (3Hofsteenge J. Huwiler K.G. Macek B. Hess D. Lawler J. Mosher D.F. Peter-Katalinic J. C-Mannosylation and O-fucosylation of the thrombospondin type 1 module.J. Biol. Chem. 2001; 276: 6485-6498Google Scholar). The enormous complexity of those modifications often hampers a detailed analysis due to low sample amounts of individual isoforms and structural diversity. O-Glycosylations for instance may be distributed to eight different core structures with numerous possible extensions and modifications (4Van den Steen P. Rudd P.M. Dwek R.A. Opdenakker G. Concepts and principles of O-linked glycosylation.Crit. Rev. Biochem. Mol. Biol. 1998; 33: 151-208Google Scholar). Structural glycan databases comprise over 2000 different sugar modifications described so far on proteins (5Cooper C.A. Harrison M.J. Wilkins M.R. Packer N.H. GlycoSuiteDB: a new curated relational database of glycoprotein glycan structures and their biological sources.Nucleic Acids Res. 2001; 29: 332-335Google Scholar). However, even basic data upon glycosylation of platelet proteins are still rare despite the importance of glycosylations e.g. in cell-cell recognition (6Gu J. Taniguchi N. Regulation of integrin functions by N-glycans.Glycoconj. J. 2004; 21: 9-15Google Scholar), ligand binding, antigen presentation, pathogen binding, and their role in disease. The site of carbohydrate attachment to the peptide backbone is of primary interest. Furthermore, the detailed structure of the attached glycan and the distribution of isoforms over a range of glycosylation sites is a potential research topic. For N-glycosylation the search for glycosylation sites is supported by a known consensus sequence (NX(S/T) where X is not equal to Pro). In combination e.g. with structural data derived from crystallization studies and experimental details regarding the type of glycosylation, this information will aid in confirmation of previous findings, e.g. protein-glycan interaction studies. The presence of modification sites on interaction surfaces may reveal completely new features in this context. For elucidation of N-glycosylation sites a very convenient method makes use of the properties of PNGaseF 1The abbreviation used is: PNGaseF, peptide:N-glycosidase F. 1The abbreviation used is: PNGaseF, peptide:N-glycosidase F. (7Tarentino A.L. Gomez C.M. Plummer Jr., T.H. Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F.Biochemistry. 1985; 24: 4665-4671Google Scholar). This enzyme not only cleaves all types of N-glycans, with few exceptions (8Tretter V. Altmann F. Marz L. Peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached α1→3 to the asparagine-linked N-acetylglucosamine residue.Eur. J. Biochem. 1991; 199: 647-652Google Scholar), from the polypeptide backbone but also possesses additional amidase activity during this process. Therefore, PNGaseF converts asparagine to aspartic acid during the cleavage reaction. This results in a 1-Da mass shift detectable by most of the recently available mass spectrometers. A major drawback regarding analysis is the high number of non-glycosylated peptides in a complete cell digest. Usually only the main structural protein compounds, e.g. actin and filamin, are identified rather than low abundance molecules, which are of superior interest. For specific enrichment of glycopeptides two methods are used. Because mainly N-glycosylated proteins are targeted in this approach we use concanavalin A for the trapping of a broad range of N-glycans (9Baenziger J.U. Fiete D. Structural determinants of concanavalin A specificity for oligosaccharides.J. Biol. Chem. 1979; 254: 2400-2407Google Scholar, 10Bhattacharyya L. Haraldsson M. Brewer C.F. Concanavalin A interactions with asparagine-linked glycopeptides. Bivalency of bisected complex type oligosaccharides.J. Biol. Chem. 1987; 262: 1294-1299Google Scholar). A 2-fold lectin affinity chromatography yields mainly glycopeptides as the dominant species. Thereby, trapped glycoproteins are eluted from the lectin column and digested with trypsin. After removal of non-glycosylated peptides by a second lectin affinity chromatography a derivatization is performed using PNGaseF. A less specific approach, not limited to certain types of N-glycans, is included in the study by use of chemical derivatization of the glycan residues and trapping of glycosylated proteins on hydrazide-functionalized resins. Thereby, reactive aldehyde groups are introduced into the sugar side chains by periodic acid oxidation of vicinal diols present in most hexoses. Those aldehydes can be covalently coupled to hydrazide-derivatized agarose beads. Elution of glycopeptides is performed by use of PNGaseF. Due to the complexity of the resulting peptide mixtures a separation prior to mass spectrometry is necessary. We used nano-reversed-phase HPLC in combination with ESI-MS/MS. The instruments used combine high sensitivity and selectivity as well as the necessary resolution and mass accuracy for the analysis of derivatized glycopeptides. Agarose-bound concanavalin A was purchased from Vector Laboratories, Burlingame, CA, and hydrazide beads were from Perbio Science, Bonn, Germany. PNGaseF was obtained from Roche Applied Science. Trypsin was ordered from Fluka, Steinheim, Germany. All other chemicals used in this study were ordered from Sigma and were of analytical or higher grade. Purified human platelets were prepared using modifications of published procedures (11Tandon N.N. Lipsky R.H. Burgess W.H. Jamieson G.A. Isolation and characterization of platelet glycoprotein IV (CD36).J. Biol. Chem. 1989; 264: 7570-7575Google Scholar) including repeated centrifugation and washing steps and apheresis-derived platelet preparations (Department of Transfusion Medicine, University of Wuerzburg, Germany). To remove plasma proteins and potentially contaminating leukocytes and erythrocytes the samples were centrifuged twice in 50-ml centrifugation tubes at 380 × g for 15 min at room temperature. Both pellets were discarded. The supernatant was centrifuged at 380 × g for 20 min, and the pellet was washed twice with 10 mm citric acid buffer containing 5 mm KCl, 145 mm NaCl, 14 mm glucose, and 1 mm MgCl2, pH 6.4. The obtained platelet pellets were frozen in liquid nitrogen until further use. Lysis of platelets was performed by adding 15 μl of 1% SDS, 100 mm NaCl, 50 mm Tris, pH 7.8 per milligram of platelet wet weight. 10 glass beads (1.2-mm diameter, VWR International, Nuernberg, Germany) were added for a volume of 1000 μl, and lysis was supported by ultrasonication. After reduction with 25 mm dithiothreitol and alkylation using 60 mm iodoacetamide samples were dialyzed against 100 mm sodium phosphate, pH 7.0 overnight. Sodium periodate was added to a concentration of 4 mg/ml, and incubation was performed at room temperature for 40 min in the dark. After a second dialysis against 100 mm sodium phosphate buffer samples were incubated with 500 μl of hydrazide bead slurry for 7 h at room temperature. Washing the resin with 100 mm urea, 50 mm Tris, pH 7.8 was followed by proteolytic cleavage of trapped glycoproteins with trypsin (0.5 μg/μl) at 37 °C overnight. The supernatant was removed, and the beads were washed thoroughly with 1 m urea in 50 mm Tris, pH 7.8 prior to digestion with PNGaseF (0.02 units/ml) at 37 °C overnight in 100 mm sodium phosphate, pH 7.0. For lectin-based enrichment of glycopeptides platelets were lysed in 1.6% n-octylglucoside in buffer A (100 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 30 mm Tris, pH 7.4) resulting in 7 mg/ml concentrations of proteins. After 1:1 dilution with buffer A the sample was applied to a self-packed 4.6-mm-inner diameter × 100-mm-long column packed with concanavalin A-derivatized agarose beads equilibrated extensively with buffer A. Samples were eluted with mm in buffer A. repeated buffer during repeated against 20 mm pH 7.8 the sample was digested at 37 °C for h at trypsin of trypsin by to °C for min the sample was 1:1 and a second lectin affinity chromatography The was and digestion with PNGaseF (0.02 units/ml) was performed at 37 °C overnight in 100 mm sodium phosphate pH 7.0. of complex peptide mixtures was by using For nano-LC-ESI-MS/MS a of Germany) and concentration prior to separation was used. diameter × and separation diameter × were purchased from was performed using a from 5 to during a of A was acid in was followed by the column with for 5 min prior to to the separation analysis was performed on a or a both with Using was for both A was followed by the most peptide For the the time for both and was The of at and a resolution (0.5 at for of the was at for two were in the time was to for the and 30 for the derived mass were by database using the Science, search were by the and Using this all with of the were in the and the data were in the process. was or equal to to a 1-Da shift of to were using a human of the for database Trypsin was used as specific with two Furthermore, were repeated of a to for cleavage with a of were for different were used to the complexity of cell lectin affinity and trapping of glycopeptides. The were the and the peptides were to 70 individual glycosylation sites on 41 different glycoproteins were from these as in I. A with the well Swiss-Prot database a high of glycosylation sites not The database known sites with experimental present and potential sites by for the consensus Furthermore, sites can be as by site information protein or be as in all other potential and sites determined by studies of the sites have not been determined so far than of the identified sites were at N-glycosylation consensus sequence due to the that the protein was known to be Furthermore, 10 sites were on proteins not known to be glycosylated at all or for which the site of glycosylation was not sites identified within human platelet proteins in a new A of a human thrombospondin 1 glycosylation site identified during the of this study is in The a 1-Da shift of the asparagine to aspartic acid within the sequence in the protein residues in are to the peptide as well as of the peptide mass the site of glycosylation at However, only the that a complete of as well as a consensus sequence is present the of this glycosylation In combination with mass accuracy of the instruments used and proteolytic in and as few cleavage are with in a of the experimental results is Furthermore, the sample that enrichment of glycopeptides within the sample by specific affinity a for possible peptide For the most only the major structural proteins, e.g. actin and filamin, be in the abundance in with affinity of peptides, e.g. liquid chromatography not with the of the the number of be in this context. rather for the results cannot be Most were derived from that in Although a number of were used for site many were the site of glycosylation was often not Therefore, the majority of the be due to the results of the two individual methods of glycosylation site analysis a Although some is many of the sites were determined by concanavalin A or hydrazide affinity but not both This distribution certain of the affinity concanavalin A for trapping structures containing the hydrazide a This is due to the of all a vicinal This group is present in most oligosaccharides derived from biological In sites were with the lectin affinity the of determined sites per protein is in of the chemical affinity The complete of proteins and sites identified is within I. To and the are as Swiss-Prot and not as database as derived from the database Therefore, all identified protein have been against Swiss-Prot using the J. D.J. and a new of protein database search Acids Res. Scholar). in the can be into a number of different functions and e.g. and proteins. In addition to the integrins and the receptor was identified in that also and low abundance proteins are detectable using the methods for identified as glycoproteins and can be are for of Those are to in of membrane during or that 2001; Scholar). a was also identified during a study for the of glycosylation sites on human plasma proteins R. and of glycoproteins using hydrazide and mass 2003; 21: Scholar). However, only and were and in during the of study also sites and be the of the as well as the sample to a certain methods the analysis of a complex sample have to be In this a first analysis of asparagine-linked glycosylation sites of human platelet proteins is Although the number of identified proteins is to a number of and glycosylation sites. sites have not been thoroughly but were only by such as shift of and Sci. 2004; Scholar) and periodic methods the presence of glycosylation but not the type or the attachment In lectin affinity chromatography and hydrazide bead trapping of glycosylated peptides in combination with nano-LC-ESI-MS/MS provided of glycosylation type and sites The identified proteins may be into a range of functions or some proteins, and will be the that many of the proteins in are known for some their glycosylation sites have not been in and other glycoproteins are as for cell-cell and interactions as well as in this are important glycosylation sites were on integrins and of which only was Therefore, data will of the properties of these However, the of post-translational modifications in databases in the Although the Swiss-Prot database features a certain number of glycosylation a still number is as for the human protein database S. R. V. V. B. M. N. N. N. M. S. N. R. S. S. M. V. A. L. R. Steen M. S. J. P. A. A. A. of human protein database as for in Res. 2003; Scholar), also from the to the of Therefore, the combination of proteomic studies and in databases may to data storage within sequence A very of a new platelet protein in present study is is shown here as for some less known or proteins described in and is by a in the major complex and a type protein to the immunoglobulin a cell receptor with intracellular phosphorylation that with and and is in of the Ras-mitogen-activated protein kinase pathway B. proteins and are by human G6f, a of the immunoglobulin in the J. 2003; Scholar). the receptor has so far not been described in platelets. The possible role of in platelets is the of investigations in of a common platelet thrombospondin glycoprotein that and interactions J. Biochem. Biol. 29: Scholar), was also identified during present studies. to type and integrins and sites of N-glycosylation of which only has been in the Swiss-Prot database so in additional site be to as were also such as plasma proteins, which may to the plasma membrane of platelets. a well protein of the human was identified two known sites of N-glycosylation and plasma from this cannot be completely due to the platelet procedures used which are to platelet and However, some proteins are present both in plasma as well as in e.g. V. This protein is a that with to to is distributed to plasma and is distributed to of platelets P.B. K.G. of in human plasma and Scholar). that carbohydrate a substantial role in protein cleavage and of M. P.B. on the but not the derived by protein Scholar). sites of N-glycosylation of be determined of which is to the cleavage site at is to a storage protein in platelet D.F. P. R.H. is in the of platelets and is by a cell Scholar). This glycoprotein P. a of proteins within 1991; Scholar) has potential glycosylation sites of which be using a combination of hydrazide and lectin the complex is a role for 1 in and platelets prior to R.A. N. G.A. B. W.H. of the within the of human Biol. Chem. 2004; Scholar). the specificity of the methods only can be on the carbohydrate of the identified glycopeptides. of the few glycoproteins studied extensively over the was identified during the of the The protein but a different glycosylation site in the present study and in the study of R. and of glycoproteins using hydrazide and mass 2003; 21: was identified in a previous hydrazide affinity R. and of glycoproteins using hydrazide and mass 2003; 21: Scholar). The glycan extensions provided by (5Cooper C.A. Harrison M.J. Wilkins M.R. Packer N.H. GlycoSuiteDB: a new curated relational database of glycoprotein glycan structures and their biological sources.Nucleic Acids Res. 2001; 29: 332-335Google Scholar) comprise a range of complex glycans A. on the chains of human of oligosaccharides.J. Biol. Chem. Scholar). Those structures can be trapped by the hydrazide as shown and also by concanavalin A interactions as by and Fiete (9Baenziger J.U. Fiete D. Structural determinants of concanavalin A specificity for oligosaccharides.J. Biol. Chem. 1979; 254: 2400-2407Google Scholar). Furthermore, the complexity of carbohydrate within a cell has to be into is possible that of glycosylated are using the two The distribution of sites by the two methods a but also high of sites only by method or the This may be the of different of the methods but also be due to the individual sample during the two analysis for the use of glycosylation site information is by combination with structural data in of protein Therefore, a is protein database J. G. The Acids Res. Scholar) the and N-glycans to within the The the different of the to be attached to the the use of glycans is In glycosylation is the only to into structural glycosylated proteins not and are prior to The is on the use of experimental the and sugar A. S. of the N-glycosylation sites in Scholar, Dwek R.A. M.R. A analysis of and from Scholar, A.L. Dwek R.A. M.R. analysis of the protein of N-glycosylation for and 2004; Scholar). The structure of the glycan is by of the A. carbohydrate by Mol. 1998; Scholar, A. of and Scholar). possible the data of proteins and the structural features of carbohydrate additions to the polypeptide In A and the in glycosylated structure of the J. into the basis of and by structures of and bound to and is with the The of has been to cell A. B. A role for the of in ligand recognition and cell Biol. Scholar, sites on lectin and are in the of to J. Scholar) in the at completely the interaction of the The attached is a high glycan in this for a cell glycoprotein this structure may be R. C. J. Platelet acid as a potential in Res. Scholar). due to acid attachment have to be into account as well interaction surfaces or experimental those is that the elucidation of glycosylation site information yields substantial for research in this field by adding e.g. for the of interaction studies. the are applied to a cell type of for Because platelets the methods for glycosylation e.g. of consensus are not at In this analysis of protein modifications is necessary as in this is a for this sites of glycosylation have been in the of and human P. M. is for and of human Biol. Chem. 2004; Scholar) but not in platelets. Therefore, results in the may not be to the A major drawback of the analysis methods is the substantial of information due to the removal of the glycan few can be made on the of the Because PNGaseF cleaves all N-glycans is not possible to additional specificity during this and additional information on the However, this can be by the range of and used. with specificity for e.g. or complex structures or specific of glycans can be used. a using e.g. and can be cleaves complex and high is against and glycans in Therefore, the of the glycan can be by of two In studies of platelet proteins are to of established and human platelet proteins that will be to some of the of platelet in and disease.