Glycobiology Advance Access originally published online on February 23, 2006
Glycobiology 2006 16(6):514-523; doi:10.1093/glycob/cwj091
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Approaches to the study of N-linked glycoproteins in human plasma using lectin affinity chromatography and nano-HPLC coupled to electrospray linear ion trap—Fourier transform mass spectrometry
Barnett Institute, Northeastern University, Boston, MA 02115
1 Present address: Centocor R&D, Inc., 145 King of Prussia Road, Radnor, PA 19087.
2 To whom correspondence should be addressed; e-mail:wi.hancock{at}neu.edu
Received on November 13, 2005; revised on January 23, 2006; accepted on February 13, 2006
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Abstract |
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 Top Abstract IntroductionResults and discussionConclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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In this publication, we will describe the combination of lectin affinity chromatography with nano high performance liquid chromatography (HPLC) coupled to a linear ion trap Fourier transform mass spectrometer (capillary LC-LTQ/FTMS) to characterize N-linked glycosylation structures in human plasma proteins. We used a well-characterized glycoprotein, tissue plasminogen activator (rt-PA), which is present at low levels in blood, as a standard to determine the dynamic range of this approach. N-linked glycopeptides derived from rt-PA could be characterized at a ratio of 1:200 in human plasma (rtPA: total plasma protein, w/w) by accurate mass measurement in the FTMS and fragmentation (MSn) in the linear ion trap. We demonstrated that this platform has the potential to characterize the general N-linked glycosylation structures of abundant glycoproteins present in human plasma without the requirement for antibody-based purification, or additional carbohydrate analytical protocols. This conclusion was supported by the determination of carbohydrate structures for three glycoproteins, IgG, haptoglobin, and alpha-1-acid glycoprotein, at their natural levels in a human plasma sample, but only after the lectin enrichment step.
Key words: Chinese hamster ovary / glycoprotein / mass spectrometry / plasma / lectin
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Introduction |
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TopAbstractIntroductionResults and discussionConclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Elsewhere we have reported on the development of a platform that combined lectin chromatography to a linear ion trap Fourier transform mass spectrometer (capillary LC-LTQ/FTMS) to enrich and characterize glycoproteins from cell culture, such as Chinese Hamster Ovarian cell lines (CHO) (Y. Wang, S.L. Wu, and W.S. Hancock, unpublished data). This study showed that combinations of lectins could be used to enrich glycoprotein pharmaceuticals significantly from a lysate and the fermentation process could be monitored by characterization of the glycoprotein structures. In this application the new hybrid MS platform was crucial to our ability to perform this detailed characterization even at relatively low product levels.
In this report, we describe application of this platform to another important sample, human plasma. Representing the largest human proteome, plasma/serum is the primary clinical specimen for disease diagnosis. Plasma is of extreme complexity with a dynamic range in concentration of more than 10 orders of magnitude (Anderson and Anderson, 2002
). By the use of a multi-lectin affinity column (M-LAC), it was found that at least 50% of plasma proteins are glycosylated (Yang and Hancock, 2004).
Glycosylation changes are associated with diseases such as cancer, and thus monitoring such changes could be of diagnostic significance. In the studies of well-characterized plasma proteins, such as transferrin and haptoglobin, the structure of the oligosaccharide has been shown to change in disease (Turner, 1995; Georgieff et al., 1997; Matei, 1997; Goodarzi and Turner, 1998; Nihlen et al., 2001; van Rensburg et al., 2004). Current approaches of studying changes in glycoproteins are mostly limited to the study of detailed glycosylation forms in a single target protein of biological or clinical interest, such as the comprehensive structural study of tissue plasminogen activator (Spellman et al., 1989; Wu et al., 1990; Guzzetta et al., 1993). There are at present few methods which can be used to screen glycoproteins for glycosylation pattern changes in complex biological samples, such as serum, apart from 2-D gels, which has a limited dynamic range.
Due to the complexity of plasma, most studies of glycoproteins have been performed on samples isolated by affinity chromatography or other chromatographic techniques (Zhang et al., 2003; Bunkenborg et al., 2004; Hagglund et al., 2004; Wada et al., 2004). One global approach, however, captures the glycopeptides present in a proteolytic digest on a solid phase support and then one can identify the corresponding peptide after cleavage of the carbohydrate linkage (Zhang et al., 2003). While this approach can identify many peptide sequences, the information about which glycan is attached to a site is lost and must be obtained in a separate procedure.
The approach which we report in this paper involves the identification of N-linked glycopeptides generated by trypsin digestion of the glycoprotein fraction captured on a lectin column, shown in Figure 1. A key step is the appearance of new peptide sequences after N-glycanase digestion of the tryptic digest. Once the parent peptide sequence has been identified the presence of potential glycoforms can be confirmed by observation of the appropriate precursor mass in the FTMS. The final step in the analysis is to study the glycan composition by fragmentation in the linear ion trap. This approach was demonstrated by the confirmation of known glycosylation sites of a standard protein, recombinant tissue plasminogen activator (rt-PA) in an add-back to human plasma. In addition, the standard was used to evaluate the efficiency of the lectin enrichment step as well as the effectiveness of the MS system. This protein is present naturally at low levels in plasma, and thus endogenous levels would not interfere with the add-back study.
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In this study we demonstrated that the lectin enrichment procedure has the potential to characterize many of the glycosylation structures of abundant glycoproteins in human plasma without the requirement for antibody-based purification or additional carbohydrate protocols. This paper illustrates this approach with the characterization of the carbohydrate structures of three plasma proteins, IgG, haptoglobin, and alpha-acid glycoprotein.
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Results and discussion |
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TopAbstractIntroductionResults and discussionConclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Strategy of multi-lectin approach
This report is part of a systematic study to explore the potential of the use of lectin mixtures to provide a more global approach to the study of glycosylation changes in biological samples. As reported earlier the use of lectin mixtures (M-LAC) gave a comprehensive capture of glycoproteins from complex samples such as serum by selecting lectins that have complementary specificities for different glycosylation structures (Yang and Hancock, 2004
). Another advantage of this approach is that the combination of lectins is more likely to capture modified glycosylation motifs so that a comparative analysis is not flawed by the loss of modified forms into the non-bound fraction. In addition, the non-bound fraction is routinely subjected to a proteomic analysis (LC/MS of the tryptic digest), and in these studies a shift in the distribution of the glycoproteins under analysis was not observed (data not shown). In numerous studies of the multi-lectin format we have not observed any of the abundant glycoproteins (Table 1) in the unbound fraction, except for small amounts of transferrin and antitrypsin (less than 25% as measured by the number of peptides identified in the LC/MS analysis). In these cases the unbound material could be attributed to non-glycosylated variants of these proteins, particularly as the lectin mixtures are used in a significant excess over the amount of glycoprotein in the sample (Yang and Hancock, 2004).
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A key aspect of this study was the proposal that the M-LAC approach could detect changes in glycosylation motifs in different samples by the use of specific displacers for each of the lectins (Yang and Hancock, 2005). In this manner an M-LAC column with three lectins would yield three different displacer fractions that could be subjected to proteomic analysis. The hypothesis that a change in glycosylation could result in a shift of the proportion of a given protein in the different displacer fractions was demonstrated by the study of several glycoproteins both as standards and then in the native state in plasma before and after treatment with glycosidases. For example, after removal of sialic acid with neuraminidase the amount of a sialyated glycoprotein eluted in the displacer used for a lectin with affinity for sialic acid (WGA) was decreased (Yang and Hancock, 2005).
The next step in this approach was to develop methodology to obtain structural information on glycosylation motifs that would extend the information from the displacer shift studies. A requirement of our approach was to develop analytical methods that were relatively high throughput and could be used on small sample amounts and thus be useful for clinical studies. We believe that it is unreasonable at this stage of technology development to expect that such an approach can yield complete structural information on glycosylation changes between different samples and rather it is our goal to identify candidate glycoproteins for further studies that would use targeted isolation procedures such as immuno-affinity columns. In this manner, transition can be done from global studies to more detailed analysis of individual biomarkers. After the in-depth characterization phase and the follow on focused biomarker studies, one could expect another development step where a technology platform suitable for large number of clinical assays is identified.
The challenge of glycoprotein characterization in a human plasma sample
Representing the largest set in the human proteome, plasma is also the primary clinical specimen for disease diagnosis. In human plasma, about 50% of the total proteins are glycosylated, and several biomarkers, such as Her-2 (Lupu et al., 1991), are present at low levels (approximately 10 ng/mL). To detect and characterize such low abundance glycoproteins in a complex biological sample, an enrichment step before LC/MS analysis is essential. After demonstrating the successful enrichment and characterization of a product glycoprotein in a cell culture lysate in a previous study (Y. Wang, S.L. Wu, and W.S. Hancock, unpublished data), we then extended use of this platform to the analysis of glycoproteins in human plasma.
It is very difficult to characterize the nature of glycosylation of even major glycoproteins in plasma without direct isolation of the protein. By two-dimensional gel electrophoresis (2-D-GE), glycoproteins can be separated as different spots, but the characterization of structural motifs in the glycosylation pattern is a challenge. In many cases, both the site of glycosylation and the corresponding structures are unknown or poorly characterized, particularly in studies of disease. We believed that when the lectin affinity chromatography was combined with a powerful new MS platform one could not only enrich the glycoproteins from complex biological samples but also gain structural information on some of the glycosylation motifs present in the more abundant glycoproteins.
As discussed in a previous study (Y. Wang, S.L. Wu, and W.S. Hancock, unpublished data), at a ratio 1:200 (rt-PA: CHO cell lysate protein), after lectin enrichment, direct analysis of the glycopeptide could be achieved without further enrichment and/or derivatization. With this approach, not only was the isotopic peak distribution of the parent ion clearly observed in the FTMS but also subsequent fragmentation information (MS2 and MS3) could be obtained in the linear ion trap. In this study, the characterization of a high-mannose glycopeptide was reported as an example. Despite the much greater complexity of plasma versus a cell lysate, we report here a similar degree of success for a human plasma sample, but only after modification of our approach.
After lectin capture the glycoproteins in the bound fraction were identified by database searching of the non-glycosylated peptides observed in the LC/MS analysis of a tryptic digest. Despite the successful lectin enrichment step it is not usually possible to identify unknown glycosylated peptides in a plasma digest due to the complexity of the sample and the heterogeneity of glycosylation. We, therefore, used the process outlined in Figure 1 to characterize such glycopeptides in a multi-step approach. A key step is to use a glycosidase to generate the corresponding non-glycosylated peptide, which can then be identified as new species in a replicate LC/MS analysis. The results of this approach will be described below. It should be noted that none of the identified abundant glycoproteins were present at significant levels in the unbound fraction except for the case of transferrin and antitrypsin which are known to contain non-glycosylated forms (Mills et al., 2001; van Rensburg et al., 2004).
The use of a double-lectin column for glycoprotein enrichment from human plasma
Glycoprotein enrichment
In the human plasma sample, rt-PA was spiked at ratios (wt/wt) of 1:1000 and 1:200 before loading the sample onto the double-lectin column. The purified glycoprotein fractions were then subjected to trypsin digestion and capillary LC -LTQ/FTMS. In both cases an improvement in the number of peptide identifications (and hence sequence coverage) was observed for rt-PA captured on the lectin columns versus direct plasma analysis. Seven rt-PA peptides were identified in plasma at a spiking ratio of 1:1000 after the lectin enrichment step, while the protein was not identified by direct analysis (without the use of lectins). For plasma with rt-PA spiked at a ratio of 1:200 (w/w), 5 and 15 unique peptides were identified for rt-PA before and after lectin enrichment respectively.
Again, we compared the intensity of the MS signal for samples before and after lectin enrichment. After the lectin enrichment, the selected rt-PA peptide, which could barely be seen without lectin enrichment at a ratio of 1:1000, was clearly observed, as shown in Figure 2. Also, at a ratio 1:200, the intensity of this peptide was improved by 10-fold with the double-lectin enrichment. These results were consistent with previous studies (Y. Wang, S.L. Wu, and W.S. Hancock, unpublished data), on mixtures of lectins and showed that the enrichment step could improve the ability to identify a plasma glycoprotein (higher sequence coverage) and detection sensitivity (higher peptide intensity).
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Although not glycosylated, we have previously observed (Yang and Hancock, 2004) that a small amount of albumin is bound to the lectin column, presumably as a result of binding to glycosylated immunoglobulins (see Table 1 for typical results). Apart from albumin, all other glycoproteins captured by the lectin column are known to be glycosylated. Since the software ranking is not directly related to quantitation, the listing in Table 1 does not indicate changes in relative concentration after enrichment, but rather gives an idea of abundant glycoproteins that are present at a sufficient concentration for characterization of glycosylation motifs by MS/MS fragmentation. Another abundant glycoprotein identified in this study is alpha-1 acid glycoprotein (listed as number 25 by the Sequest algorithm), which was subsequently identified by the N-glycanase digestion.
The characterization of glycostructures in a human plasma sample
To detect and identify unknown N-linked glycopeptides present in the glycoprotein fraction we used an N-glycanase (PNGase A) digestion of the corresponding tryptic digest. This step removed N-linked glycans from the corresponding glycopeptide, and we then analyzed both the original and deglycosylated digest with capillary LC -LTQ/FTMS (see Figure 3 for a typical example). The N-glycanase digest also resulted in a change of amino acid sequence from the corresponding genomic sequence (residue N changed to residue D) (Plummer et al., 1981). After a database search of the MS output (addition of 1 Da to residue Asn), we identified 25 peptide candidates that were detected only after PNGase A treatment and also contained the consensus NXS/T sequence (Table 2). Out of the 25 peptides containing 26 consensus NXS/T sequences, 20 N-linked glycosylation sites were confirmed by SwissProt annotation and 6 other sites have not been reported to be glycosylated. Not surprisingly, the 25 glycopeptides were derived from high abundance glycoproteins (total of 23 proteins) present in human plasma.
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In the following examples we demonstrate that this approach does enable the structural characterization of glycopeptides in the LTQ/FTMS. In the LC/MS study, the peptides were first scanned by FTMS with high mass accuracy (
2ppm) and resolution (= 100,000) and then the selected target peptides were fragmented by collision-induced dissociation in MS2 and MS3 scans. As the full mass spectrum of the precursor ion does not yield an unequivocal structural determination, even with an accuracy of less than 2 ppm, tandem MS studies are necessary to provide fragments that correspond to loss of oligosaccharide moieties and to determine site of attachment to the peptide sequence. We have previously shown that glycopeptides usually elute from a reversed phase column slightly ahead of the non-glycosylated peptide (Guzzetta et al., 1993).
Figure 3 shows the LC/MS analysis of the glycopeptide (EEQYN*STYR) that is derived from enzymatic digestion of the IgG tryptic peptides (retention time of 16.79 min versus 17.89 min for the deglycosylated peptide). The deglycosylation step resulted in an approximately 10-fold increase in the MS signal, which aided identification of the peptide. With knowledge of the peptide sequence it was possible to predict the mass of potential glycopeptide structures and search for the corresponding ions in the MS analysis at a retention time window adjacent to the elution point of the deglycosylated peptide. In the case of IgG the complex biantennary glycan structure was identified by a full MS scan in the FTMS (precursor mass 1083.755) and tandem MSn scans in the linear ion trap (see Figure 4b–d). In part b, the isotope cluster of the peptide molecular ion (charge state 3+) is shown (as measured in the FTMS) together with the glycopeptide structure that is consistent with the observed mass. This ion was selected for CID fragmentation (MS2) and some of the major fragments, together with the charge state, are shown in Figure 4c. In the next stage (MS3), the major ion (m/z 1297.72) was further fragmented in the linear ion trap and the result is shown in Figure 4d. For simplicity, not all fragments are annotated in the figure, but the mass of all major fragments was shown to be consistent with the proposed structure (data not shown). In addition, the presence of glycoforms that contained a biantennary structure with terminal sialic acid and galactose was consistent with the expected specificity of the double lectin column. In this sample, which reflects the natural state of IgG present in the plasma sample, it would be expected that glycan heterogeneity is observed at this site. As the purpose of this initial report is to focus on the methodological aspects of our approach, we have not attempted to determine if this lack of glycan heterogeneity is due to the composition of the sample or to the specificity of the mixtures of lectins used in this M-LAC configuration. We are planning to perform follow-on disease studies and in this case we will confirm the initial identification of changes in a candidate biomarker with targeted antibody capture approaches to ensure that we characterize the full set of glycosylation structures. Also we have noted in an earlier publication that some disease studies will require the optimization of the combination of lectins and displacers to the panel of glycoproteins that is targeted in the study (Yang and Hancock, 2004).
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represents N-acetyl-glucosamine; 
represents fucose; and ‘O’ represents galactose.In the following examples of identification of glycosylation structures for other abundant plasma glycoproteins present in the plasma sample the same analytical process as for IgG was used. The lectin bound fraction was successively digested with trypsin and PNGase A, and the new deglycosylated peptide was identified in the MS. The corresponding ion chromatograms were similar to those in Figure 3 and showed a significant increase in ion intensity on removal of the glycosylation heterogeneity (data not shown). The identification of a glycopeptide derived from haptoglobin is shown in Figure 5. In this case, the observed structure was a triantennary glycan with terminal sialic acid and galactose, and the fragmentation of the glycoforms was consistent with the proposed glycan structures. In this analysis only three of the four known glycosylation sites of transferrin were observed. While this result could be related to missing glycosylation in this plasma sample rather than failure of the LC/MS measurement, future work will be directed at increasing the dynamic range of the proteomic measurement to allow detection of peptides that do not ionize or fragment with high efficiency. A third example is the identification of the glycopeptide derived from alpha-1-acid glycoprotein. A triantennary glycan with three sialic acids was characterized by our approach, as shown in Figure 6.
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represents N-acetyl-glucosamine; ‘O’ represents galactose; and 
represents sialic acid.
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represents N-acetyl-glucosamine; ‘O’ represents galactose; and 
represents sialic acid.In summary, complex glycoforms which contain sialic acid could be significantly enriched by the double-lectin column system from plasma samples. Furthermore, the new hybrid MS platform, a combination of a linear ion trap and a FTMS, is particularly suitable for the characterization of a glycopeptide in a complex tryptic digest. The glycopeptide identifications demonstrated that this platform has the potential to monitor structural changes associated with shifts in glycosylation in disease and will be the subject of future studies. While it is unreasonable to expect such an approach to identify all glycoforms present in the target proteins, the information from the global study can then be used to direct a more focused characterization of selected glycoproteins.
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Conclusions |
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TopAbstractIntroductionResults and discussionConclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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In this study, we extended our platform of multi-lectin enrichment with capillary LC -LTQ/FTMS of a tryptic digest of human plasma. An LC/MS analysis of a PNGase digest of the peptide mixture was developed to identify novel glycopeptides via the appearance of a deglycosylated peptide. In a replicate LC/MS analysis the precursor mass of the corresponding N-linked glycosylated structures was measured in the FTMS, and the glycosylation structure was studied by fragmentation in the ion-trap part of the mass spectrometer. While the current results are limited to N-linked glycosylation sites we are currently exploring approaches to address the characterization of O-linked structures. We have demonstrated that the use of Jacalin (JAC) lectin can give a capture of O-glycosylated proteins in the M-LAC format (Yang and Hancock, 2004
) and we plan to combine this with specific chemistry and mass spectrometric approaches. We believe that this approach, when fully developed, will have significant potential for biomarker discovery and more global characterization of changes in glycoprotein motifs. Future research will be focused on extending the dynamic range of this approach for the characterization of lower-level glycoproteins. |
Materials and methods |
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TopAbstractIntroductionResults and discussionConclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Lectin affinity column preparation
Single lectin columns, concanavalin A (ConA), wheat germ agglutinin (WGA), and JAC were prepared by adding 0.5 mL of the agarose bound lectin (Vector Laboratories, Burlingame, CA) into empty polypropylene columns (Pierce Biotechnology, Inc., Rockford, IL). The double-lectin column was prepared by mixing 0.25 mL of agarose-bound ConA and 0.25 mL of agarose-bound WGA in an empty polypropylene column. The agarose gel was fixed with two frits. The columns were equilibrated with bind/wash buffer (20mM Tris, 0.15M NaCl, 1mM Mn2+ and 1mM Ca2+, pH 7.4) before use.
Lectin enrichment
Protein samples (
30µg) were diluted with bind/wash buffer (1:10 v/v) before loading onto the lectin columns. The samples were incubated on the column for at least 15 min. The unbound proteins were washed with 1–2 mL bind/wash buffer and then the captured proteins were eluted with 1–2 mL elution buffer (20 mM Tris, 0.5 M NaCl, pH 7.4 with 0.5 M methyl-
-D-mannopyranoside [ConA] or 0.5 M N-acetyl-glucosamine [WGA] or 0.8 M galactose [JAC]). For the double-lectin column, the elution buffer included 0.25 M methyl--D-mannopyranoside and 0.5 M N-acetylglucosamine in 20 mM Tris and 0.5 M NaCl. The bound and unbound fractions were collected and concentrated with 4-mL 10 kDa MWCO Amico filters (Millipore, Billerica, MA). The total recovery of the lectin column was based on the Bradford assay.
Tryptic digestion
The protein samples were diluted in 0.1 M ammonium bicarbonate buffer with 6 M guanidine chloride, pH 8.0. The denatured samples were reduced by incubating with 5 mM dithiothreitol (DTT) at 75°C for 1 h and then alkylated by incubating with 20 mM iodoacetamide at room temperature in dark for 2 h. After the buffer exchange with a 10 kD MWCO filter (Millipore, MA), the samples were reconstituted with 0.1 M ammonium bicarbonate buffer. Trypsin (Promega, Madison, WI) was then added at 1:100 ratio (w/w) and the samples were incubated at ambient temperature for overnight digestion. Another equal amount of trypsin was added for another 6 h period to ensure complete digestion.
Deglycosylation of tryptic glycopeptides by PNGase A
The solution of rt-PA digest was adjusted to pH 5.0 with 1.0% formic acid (v/v) and was treated with PNGase A (Sigma-Aldrich, St. Louis, MO) for 3 h at 37 °C.
LC/MS analyses
All the LC/MS experiments were performed on an Ultimate capillary LC system (Dionex, Sunnyvale, CA) interfaced to a Finnigan LTQ-FT mass spectrometer (ThermoElectron, San Jose, CA). Mobile phases were (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. A PicoFrit BioBasic C18 column (75 µm i.d x 15 cm; New Objective, Woburn, MA) was used for all the analyses with a 50 min gradient (2–35% B in 40 min and 35–80% B in 10 min). The flow rate was maintained at around 300 nL/min after flow splitting. The electrospray ionization conditions were: ion capillary transfer tube temperature, 245°C; needle voltage, +2.4 kV; normalized collision energy, 35%. Spectra were acquired with the instrument operating in the data-dependent mode of operation. Four MS2 and four MS3 were performed after each full mass scan. The LTQ-FT acquired a full mass spectrum with the FTMS as the analyzer. The MS/MS and MS/MS/MS spectra were acquired with the LTQ as the analyzer. Mass measurements made with the FTMS in the full mass spectrum were all within 2–3 ppm of the theoretical masses. All MS/MS and MS/MS/MS spectra were interpreted manually.
Peptide identification
Peptide identification was obtained through a human rapido database search using the SEQUEST algorithm incorporated into the BioWorks software (version 3.1SR1). Peptides were automatically identified if the peptide has an Xcorr score (+1 > 1.5; +2 > 2.0; +3 > 2.5) with trypsin cleavage at both ends. In addition, manual inspections were performed for the MS/MS spectra. Glycopeptide backbones were identified only after PNGase A treatment. The N-linked glycosylation sites were confirmed with the consensus sequence NXS/T, where X could be any amino acid except proline.
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Conflict of interest statement |
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TopAbstractIntroductionResults and discussionConclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResults and discussionConclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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The authors thank Prof. Barry L. Karger, Dr. Jianmin Ren, Dr. JeongKwon Kim, and Dr. Marina Hincapie for helpful discussion. Barnett Institute contribution number 875.
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Abbreviations |
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2-D-GE, two dimensional gel electrophoresis; CHO, Chinese hamster ovary; ConA, concanavalin A; FT, Fourier transform; HPLC, high performance liquid chromatography; JAC, jacalin; LTQ, linear ion trap; MS, Mass Spectrometry; rt-PA, recombinant tissue plasminogen activator
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References |
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TopAbstractIntroductionResults and discussionConclusionsMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Anderson, N.L. and Anderson, N.G. (2002) The human plasma proteome: history, character, and diagnostic prospects. Mol. Cell Proteomics, 1, 845–867.
Bunkenborg, J., Pilch, B.J., Podtelejnikov, A.V., and Wisniewski, J.R. (2004) Screening for N-glycosylated proteins by liquid chromatography mass spectrometry. Proteomics, 4, 454–465.[CrossRef][Web of Science][Medline]
Georgieff, M.K., Petry C.D., Mills, M.M., McKay, H., and Wobken, J.D. (1997) Increased N-glycosylation and reduced transferrin-binding capacity of transferrin receptor isolated from placentae of diabetic women. Placenta, 18, 563–568.[CrossRef][Medline]
Goodarzi, M.T. and Turner, G.A. (1998) Reproducible and sensitive determination of charged oligosaccharides from haptoglobin by PNGase F digestion and HPAEC/PAD analysis: glycan composition varies with disease. Glycoconj. J., 15, 469–475.[CrossRef][Web of Science][Medline]
Guzzetta, A.W., Basa, L.J., Hancock, W.S., Keyt, B.A., and Bennett, W.F. (1993) Identification of carbohydrate structures in glycoprotein peptide maps by the use of LC/MS with selected ion extraction with special reference to tissue plasminogen activator and a glycosylation variant produced by site directed mutagenesis. Anal. Chem., 65, 2953–2962.[Medline]
Hagglund, P., Bunkenborg, J., Elortza, F., Jensen, O.N., and Roepstorff, P. (2004) A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation. J. Proteome Res., 3, 556–566.[CrossRef][Medline]
Lupu, R., Dickson, R.B., and Lippman, M.E. (1991) The role of erbB-2 and its ligands in growth control of malignant breast epithelium. Princess Takamatsu Symp, 22, 49–60.[Medline]
Matei, L. (1997) Plasma proteins glycosylation and its alteration in disease. Rom. J. Intern. Med., 35, 3–11.[Medline]
Mills, K., Mills, P.B., Clayton, P.T., Johnson, A.W., Whitehouse, D.B., and Winchester, B.G. (2001) Identification of alpha (1)-antitrypsin variants in plasma with the use of proteomic technology. Clin. Chem., 47, 2012–2022.
Nihlen, U., Montnemery, P., Lindholm, L.H., and Lofdahl, C.G. (2001) Increased serum levels of carbohydrate-deficient transferrin in patients with chronic obstructive pulmonary disease. Scand. J. Clin. Lab. Invest, 61, 341–347.[Medline]
Plummer, T.H., Jr. and Tarentino, A.L. (1981) Facile cleavage of complex oligosaccharides from glycopeptides by almond emulsin peptide: N-glycosidase. J. Biol. Chem., 256, 10243–10246.
van Rensburg, SJ., Berman, P., Potocnik, F., MacGregor, P., Hon D., and de Villiers, N. (2004) 5- and 6-glycosylation of transferrin in patients with Alzheimer’s disease. Metab. Brain Dis 19, 89–96.[Medline]
Spellman, M.W., Basa, L.J., Leonard, C.K., Chakel, J.A., O’Connor, J.V., Wilson, S., and van Halbeek H. (1989) Carbohydrate structures of human tissue plasminogen activator expressed in Chinese hamster ovary cells. J. Biol. Chem., 264, 14100–14111.
Turner, G.A. (1995) Haptoglobin. A potential reporter molecule for glycosylation changes in disease. Adv. Exp Med. Biol., 376, 231–238.[Medline]
Wada, Y., Tajiri, M., and Yoshida, S. (2004) Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics. Anal. Chem., 76, 6560–6565.[Medline]
Wu, SL., Teshima, G., Cacia, J., and Hancock, WS. (1990) Use of high-performance capillary electrophoresis to monitor charge heterogeneity in recombinant-DNA derived proteins. J. Chromatogr., 516, 115–122.[Medline]
Yang, Z. and Hancock, W.S. (2004) Approach to the comprehensive analysis of glycoproteins isolated from human serum using a multi-lectin affinity column. J. Chromatogr. A, 1053, 79–88.[CrossRef][Web of Science][Medline]
Yang, Z. and Hancock, W.S. (2005) Monitoring glycosylation pattern changes of glycoproteins using multi-lectin affinity columns. J. Chromatogr. A, 1070, 57–64.[CrossRef][Web of Science][Medline]
Zhang, H., Li, XJ., Martin, DB., and Aebersold, R. (2003) Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat. Biotechnol., 21, 660–666.[CrossRef][Web of Science][Medline]
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