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Comparative Glycoproteomics of N-Linked Complex-Type Glycoforms Containing Sialic Acid in Human Serum Ruiqing Qiu and Fred E. Regnier*
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907
This study describes a simple and efficient approach for comparative analysis of sialylated glycoforms of proteins containing differentially branched complex-type glycans. The analytical protocol is based on glycopeptide selection from tryptic digests with serial lectin affinity chromatography (SLAC), quantification with global internal standard technology, fractionation of deglycosylated peptides with reversed-phase chromatography, and peptide sequencing with tandem mass spectrometry. Fractionation of complex tri- and tetraantennary N-linked glycoforms from biantennary N-linked glycoforms bearing terminal sialic acid residues was achieved using a set of serial lectin columns with immobilized Sambucus nigra agglutinin and concanavalin A. These two fractions from the affinity selection were differentially labeled, mixed, and then deglycosylated with the enzyme PNGase F. The deglycosylated sample was further fractionated by reversed-phase chromatography and analyzed by electrospray ionization mass spectrometry. The SLAC strategy was applied to tryptic digests of human serum, and it was found that most sialylated glycopeptides identified carry more biantennary glycans than tri- and tetraantennary glycans, and the relative amount of biantennary glycan versus tri- and tetraantennary glycans was different at separate glycosylation sites within the same glycoprotein. Glycosylation is one of the most common posttranslational modifications of proteins. Glycoproteins play an important role in many cellular processes. One is at the cell surface where glycoproteins are involved in protein targeting along with cellcell and cell-matrix interactions.1,2 Deletion of genes responsible for glycosylation can be lethal. More than half of all proteins have been estimated to be glycosylated.3 Protein glycosylation is determined by the combination of glycosyltransferases and glycosidases present in a cell at any particular time. Accordingly, glycosylation is a highly complex and dynamic process resulting in the production of multiple glycoforms of the same protein. A major contributor to glycan microheterogeneity is the fact that the reactions involved in * To whom correspondence should be addressed. E-mail: fregnier@ purdue.edu. (1) Drickamer, K.; Taylor, M. E. Trends Biochem. Sci. 1998, 23, 321-324. (2) Kim, Y. J.; Varki, A. Glycoconjugate J. 1997, 14, 569-576. (3) Van den Steen, P.; Rudd, P. M.; Dwek, R. A.; Opdenakker, G. Crit. Rev. Biochem. Mol. Biol. 1998, 33, 151-208. 10.1021/ac050554q CCC: $30.25 Published on Web 10/08/2005
© 2005 American Chemical Society
glycosylation and deglycosylation at specific sites in glycans are frequently incomplete. This gives protein glycosylation a combinatorial character. Glycans in the wide variety of glycoforms seen in most proteins are often capped with sialic acid residues, generally linked via an R-2,3 or an R-2,6 bond to Gal/GalNAc. But sialylation is reversible, generally being removed at some point in the life cycle of a glycoprotein. The fact that sialic acid carries a negative charge at physiological pH affects glycoconjugate conformation and plays a crucial role in cell surface interactions. The presence of sialic acid on the surface of a cell is a recognition determinant in cellcell interactions4 and protects cells from membrane proteolysis.5 Aberrations in sialylation have even been associated with disease. Increased sialylation on the surface of tumor cells is well known and is due to either (1) increased activity of a sialyltransferase, such as R-2,6-sialyltransferase, or (2) increases in the number of termini available for sialylation as a result of an upregulation of branching in N-linked glycans. Although lectins have been used for decades in the histochemical detection of sugar chains on cell surfaces,6 they are becoming increasing important as analytical tools in glycoproteomics.7 Lectin affinity chromatography has recently been used to purify glycoproteins and to examine their microheterogeneity to the level of a single sugar residue.8,9 Yang and Hancock10 developed a multi-lectin affinity column, which contains concanavalin A (Con A), wheat germ agglutinin, and jacalin to capture sialic acid and fucosylated glycoproteins from serum. The use of a series of different lectin columns whose binding specificities have been precisely elucidated enables the fractionation of oligosaccharides or glycoproteins into structurally distinct groups that give a general picture of structure distribution in a sample. Since the introduction of serial lectin affinity chromatography (SLAC) by Cummings and Kornfeld,11 SLAC has been widely used for the fractionation of complex N-linked glycan mixtures derived from various glycoproteins. SLAC fractionation and characterization has been performed on N-glycan structures derived from (4) Paulson, J. C. Trends Biochem. Sci. 1989, 14, 272-276. (5) Gorog, P.; Pearson, J. D. J. Pathol. 1985, 146, 205-212. (6) Schrevel, J.; Gros, D.; Monsigny, M. Prog. Histochem. Cytochem. 1981 14, 1-269. (7) Lis, H.; Sharon, N. Annu. Rev. Biochem. 1986, 55, 35-67. (8) Satish, P. R.; Surolia, A. J. Biochem. Biophys. Methods 2001, 49, 625-640. (9) Xiong, L.; Andrews, D.; Regnier, F. J. Proteome Res. 2003, 2, 618-625. (10) Yang, Z.; Hancock, W. J. Chromatogr., A 2005, 1070, 57-64. (11) Cummings, R. D.; Kornfeld, S. J. Biol. Chem. 1982, 257, 11235-11240.
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human immunoglobulin G,12 natural and recombinant blood coagulation factor VIII,13 and human serum transferrin14 and in cancer studies. Altered N-linked glycan structures have been detected in prostate-specific antigen from human prostate carcinoma15 and N-acetyl β-D-hexosaminidase in human renal oncogenesis16 using SLAC. This study describes a simple and rapid method based on serial lectin affinity chromatography for fractionation and comparison of glycan site heterogeneity on glycoproteins derived from human serum. The objective of this work was to develop a proteomescale method that would recognize and quantify differences or changes in the degree of branching between sialic acid-bearing glycan isoforms from specific glycosylation sites on proteins. The resulting method (1) recognizes and selects sialylated glycopeptides, (2) fractionates these sialylated glycopeptides based on their degree of branching, and (3) quantifies differences in the amount of glycoforms. MATERIALS AND METHODS Materials. Human serum, tris(hydroxymethyl)aminomethane (Tris base), tris(hydroxymethyl)aminomethane hydrochloride (Tris acid), l-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin, iodoacetic acid, cysteine, dithiothreitol (DTT), nitrosyl-L-lysine chloromethyl ketone, manganese chloride, calcium chloride, N-hydroxysuccinimide, and lactose were purchased from Sigma (St. Louis, MO). The d0 and d6 acetic anhydride were purchased from Aldrich (Milwaukee, WI). PeptideN-glycosidase F (PNGase F) and methyl R-D-mannopyranoside (R-MM) were obtained from Calbiochem (San Diego, CA). HPLCgrade acetonitrile (ACN) and hexane were purchased from Mallinckrodt Baker (Phillipsburg, NJ). Con A agarose was purchased from Amersham Biosciences, (Piscataway, NJ), and Sambucus nigra (SNA) agarose was purchased from Vector Laboratories (Burlingame, CA). Proteolysis. A 1-mL aliquot of human serum was reduced in 0.1 M phosphate buffer (pH 8.0) with 8 M urea and 10 mM DTT. After incubation at 37 °C for 2 h, iodoacetic acid was added to a concentration of 20 mM and the resultant mixture incubated in darkness on ice for additional 2 h. The alkylation reaction was quenched for 30 min at room temperature by the addition of cysteine to a final concentration of 20 mM. After diluting the sample with 0.1 M phosphate buffer to a final concentration of 2 M urea, TPCK-treated trypsin was added to the sample at a 50:1 protein-to-trypsin mass ratio. The sample was incubated at 37 °C for 24 h. Proteolysis was terminated by adding tosyllysine chloromethyl ketone protease inhibitor at a molar concentration exceeding that of trypsin by 2-fold. The sample was then frozen in liquid nitrogen for 10 min. Synthesis of N-Acetoxy-d3-succinimide. A solution of 4.0 g (34.8 mmol) of N-hydroxysuccinimide in 11.4 g (105 mmol) of acetic-d6-anhydride was stirred at room temperature. White (12) Harada, H.; Kamei, M.; Tokumoto, Y.; Yui, S.; Koyama, F.; Kochibe, N.; Endo, T.; Kobata, A. Anal. Biochem. 1987, 164, 374-381. (13) Hironaka, T.; Furukawa, K.; Esmon, P. C.; Fournel, M. A.; Sawada, S.; Kato, M.; Minaga, T.; Kobata, A. J. Biol. Chem. 1992, 267, 8012-8020. (14) Fu, D. T.; van Halbeek, H. Anal. Biochem. 1992, 206, 53-63. (15) Sumin, S.; Arai, K.; Kitahara, S.; Yoshida, K. J. Chromatogr., B 1999, 727, 9-14. (16) Yoshida, K.; Moriguchi, H.; Sumi, S.; Horimi, H.; Kitahara, S.; Umeda, H.; Ueda, Y. J. Chromatogr., B 1999, 723, 75-80.
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Figure 1. Analytical strategy for fractionation and comparison of differentially branched complex-type glycoforms that contain terminal sialic acid residues.
crystals began to deposit in 10 min. After 15 h, the reaction mixture was filtered. The crystals were washed with hexane and then dried under vacuum. The product yield of N-acetoxy-d3succinimide was 5.34 g (100%), mp 133-134 °C. Acetylation of the Peptides. A 5-fold molar excess of N-acetoxysuccinamide and N-acetoxy-d3-succinimide were added individually to two equal aliquots of tryptic digests. The reaction was allowed to proceed for 4-5 h at room temperature. NHydroxylamine was then added to the mixture in excess to the amount of labeling reagents, and the pH was adjusted to 11-12. Incubation with hydroxylamine was allowed to proceed for 30 min. The function of the hydroxylamine reaction was to hydrolyze esters that might have been formed during the acetylation reaction. The labeled samples were adjusted to pH 7-8 before affinity selection. Chromatography. All chromatographic separations were performed using a Biocad 60 workstation from PerSeptive Biosystems (Framingham, MA). (a) SNA and Con A Serial Affinity Selection. SNA agarose beads (Vector Laboratory) were packed into a 50 × 10 mm i.d. peek column. A 1-mL sample was applied to the SNA column after it had been equilibrated with 10 mM PBS. Unbound peptides were washed away with PBS, and the bound glycopeptides were eluted from the column with 0.2 M lactose in 10 mM PBS. Con A agarose beads (Amersham Biosciences) were packed into a 50 × 10 mm i.d. PEEK column. The eluent from the SNA column was applied to a Con A column after it had been equilibrated with 0.2 M Tris (pH 7.5) loading buffer containing 0.1 M NaCl, 1 mM CaCl2, and 1 mM MnCl2. After the unbound peptides were washed away, 10 mM R-MM in loading buffer was used to elute the glycopeptides.
Figure 2. Serial lectin affinity chromatogram of tryptic digest of human serum. A tryptic digest of human serum was applied to an SNA and Con A serial column set and used according to the protocol shown in Figure 1. Fraction I is the flow-through fraction from the Con A column while fraction II was eluted with 10 mM R-MM as indicated by the arrow.
Figure 3. Reversed-phase chromatograms of glycopeptides from human serum. The top trace is from the flow-through fraction from Con A column (fraction I in Figure 2). The bottom trace is from the fraction eluted with 10 mM R-MM from Con A column (fraction II in Figure 2).
(b) HPLC Fractionation. Fractionation of peptides on a 250 × 4.6 mm i.d. Vydac C18 column was achieved with a 90-min gradient from 100% buffer A (5% ACN, 95% H2O containing 0.1% TFA) to 60% buffer B (95% ACN, 5% H2O containing 0.1% TFA) at a flow rate of 1 mL/min. Eluted peptides were monitored at 215 nm, and fractions were manually collected for ESI-MS analysis.
Deglycosylation by PNGase F. Glycopeptides were vacuumdried and redissolved in 100 µL of 100 mM sodium phosphate buffer (pH 7.5). The reaction mixture was incubated with 2 µL (10 units) of PNGase F for 24 h at 37 °C. ESI-MS Analysis. Electrospray mass spectra were obtained on a QSTAR quadrapole time-of-flight mass spectrometer (Applied Analytical Chemistry, Vol. 77, No. 22, November 15, 2005
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Figure 4. Reversed-phase chromatogram of deglycosylated peptides from human serum analyzed according to the protocol in Figure 1.
Figure 5. ESI tandem mass spectrum of the peptide EHEGAIYPDNTTDFQR from ferroxidase precursor. This peptide had a [M + 2H]2+ precursor ion at m/z 969.92. The y and b ion series are indicated.
Biosystems, Framingham, MA) equipped with an API ion source. The instrument was operated in the positive ion TOF mode (m/z 300-2000). Samples were dissolved in 100 µL of MeOH/H2O (50/50, v/v) containing 1% (v/v) formic acid and infused at 8 µL/min. The ion spray voltage used was set at 5500 V. Tandem Mass Spectrometry. Tandem MS analysis was performed by transmitting the appropriate parent ion from the quadrapole to the collision cell. Collision gas used was nitrogen at a pressure of (4-6) × 10-6 Torr. Peptides were fragmented with appropriate collision energy (25-60 eV). Proteins were identified by searching the MS/MS spectra against the NCBI 7228 Analytical Chemistry, Vol. 77, No. 22, November 15, 2005
database (www.ncbi.nlm.nih.gov) using the Mascot search engine (Matrix Science, London, U.K.). RESULTS AND DISCUSSION Analytical Strategy. The analytical strategy used in this study is shown in Figure 1. Human serum samples were digested with trypsin and the digests applied to an SNA affinity column to select sialic acid-containing glycopeptides. SNA is a lectin that recognizes terminal R-2,6 linked sialic acid residues. In contrast, Con A can bind complex biantennary N-linked, hybrid-type, and high-mannose-type glycans. After the unbound peptides passed through
Table 1. Peptides Identified from Human Serum Together with the Relative Amount of Differentially Branched Glycoforms of These Peptides SWISS-PROT accession no.
protein name
P01009 P01860 P02787 P19827 P08603
R-1-antitrypsin Ig γ-3 heavy chain disease proteins transferrin inter-R (globulin) inhibitor complement H factor
P02790
hemopexin precursor
P04004
vitronectin
P00450
ferroxidase precursor
P00737
haptoglobin-1 precursor
P04114 P01591 P02748 P01019 P02679 P02765
apolipoprotein B-100 Ig J chain complement 9 angiotensinogen fibrinogen γ-A chain precursor R-2-HS glycoprotein
Q96PD5
N-acetylmuramoyl-L-alanine amidase precursor histidine-rich glycoprotein precursor Ig R-2 chain C region
P04196 P01877
P36955 P02749
EPC-1 gene product β-2-glycoprotein I precursor apolipoprotein H)
P01023
R-2-macroglobulin precursor
P03952 P05546 P25311 Q14624 P08185 P04217 P08709 P10909 P01871 P19652
plasma kallikrein B1 precursor leuserpin 2 (hLS2) precursor Zn-R-2-glycoprotein PK-120 precursor corticosteroid binding globulin precursor R-1-B-glycoprotein coagulation factor VII precursor apolipoprotein J Ig mu chain precursor, membrane-bound (clone 201) orosomucoid 2
P01011
R-1-antichymotrypsin precursor
P02763 P29622
orosomucoid 1 kallistatin precursor
sequence of identified peptidea
isotope ratio (d0/d3)
YLGNATAIFFLPDEGK EQQFNSTFR CGLVPVLAENYNK ANLSSQALR IPCSQPPQIEHGTINSSR MDGASNVTCINSR ALPQPQNVTSLLGCTH SWPAVGNCSSALR NGSLFALR NISDGFDGIPDNVDAALALPAHSYSGR EHEGAIYPDNTTDFQR ENLTAPGSDSAVFFEQGTTR ELHHLQEQNVSNAFLDK MVSHHNLTTGATLINEQWLLTTAK VVLHPNYSQVDIGLIK NLFLNHSENATAK FNSSYLQGTNQITGR ENISDPTSLR AVNITSENLIDDVVSLIR HLVIHNESTCEQLAK VDKCLQSLEDILHQVENK AALAAFNAQNNGSNFQLEEISR KVCQDCPLLAPLNDTR NAQNNGSNFQLEEISR GFGVAIVGNYTAALPTEAALR
0.04 0.05 0.07 0.09 0.10 0.26 0.11 0.26 0.12 0.64 0.15 0.53 1.1 0.16 0.26 0.53 0.16 0.16 0.17 0.18 0.19 0.20 0.30 0.44 0.20
IADAHLDRVENTTVY LSLHRPALEDLLLGSEANLTCTLTGLR HYTNSSQDVTVPCR TPLTANITK SVTWSESGQNVTAR VTQNLTLIEESLTSEFIHDIDR VYKPSAGNNSLYR
0.26 0.29 0.69 0.43 0.56 0.31 0.34
LGNWSAMPSCK GCVLLSYLNETVTVSASLESVR VSNQTLSLFFTVLQDVPVR IYSGILNLSDITK NLSMPLLPADFHK DIVEYYNDSNGSHVLQGR LPTQNITFQTESSVAEQEAEFQSPK AQLLQGLGFNLTER AIFYETQPSLWAESESLLKPLANVTLTCQAR YPHKPEINSTTHPGADLQENFCR LANLTQGEDQYYLR YKNNSDISSTR
S_Hc 0.44 0.50 0.45 0.48 0.67 0.94 0.94 S_Hc S_Hc 1.1 1.2
SVQEIQATFFYFTPNKTEDTIFLR CANLVPVPITNATLDR GLKFNLTETSEAEIHQSFQHLLR FNLTETSEAEIHQSFQHLLR CANLVPVPITNATLDQITGK SQILEGLGFNLTELSESDVHR
3.2 S_Ld 7.5 S_Ld 8.2 S_Ld
a The consensus motif for N-linked glycosylation is highlighted and in italic style. b The relative standard deviation of this method has been established to be 4%.21 c S_H represents heavily labeled singlet. d S_L represents lightly labeled singlet.
the column, sialylated glycopeptides were eluted from the SNA column with 0.2 M lactose in 10 mM PBS. The selected glycopeptides were then applied to a Con A column. It has been shown that glycopeptides with tri- and tetraantennary complex-type oligosaccharides pass through Con A sepharose, whereas glycopeptides with biantennary complex-type glycans can be eluted from the Con A column by 10 mM R-MM.17-19 The flow-through (17) Krusius, T.; Finne, J.; Rauvala, H. FEBS Lett. 1976, 71, 117-120. (18) Ogata, S.; Muramatsu, T.; Kobata, A. J. Biochem. (Tokyo) 1975, 78, 687696.
fraction (fraction I) was collected and labeled with the light (d0) form of N-acetoxysuccinimide. Ten millimolar R-MM at pH 7.5 was used to elute the glycopeptides bearing complex biantennary N-linked glycans from the Con A column (fraction II). This fraction was labeled with the heavy (d3) form of N-acetoxysuccinimide (NAS). This reagent brings about acetylation of primary amines in peptides through nucleophilic displacement of acetate from NAS by amines in peptides. Fractions I and II were then mixed and (19) Kornfeld, K.; Reitman, M. L.; Kornfeld, R. J. Biol. Chem. 1981, 256, 66336640.
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Figure 6. Mass spectra of peptide isotopomers from ferroxidase precursor that carried N-linked glycans. The do/d3 isotope ratio is 0.15 in (a) and 1.10 in (b). (a) The mass spectrum of the isotopomers of peptide EHEGAIYPDNTTDFQR in which the first three monoisotopic ions of nondeuterated peptide are seen at m/z 968.40, 968.91, and 969.41, and the first three monoisotopic ions of the deuterated peptide are seen at m/z 969.92, 970.42, and 970.92. (b) The mass spectrum of the isotopomers of peptide ELHHLQEQNVSNAFLDK. The first three monoisotopic ions of the nondetuerated peptide are seen at m/z 1054.00, 1054.50, and 1055.00 and the first three monoisotopic ions of the deuterated peptide are seen at m/z 1057.01, 1057.52, and 1058.52.
deglycosylated with the enzyme PNGase F. After deglycosylation, the peptides were fractionated by reversed-phase chromatography (RPC) and the isotope ratios were determined from ESI-MS spectra. The parent proteins were identified by peptide sequencing and database searches. Reversed-Phase Chromatography of Glycopeptides. Figure 2 is the affinity chromatogram of tryptic-digested human serum after serial lectin affinity selection. Fraction I is the flow-through fraction from the Con A column, containing all unbound components. Glycopeptides bearing tri- and tetraantennary complex-type glycans are in this fraction. Fraction II was eluted with 10 mM R-MM. This fraction contains glycopeptides with biantennary complex-type glycans. Fractions I and II were collected separately and further fractionated on a C18 RPC column (Figure 3). The chromatographic profiles in Figure 3 show qualitatively that, in many cases, the absorbance at 215 nm for the bottom 7230 Analytical Chemistry, Vol. 77, No. 22, November 15, 2005
trace is higher than that for the top trace at the same elution time. This indicates that most glycopeptides in human serum are biantennary sialylated glycoforms and that their concentration is higher than the sialylated tri- and tetraantennary glycoforms. This is in agreement with a previous report that sialylated biantennary N-linked oligosaccharides are typical of many serum glycoproteins.20 A more quantitative comparison of the concentration of different glycoforms was carried out in the following section. Protein Identification and Quantitative Comparisons of Sialylated Complex-Type Glycoforms. Human serum was examined using the serial lectin affinity protocol for quantitative analysis according to the scheme shown in Figure 1. A reversedphase chromatogram of the deglycosylated peptides derived from (20) Kornfeld, R.; Kornfeld, S. Annu. Rev. Biochem. 1985, 54, 631-664.
this protocol is seen in Figure 4. Fractions from the RPC columns were collected, and peptides were identified by ESI-MS. The isotope ratio of peptide isoforms in mass spectra was determined by comparing the total area of the first three monoisotopic peaks from the nondeuterated and deuterated forms of the peptide. This allowed the relative concentration of tri- and tetraantennary glycoforms of a peptide to be compared to biantennary glycoforms. The typical mass difference in peptide isotopic pairs is 3 or 6 amu, depending on whether the amino acid at the C-terminus of the peptide is arginine or lysine. Based on previous studies with SLAC columns, the relative standard deviation of this method is 4%.21 Peptide identification was achieved by tandem mass spectrometry. The parent ion of peptides was selected in the quadrapole section on a tandem quadrapole/time-of-flight mass spectrometer and subjected to collision-induced dissociation (CID) prior to analysis in the time-of-flight section of the instrument. The resulting CID spectra were searched against the human National Center for Biotechnology Information (NCBI) sequence database using Mascot. In some cases, the glycoproteins were identified by more than one peptide because of miscleavages by trypsin during digestion. Table 1 lists the identified peptides, the proteins from which they originated, and the observed d0/d3 isotope ratio for each identified peptide. All the peptides identified contained the conserved N-linked glycosylation motif (Asn-X-Ser/Thr), indicating that N-glycosylated peptides were isolated with high selectivity. It can be seen from Table 1 that the isotope ratio for most peptides was less than 1. This indicates that, for most glycopeptides obtained from human serum, the concentration of sialylated complex biantennary N-linked glycoforms is higher than that of tri- and tetraantennary glycoforms. However, the isotope ratios of peptides from three proteins, R-1-antichymotrypsin precursor, orosomucoid 1, and orosomucoid 2 were found to be higher than 3 (Table 1). This indicates that these glycopeptides contain larger amounts of sialylated tri- and tetraantennary N-linked glycans than biantennary-type glycans. A typical tandem mass spectrum is seen in Figure 5 where the precursor ion is at m/z 969.92. When subjected to a database search, the series of “b” and “y” ions in the spectrum lead to the conclusion that this peptide has the sequence EHEGAIYPDNTTDFQR and was derived from ferroxidase. The interesting thing about ferroxidase is that it has a second glycosylation site which exhibited a very different degree of branching. Whereas the isotope ratio for the peptide EHEGAIYPDNTTDFQR was 0.15, the isotope ratio for the peptide ELHHLQEQNVSNAFLDK at the (21) Qiu, R.; Regnier, F. E. Anal. Chem. 2005, 77, 2802-2809. (22) Turner, G. A. Clin. Chim. Acta 1992, 208, 149-171. (23) Seberger, P. J.; Chaney, W. G’. Glycobiology 1999, 9, 235-241.
second site was 1.10 (Figure 6). The same phenomenon was seen in other proteins from which more than one glycopeptide was identified (Table 1). This result suggests that the branching of the glycans at different sites in the same protein is independently regulated, and the method developed in this study can be used to detect the different degrees of branching. Several peptides appeared as a single cluster of monoisotopic peaks instead of the doublet cluster separated by 3 or 6 amu. In some cases, peptides were identified as being exclusively labeled with the heavy isotope form of the coding agent. The peptide LGNWSAMPSCK from β-2-glycoprotein I precursor, peptide AIFYETQPSLWAESESLLKPLANVTLTCQAR from R-1-B-glycoprotein, and peptide YPHKPEINSTTHPGADLQENFCR from coagulation factor VII precursor (Table 1) were of this type. This means the glycans attached to these peptides are exclusively biantennary and the peptides are either not conjugated with either tri- or tetraantennary-type complex glycans or the amount of these glycoforms is below the detectable range of the method. Greater than 50-fold differences are difficult to detect with this method. On the other hand, peptides identified as lightly labeled singlets carry very low or no biantennary-type glycans. The peptide CANLVPVPITNATLDR from orosomucoid 2, peptide FNLTETSEAEIHQSFQHLLR from R-1-antichymotrypsin precursor, and peptide SQILEGLGFNLTELSESDVHR from kallistatin precursor fall in this class (Table 1). CONCLUSIONS It may be concluded that serial lectin affinity chromatography combined with the stable isotope coding strategy for quantification along with reversed-phase chromatography and mass spectrometry provides a global, high-throughput method for the comparative analysis of differential branching among complex-type Nlinked glycans. Moreover, these methods have the potential to identify aberrations in glycosylation in a wide variety of glycopathologies where the degree of branching in glycan structures changes. Among these are those in which branching and sialylation increase22 as in the case of cancer where N-linked β-1,6 branching and sialylation increases in parallel with metastatic potential.6,23 Conversely, some acute infections or acute phases of chronic diseases have the opposite effect. These should be detectable as well. ACKNOWLEDGMENT The authors gratefully acknowledge financial support from NIH Grant GM-59996. Received for review April 1, 2005. Accepted August 8, 2005. AC050554Q
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