2 Department of Medical Biochemistry, Göteborg University, 413 90 Gothenburg, Sweden, and 3 Cancer Research UK Breast Cancer Biology, Guy's Hospital, London SE1 9RT, United Kingdom
Received on June 9, 2004; revised on September 21, 2004; accepted on September 22, 2004
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Abstract |
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Key words: Edman sequencing / glycosyltransferase / mass spectrometry / mucin / O-glycosylation
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Introduction |
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The O-glycan biosynthesis is initiated by the transfer of GalNAc to Ser or Thr of the protein backbone, catalyzed by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). Multiple members of this protein family have been identified with different kinetic properties and different expression patterns both temporarily and spatially (Nehrke et al., 1998). Few studies have addressed how GalNAc-O-glycosylation proceeds in vivo and if indeed the large amount of data from in vitro studies applies in vivo. One complication with interpretation of in vivo data is that the repertoires of GalNAc-Ts in cells are unknown, as are the full capacities for O-glycosylation these have. Nehrke et al. (1998)
originally addressed this using a minigene expression construct containing a peptide substrate derived from the HIV gp120 protein. This peptide contains one single Thr, which is glycosylated in vitro only by GalNAc-T3 (Bennett et al., 1996
). Using this system it was possible to fully recapitulate in vivo the GalNAc-T3 function found in vitro (Nehrke et al., 1998
). Mutations in the gene encoding GalNAc-T3 has recently been found to cause familial tumoral calcinosis, a severe autosomal recessive metabolic disorder (Topaz et al., 2004
).
MUC1, the first mucin to be cloned, is a transmembrane mucin expressed on the apical surface of most epithelial cells, including the mammary glands, and also on hematopoietic cells and in bone marrow (Gendler, 2001). The MUC1 protein consists of a very large extracellular domain (10002200 amino acids), a transmembrane domain, and a phosphorylated cytoplasmic tail involved in intracellular signaling. The extracellular domain is dominated by a 20-amino-acid tandem repeat (TR) sequence, repeated 20120 times. This variable number of TRs (VNTR) is an inherited polymorphism. The VNTR domain carries most of the carbohydrates of the glycoprotein, with each TR containing five possible O-glycan attachment sites. MUC1 is highly overexpressed in many forms of cancer, in particular breast carcinomas (Girling et al., 1989
) when the apical polarization is lost, resulting in MUC1 being expressed over the entire cell surface (Gendler, 2001
). Aberrant O-linked glycosylation of MUC1 is also observed in carcinomas, both at the level of the composition of the glycans and in the degree of site occupancy. In lactating milk and hence presumably in normal breast tissue, the most common oligosaccharides are based on the core 2 structure GlcNAcß1-6(Galß1-3)GalNAc-polypeptide (Amano et al., 1991
; Hanisch et al., 1989
) and terminated by Lewis type epitopes (Hanisch et al., 1990
). Sialylated Lewis-type epitopes are also expressed in colon carcinomas (Baeckstrom et al., 1991
). Studies of MUC1 expressed in the human breast cancer cell line T47D show that the glycans are shorter, are core 1based (Galß1-3GalNAc-polypeptide), and can be highly sialylated (Hanisch et al., 1996
; Hull et al., 1989
; Lloyd et al., 1996
). However, some breast cancer cell lines (MCF7, MDA 231, and ZR75-1) can express core 2 O-glycans (Bäckström et al., 2003
; Muller and Hanisch, 2002
). In accordance, studies on primary breast carcinomas have shown that the glycosyltransferase responsible for adding sialic acid to core 1 glycans is elevated in breast carcinomas, whereas no consistent change was seen for C2GnT1 (Burchell et al., 1999
).
To glycosylate all five acceptor sites of the MUC1 TR (PAPGSTAPPAHGVTSAPDTR; sites underscored, numbering order) in vitro, the combined and sequential action of several GalNAc-Ts are needed. Ser5 and Thr6 (in GSTA) and Thr14 (in VTSA) are readily glycosylated by GalNAc-T1, -T2, or -T3 (Wandall et al., 1997), but only one transferase identified this far, GalNAc-T4, has the ability to use Ser15 (in VTSA) and Thr19 (in PDTR) and complete the glycosylation (Bennett et al., 1998
). However, GalNAc-T4 is not active on naked MUC1 TR peptides in vitro but requires prior glycosylation with GalNAc residues at other sites. In vitro studies showed that this specificity for GalNAc-MUC1 of GalNAc-T4 was directed by its lectin domain (Hassan et al., 2000
).
To be able to study the site occupancy of the MUC1 TR, we constructed a shorter recombinant reporter protein, MUC1(1.7TR)-IgG2a, designed for studies of O-glycosylation in vivo. The recombinant protein, containing 1.7 TR of MUC1, fused to a murine IgG2a Fc chain, was produced in Chinese hamster ovary (CHO) K1 cells, where it achieved an O-glycosylation profile similar to that found on breast cancer MUC1. The system was also used to investigate the effect of GalNAc-T4 in vivo by coexpressing the transferase with MUC1(1.7TR)-IgG2a.
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Results |
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Purified and enterokinase-digested MUC1(1.7TR)-IgG2a from wild-type CHO-K1 was subjected to Edman degradation. It was found that a majority of the amino acids at all five potential O-glycosylation sites were occupied, in addition to two non-TR Ser before the MUC1 sequence (Table III and Figure 6, black bars). The Thr6 (in GSTA) and Thr14 (in VTSA) motives were almost fully glycosylated (more than 95%), whereas the Ser5 and Ser15 of these motives showed lower occupancy (88%). However, Ser5 (in GSTA) was more occupied in the second TR (96%). This deviation could possibly be due to that this amino acid in the second repeat is more embedded in its normally surrounding amino acids and that this could have an impact on the glycosylation event. Least occupied was the Thr19 of the PDTR motif with a substitution of 75%. The glycosylation of Thr19 (in PDTR) and Ser15 (in VTSA) indicated the presence of an endogenous GalNAc-T4 (or a GalNAc-T with a similar activity) in CHO-K1 cells.
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Mutant GalNAc-T4D459H did not result in the increase in site occupancy found with wild-type GalNAc-T4. However, the effect of the mutated GalNAc-T4D459H was more complex (Table III and Figure 6, dark gray bars). The residues fully occupied in wild-type CHO were not affected. At the three remaining sites, two different effects were observed. At Ser5 (in GSTA) and Thr19 (in PDTR), the occupancy was similar to that on MUC1 TR from wild-type CHO, suggesting that the mutation inactivated the effect of the human GalNAc-T4 on these sites, in line with previous in vitro studies. At Ser15 (in VTSA), a dominant negative effect was observed, with a decreased occupancy from almost 90% in wild-type CHO to around 50%. The reason for this phenomenon is not known, but there seems to be a direct relation to the level of transferase expression as the clone with the higher expression of GalNAc-T4D459H, clone #15, had a lower occupancy of this Ser compared to clone #13 (Tables I and III).
The oligosaccharides expressed on MUC1(1.7TR)-IgG2a from the GalNAc-T4 clones were released and analyzed by LC-MS (Figure 3BE). The same oligosaccharides were produced in these cell lines as in wild-type CHO-K1, but the relative amounts of the components differed (Table II). The two major components in wild-type CHO-K1, NeuAc2-3Galß1-3GalNAcol and NeuAc
2-3Galß1-3(NeuAc
2-6)GalNAcol, were the dominating in all GalNAc-T4-expressing clones as well. Trace amounts of an additional component with [M-H] at m/z 587 was detected among the O-glycans from GalNAc-T4 expressing clone #3D9. The differences in the O-glycosylation profiles could not be correlated to the expression levels of GalNAc-T4 (Table I) or to the form of GalNAc-T4 expressed.
Validation of the results on the O-glycan occupancy
To validate the results from the Edman degradation, a second approach to determine the O-glycan site occupancy of the MUC1 TR was employed. The MUC1(1.7TR)-IgG2a was enzymatically deglycosylated, leaving only the innermost GalNAc intact and attached to the protein as a marker of substituted O-glycan sites. Then a 25-amino-acid-long glycopeptide, MUC1TR-XL containing the MUC1 TR plus the five adjacent amino acids on its N-terminal side, was cleaved out of MUC1(1.7TR)-IgG2a by sequential enterokinase and clostripain (an endo-ArgC protease) digestion. The five extra amino acids (GASSM) stem from the cassette used during the cDNA construction and have no biological significance in the context of MUC1. However, the two extra Ser residues function as O-glycan sites, giving the MUC1TR-XL peptide a total of seven possible O-glycan sites.
Nano LC-ESI-MS was then performed on the MUC1TR-XL glycopeptides to determine the number of GalNAcs attached. Using the theoretical masses of the glycopeptides of interest, we could specifically target these in the MS analysis. This showed that glycopeptides carrying four to seven GalNAcs were present in the samples produced by all the cell lines. The identities of the ions for the glycopeptides were confirmed by nano LC-ESI-MS/MS. An example of this is shown in Figure 7 by a collision-induced mass spectrum of MUC1TR-XL substituted with six GalNAcs, [M+3H]3+ at m/z 1181. The fragmentation was primarily observed only in peptide bonds at the amino acid Pro, making it difficult to accurately predict detailed site occupancy by MS. However, the average O-glycan site occupancy on the whole glycopeptide could be calculated by integrating the peak areas in the LC-MS mass chromatograms for the glycopeptides with different number of sites used (Table IV). The results were in agreement with the results from the Edman degradation, with the highest degree of glycan substitution in the CHO-K1/wild-type GalNAcT4 cell lines.
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Discussion |
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Studies on the effect and mechanism of the O-glycosylation machinery are complicated, due to both the complexity of the system in terms of the number and location of glycosyltransferases and difficulties in producing sufficient amounts of the glycoproteins. Most specificity studies of glycosyltransferases have therefore been performed in vitro, and this has produced valuable and extended insight into the mechanisms of O-glycosylation. To confirm and further study these results in vivo, a MUC1 TR O-glycosylation reporter protein was constructed allowing analysis by Edman sequencing. An IgG2a-Fc domain encoded by a genomic sequence was added to achieve maximum expression and secretion. The construct also included a myc-tag for detection, a His-tag for purification, and a carboxylic acidrich C-terminus to allow covalent attachment of the protein prior to Edman sequencing. A recombinant protein has been used before as a glycosylation reporter protein by Nehrke et al. (1996). Muller and Hanisch (2002)
have used a reporter plasmid containing six MUC1 TRs to analyze the glycans added but not the sites of glycosylation.
The O-glycosylation reporter was used to produce a recombinant protein including 34 amino acids corresponding to 1.7 TR of MUC1. The recombinant protein MUC1(1.7TR)-IgG2a was expressed in CHO-K1 cells, known to produce a rather simple O-glycosylation (Sasaki et al., 1987). The oligosaccharides were made up of four components, NeuAc
2-3Galß1-3GalNAcol, NeuAc
2-3Galß1-3 (NeuAc
2-6)GalNAcol, Galß1-3(NeuAc
2-6)GalNAcol, and Galß1-3GalNAcol, the two first being the most abundant. The glycan density of the TR domain was 4.5 glycans per TR, and all individual glycosylation sites were occupied to 7597%, seen over the reporter protein population. Least occupied was the Thr19 of the immunodominant PDTR motif, which was glycosylated in 75% of the molecules. GalNAc-T4 is the only GalNAc-T identified so far that can glycosylate the final two of the five sites (Ser15 in VTSA and Thr19 in PDTR) within the TR of MUC1, and so take the glycosylation to completion (Bennett et al., 1998
). The repertoire of GalNAc-Ts in CHO cells is unknown, but the results presented here suggest that a GalNAc-T4 ortholog or a new GalNAc-T with similar activity is expressed. A GalNAc-T4-like activity in CHO cells has indeed been implicated before, because Thr57 of PSGL-1 is glycosylated in these cells (Li et al., 1996
), a site known to be glycosylated by GalNAc-T4 (Bennett et al., 1998
). However, the endogenous GalNAc-T4 was not capable of fully glycosylating MUC1 TR.
One of the more challenging analytical problems is to determine the glycosylation sites and the specific glycans attached to each site in mucin domains. These domains are usually not degradable with proteases. This can be overcome by trimming the glycans down to GalNAc only, as shown for PSM and MUC1 (Gerken et al., 1997; Muller et al., 1997
), allowing MS determination of the total number of glycans attached to the generated glycopeptides. However, for analysis of specific glycosylation sites, Edman sequencing is at present the superior method, because it cleaves off the glycoamino acids with identical yields irrespective of glycans attached and allows better quantification. The importance of this is illustrated here, where an accurate determination of the level of occupancy revealed subtle site-specific alterations by the addition of different glycosyltransferases. The value of Edman sequencing for solving problems of this type has been shown by Gerken et al. (1997
, 1998
, 2002)
on PSM. When analyzing full mucins it is necessary to partially deglycosylate the protein to allow proteolytic cleavage, something that might generate miscleavages and biased results. This problem is circumvented with small reporter constructs, especially designed for Edman sequencing such as that described here. One could argue that these may not reflect the normal glycosylation of the full-length molecule. However, comparing the O-glycosylation of MUC1(1.7TR)-IgG2a to that of a recombinant protein containing 16 TR of MUC1 produced in CHO-K1 (Bäckström et al., 2003
), the dominating oligosaccharides were the same and the glycan densities similar. Thus the MUC1 TR seems to have the same glycosylation profile in CHO-K1 cells, irrespective of the number of TR on the protein.
Controlled manipulation of the glycosylation pattern of cell lines would also be valuable in studies of the glycosylation machinery of the secretory pathway and the importance of individual glycosyltransferases in vivo. Here we show that recombinant expression of GalNAc-T4 in vivo added to the activity of CHO-K1 endogenous GalNAc-Ts, which resulted in increased glycan occupancy at Ser5 (in GSTA), Ser15 (in VTSA), and Thr19 (in PDTR). These are the sites of human GalNAc-T4 specificity within the MUC1 TR, as predicted from in vitro studies (Bennett et al., 1998; Hanisch et al., 2001
). The present results thus supported the proposed function and specificity of GalNAc-T4 and that the in vivo effect is similar to that observed in vitro. It also demonstrated that it is possible to manipulate the glycosylation site occupancy by the introduction of new glycosyltransferases.
GalNAc-T4 contains a ricin-like lectin domain with three repeats named , ß, and
using the nomenclature of Hagen et al. (1999)
. Point-mutation from Asp to His in the
-repeat generates the GalNAc-T4D459H mutant, incapable of glycosylating MUC1 TR glycopeptides in vitro (Hassan et al., 2000
). When MUC1(1.7TR)-IgG2a was coexpressed with GalNAcT4D459H, the glycan occupancies of the TR glycosylation sites were as expected similar to those found in wild-type CHO-K1 cells, supporting that the mutation inactivates the GalNAc-T4. Surprisingly, one exception to the predicted effect was that Ser in VTSA was less glycosylated than in wild-type CHO-K1. This competition between the mutant GalNAcT4D459H and the CHO-K1 endogenous GalNAc-T glycosylating this particular Ser is currently not understood. This dominant negative effect suggests that the GalNAcT4D459H can bind the glycopeptide while still being unable to glycosylate this site or is related to the two other nonmutated lectin domain repeats (ß and
).
In conclusion, the O-glycosylation reporter protein, MUC1(1.7TR)-IgG2a, has been used to show that the sites for glycosylation can be modulated in vivo by overexpressing GalNAc-T4 but also that the in vivo situation can be more complex. This work opens for engineering cell lines for the manipulation of O-glycans to specific sites, further improving the possibility to produce recombinant proteins with specific glycosylation.
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Materials and methods |
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Cell culture and transfections
CHO-K1 cells (ATCC, Manassas, VA) were cultured in Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 10% fetal bovine serum, penicillin, and streptomycin. Geneticin (Invitrogen) was used at 250 mg/L and zeocin (Invitrogen) at 50 mg/L when appropriate. The plasmids encoding the human full-length GalNAc-T4neo and were kindly provided by Drs. Henrik Clausen and Eric Bennett, Copenhagen University (Hassan et al., 2000
). CHO-K1 cells stably expressing these transferases were generated by transfection of CHO-K1 cells. DNA was produced in XL-10 Gold bacteria (RecA, Stratagene) and prepared using the Megaprep Tip 2500 protocol from Qiagen (Hilden, Germany). The pSecTagBO18/MUC1(1.7TR)/IgG2a vector was transfected into wild-type and GalNAc-T4 CHO-K1 cells using DMRIE-C (Invitrogen). Stable clones were generated by adding selection pressure after 24 h. Cells were passaged after 8 days of confluency. Spent culture medium containing MUC1(1.7TR)-IgG2a was collected every 5 days, cleared by centrifugation (4500 x g, +4°C, 15 min) and after addition of 0.05% NaN3 stored at +4°C. Cell lysates were prepared as described previously (Axelsson et al., 1998
), in the presence of N-ethylmaleimide (Sigma, St. Louis, MO), phenylmethylsulfonyl fluoride (Calbiochem, San Diego, CA), aprotinin (Trasylol, Bayer, Leverkusen, Germany), and leupeptin (Sigma).
Purification and on-column enterokinase digestion of MUC1(1.7TR)-IgG2a
Spent culture medium containing MUC1(1.7TR)-IgG2a fusion protein was dialyzed against H2O. NaCl (0.3 M) was added and pH adjusted to 7.4 before application onto a 1 ml HiTrap chelating column (Amersham Bioscience, Uppsala, Sweden), loaded with Co2+ according to the manufacturer's instructions. The column was sequentially washed with start buffer (20 mM sodium phosphate, pH 7.4, 0.5 M NaCl), with 4 M guanidinium-HCl in start buffer and with 20 mM imidazole in start buffer, and then equilibrated with EKMax buffer (50 mM TrisHCl, pH 8.0, 1 mM CaCl2, 0.1% Tween-20). Enterokinase Max (Invitrogen, 5 U in 2 ml EKMax buffer) was applied, and the column was incubated at 37°C for 20 h. After washing the column with EKMax buffer and with 20 mM imidazole in start buffer, MUC1(1.7TR)-IgG2a was eluted with 100 mM imidazole in start buffer. The eluates were desalted on a PD-10 column (Amersham Bioscience), eluted in H2O, lyophilized, and dissolved in H2O.
The amount of MUC1(1.7TR)-IgG2a in different fractions was determined by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), as described (Baeckstrom et al., 1991). PolySorp ELISA plates (Nunc, Roskilde, Denmark) were coated with rabbit anti-mouse Ig antibodies (0.5 µg/well; Dako, Glostrup, Denmark), samples were applied, and bound MUC1(1.7TR)-IgG2a was detected with europium-conjugated goat anti-mouse IgG antiserum (Jackson Immunoresearch, West Grove, PA). Obtained reactivities were compared to those from a MUC1(1.7TR)-IgG2a standard sample with known concentration, determined from Edman degradation analysis data. Fractions were also analyzed with SDSPAGE and western blotting. Some MUC1(1.7TR)-IgG2a was eluted in the 4 M guanidinium-HCl and 20 mM imidazole wash fractions. These fractions were pooled, dialyzed against H2O, and repurified in the same procedure just described after addition of NaCl and adjustment of pH. Eluates from such repurifications were pooled with the eluate from the corresponding first round.
SDSPAGE and western blotting
Proteins were analyzed by SDSPAGE with 0.1% SDS using reducing conditions. Equal amounts of protein were loaded in all lanes, as determined by the BCA protein determination assay (Pierce, Rockford, IL). Proteins were either visualized with silver staining or Alcian blue (0.125% in 25% ethanol, 10% acetic acid; Sigma), or blotted to 0.45 µM nitrocellulose membranes (Bio-Rad, Hercules, CA). Western blotting was performed in a Trans-blot SD semi-dry transfer cell (Bio-Rad) at a current of 1.25 mA/cm2 for 50 min, with 20% MeOH, 25 mM Tris, 192 mM glycin as transfer buffer. IgG2a-tagged proteins were immunodetected with AP-conjugated goat anti-mouse immunoglubulins diluted 1:1000 (Dako). GalNAc-T4 was immunodetected with UH6 anti-GalNAc-T4 antibody (Bennett et al., 1998) diluted 1:10 followed by AP-conjugated goat anti-mouse Igs antibody diluted 1:1000.
Miniscale release of O-linked oligosaccharides
O-linked oligosaccharides were released from 100 pmol of MUC1(1.7TR)-IgG2a by miniaturized reductive ß-elimination (Schulz et al., 2002). In short, glycoproteins were incubated in 40100 µl 0.05 M KOH and 1 M NaBH4 at 45°C for 16 h, in 200-µl tubes with perforated lids to allow the H2 formed to evaporate. After incubation, the solution was neutralized with
0.05 vol glacial acetic acid, and desalted with 50 µl AG50Wx8 cation exchange resin (Bio-Rad) packed in a Zip-tip (Millipore, Bedford, MA). The desalted material was eluted in H2O and dried by vacuum centrifugation in a Hetovac (Heto-Holten, Allerød, Denmark). Borate was removed as its methyl ester by the addition of 50 µl 2% HAc in methanol and evaporation in the Hetovac, repeated five times. Finally, the samples were dissolved in 10 µl H2O.
Preparation of oligosaccharide standards
O-linked oligosaccharides were released by reductive ß-elimination (Carlstedt et al., 1993) from approximately 50 mg of recombinant MUC1(16TR)-IgG (Bäckström et al., 2003
) produced in CHO-K1 cells. Released and desalted oligosaccharides were separated on a Hypercarb 5-µm column (100 x 4.6 mm; Thermo Hypersil-Keystone, Bellefonte, PA) using the ÄKTA prime HPLC system (Amersham Biosciences) and water-acetonitrile gradients with 20 mM NH4HCO3 (040% acetonitrile in 40 min at 0.5 ml/min flow collecting 0.15-ml fractions to isolate Gal-GalNAcol and NeuAc-Gal-GalNAcol; 040% acetonitrile in 80 min at 0.5 ml/min flow collecting 0.4-ml fractions to isolate NeuAc-Gal-(NeuAc-)GalNAcol). Eluted oligosaccharides were detected by UV absorption (206 nm) and fractions corresponding to individual peaks in the chromatograms were pooled. Identity and purity of the oligosaccharides were controlled by nano ESI-MS, and their concentration determined by monosaccharide composition analysis (Karlsson and Hansson, 1995
). The mean of the Gal and the GalNAcol molar concentrations (mean of two analyses) was used to calculate the oligosaccharide concentration.
N-terminal sequencing and evaluation of site-specific glycan substitution
N-terminal sequencing of purified enterokinase-digested MUC1(1.7TR)-IgG2a by Edman degradation was performed on a 492 Procise Sequencer (Applied Biosystems, Foster City, CA). Samples were applied by adsorption of sample solution (0.33 pmol/µl) to TFA-resistant filters (Applied Biosystems). A pulsed-liquid standard procedure was used, with exception of cycles 6, 8, 13, 14, 22, 26, 28, 33, and 34, where TFA release was performed at 53°C to increase the yield of proline. The amount (in pmoles) of nonglycosylated amino acid resulting from each cycle was obtained from the lag-corrected values given by the machine's software. A hypothetical expected amount was calculated for each cycle corresponding to the amount that would result from a totally nonglycosylated amino acid population. This value was based on the repetitive yield and on the amount of sample applied at the start. The level of glycan substitution at each glycosylation site was determined from the loss of Ser or Thr signal, judged by the ratio of obtained signal to expected signal.
Preparation of MUC1TR-XL glycopeptides containing one TR of MUC1 and five adjacent amino acids
Three hundred picomoles of MUC1(1.7TR)-IgG2a in spent culture medium was bound to 100 µl anti-mouse Ig agarose beads (Sigma-Aldrich) and then proteolytically digested and deglycosylated using sequential incubations at 37°C with 2 U Enterokinase Max in 50 mM TrisHCl (pH 8.0) with 1 mM CaCl2 and 0.1% Tween-20 for 22 h; 4 mU Vibrio cholerae neuraminidase (Roche, Indianapolis, IN) in 0.05 M sodium acetate (pH 5.5) with 4 mM CaCl2 and 100 µg/ml bovine serum albumin for 22 h; and 4 mU ß-galactosidase (bovine testes, Sigma-Aldrich) in 0.1 M citrate phosphate buffer (pH 5.0) for 44 h with 4 mU fresh enzyme added after 22 h. The partially deglycosylated protein was released from the agarose beads using 0.1 M glycin-HCl, pH 2.4, 0.15 M NaCl, neutralized using 2.5 M TrisHCl pH 10, dialyzed against H2O in a Slide-A-Lyzer cassette (Pierce), lyophilized, and dissolved in 25 mM sodium phosphate (pH 7.6), 0.2 mM CaCl2. One unit Clostripain (Sigma-Aldrich), which had been preactivated with 2.5 mM dithiothreitol for 4 h at 2025°C, was added and allowed to digest the protein for 20 h at 37°C. The reaction mixture was then stored in 20°C until analyzed by nano LC-ESI-MS.
LC-MS
Desalted oligosaccharide alditols were subjected to nano LC-ESI-MS or LC-ESI-MS/MS on a 150 x 0.25 mm ID column packed with 5 µm Hypercarb particles (Thermo-Hypersil, Runcorn, UK). HPLC was performed using an HP1100 binary pump (Hewlett-Packard, Palo Alto, CA) delivering 200 µl/min, and the flow was splitted in a Valco Tee (VICI, Schenkon, Switzerland) using a 40 cm x 50 µm ID fused silica tubing as a restrictor, down to a flow rate of 5 µl/min through the column. A water-acetonitrile gradient (040% acetonitrile in 40 min) with 5 mM NH4HCO3 was used. The column eluent was transferred via a 50 µm ID fused silica tubing into a nano-LC interface (Micromass, Manchester, UK). ESI-MS and ESI-MS/MS were performed on a Q-TOF1 mass spectrometer (Micromass) in the negative ion mode. Electrospray voltage was 3.5 kV; sample cone voltage was 38 V; sample cone temperature was 100°C; compressed air was used as cone gas at a flow rate of 300 L/h and as nebulizer gas at 3 bar. In LC-MS, full scans were performed over the range m/z 2001500. The TOF spectrometer was calibrated in the negative ion mode using NaI. For LC-MS/MS, automatic function switching was used for colliding [M-H] ions at intensities higher than 10 counts/s. Argon was used as collision gas with a collision energy of 22 eV for m/z 300600, 30 eV for m/z 600900, 45 eV for m/z 9001100, and 60 eV for m/z 11002000. Full scans were performed over the range m/z 1001500.
MUC1TR-XL glycopeptides were analyzed by nano LC-ESI-MS in the positive ion mode. Glycopeptides, 0.4 µl of 20 µl, were injected using a Microinjection valve M-435 (Upchurch Scientific, Oak Harbor, WA) into a fused-silica 120 x 0.075 mm ID column, packed with Kromasil C18-bonded 3.5 µm porous particles (100 Å, Eka Chemicals, Bohus, Sweden). PEEK tubing 400 µm ID (Upchurch Scientific) was used to seal the fused-silica column in a 1.58-mm Valco through-bore union (Valco Instruments, Houston, TX) against a 2 µm steel screen (Valco). A tapered emitter tip made by fused-silica (150 µm OD, 20 µm ID, Polymicro Technologies, Phoenix, AZ) was connected to the union by an in-house interface using 150 µm ID PEEK tubing. The flow rate was splitted down to 250 nl/min. A gradient of 050% acetonitrile in 0.1% formic acid (1% acetonitrile/min) was used. The electrospray capillary voltage was +3.2 kV; sample cone voltage was +40 V; sample cone temperature was 80°C; nitrogen was used as cone gas at a flow rate of 250 L/h and as nebulizer gas at 2 bar. Full scans were performed over the range m/z 4001500. Mass chromatograms for [M+3H]3+ for the different MUC1 glycopeptides were generated and the peaks integrated using MassLynx 3.4. For LC-MS/MS, selected ions were collided using argon as collision gas with a collision energy of 35 eV for m/z 1045 and m/z 1113 and 40 eV for m/z 1181 and m/z 1248. Full scans were performed over the range m/z 1002000.
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Abbreviations |
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References |
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