Abstract

the terminal monosaccharide of cell surface glycoconjugates is typically a sialic acid (sa), and aberrant sialylation is involved in several diseases. several methodological approaches in sample preparation and subsequent analysis using mass spectrometry (Ms) have enabled the identification of glycosylation sites and the characterization of glycan structures. In this paper, we describe a protocol for the selective enrichment of sa-containing glycopeptides using a combination of titanium dioxide (tio 2 ) and hydrophilic interaction liquid chromatography (HIlIc). the selectivity of tio 2 toward sa-containing glycopeptides is achieved by using a low-pH buffer that contains a substituted acid such as glycolic acid to improve the binding efficiency and selectivity of sa-containing glycopeptides to the tio 2 resin. By combining tio 2 enrichment of sialylated glycopeptides with HIlIc separation of deglycosylated peptides, a more comprehensive analysis of formerly sialylated glycopeptides by Ms can be achieved. in enriching glycopeptides. The drawback of this strategy is the lack of specificity toward any specific subset of glycostructures, even though a zwitterionic type of HILIC has been shown to be effective in separating intact SA glycopeptides from a single glycoprotein The enriched sialylated glycopeptides can be analyzed directly to determine the glycosylation site and the glycan structure attached to it or they can be subjected to enzymatic deglycosylation to map the glycosylation sites in a large-scale manner. The determination of site occupancy and all possible glycan structures attached to the individual sites is still a challenging task, and currently this can only be performed on purified glycoproteins. We have successfully used the TiO 2 enrichment strategy to identify sialylated glycopeptides from several biological sources, including saliva and plasma The TiO 2 /HILIC strategy requires 2 d, including the enzymatic deglycosylation process, and the entire procedure from harvesting cells to compiling the data set can usually be performed in <5 d, depending on the number of fractions to be analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Experimental design In this protocol, we describe the analysis of N-linked sialylated glycoproteins from HeLa cells. The following points reflect our current setup and can be changed according to the user's needs: 1. The efficiency of the enrichment of sialylated glycopeptides should be tested and optimized using peptides originating from a tryptic digestion of bovine fetuin. The expected results are shown in reference 34. 2. The amount of starting material is not crucial. The protocol described below has been optimized for 100-500 µg of starting material (i.e., isolated protein). 3. As the TiO 2 chromatography strongly binds phosphorylated peptides, the protocol is performed on phosphatase-treated peptide mixtures to improve the recovery of sialylated glycopeptides; the sample has to be dephosphorylated to avoid copurification of phosphopeptides using TiO 2 resin. 4. The gradient for HILIC fractionation is optimized according to sample complexity. is dependent on the complexity of the sample, as well as on the MS instrument time available. A tryptic digest of fetuin can be used as a positive control sample to start practicing with the TiO 2 enrichment, and a mixture of peptides from standard proteins can be spiked into the sample to validate the specificity of the method for complex mixtures. As a negative control, RNaseB or a neuramidase (sialidase)-treated tryptic peptide sample from fetuin can be used to show that the method is selective for SA-specific glycopeptides. The protocol is illustrated here for the qualitative mapping of formerly sialylated glycosylation sites present on HeLa cell membranes. This method is also applicable to different quantitative strategies, e.g., isobaric tag for relative and absolute quantitation (iTRAQ), stable isotope labeling by amino acids in cell culture (SILAC) or isotope-coded affinity tags (ICATs). The present protocol can be used to explore the qualitative and quantitative content of sialylated glycosylation sites in cells, tissues or biofluids in order to find differences induced by treatment of cells with various reagents or to find differences induced by cell differentiation or development. Magnetic stirrer SpeedVac vacuum concentrator (Thermo Scientific) Vertical high-pressure column-packing bomb (Proxeon) Agilent 1200 HPLC (Agilent) LTQ Orbitrap XL mass spectrometer (Thermo Scientific) equipped with an EASY-nLC nanoHPLC (Proxeon) Reprosil-Pur C18-AQ (3 µm, Dr. Maisch GmbH) Polycarbonate centrifuge tube (Hitachi Koki, cat. no. S404332A) Proteome Discoverer software 1.2 (Thermo Scientific) Qubit Quantitation Platform fluorometer (Invitrogen) Analytical tools (e.g., MASCOT (Matrix Sciences), Proteome Discoverer 1.2 (Thermo Scientific) or equivalent) REAGENT SETUP HeLa cells HeLa cells (human cervix epithelial adenocarcinoma cells) were grown in DMEM; ~10 7 cells were used as starting material per sample. Lysis buffer Lysis buffer is composed of Na 2 CO 3 (100 mM) and protease inhibitor cocktail. (pH 11).  crItIcal Prepare 50 ml of Na 2 CO 3 solution and store it at -20 °C for 2-3 months. Add the protease inhibitors immediately before use. Denaturing buffer Denaturing buffer is composed of urea (6 M), 2 M thiourea and 50 mM NH 4 HCO 3 (pH 7.9). Prepare 50 ml of denaturing buffer solution and store it at -20 °C for 2-3 months. TiO 2 buffers Loading buffer: 1 M glycolic acid in 80% (vol/vol) acetonitrile, 5% (vol/vol) TFA. ! cautIon TFA is highly corrosive acid. Handle in a fume hood while wearing gloves.  crItIcal The concentration of glycolic acid and TFA is critical to obtain high selectivity in the enrichment. Washing buffer 1: 80% (vol/vol) acetonitrile, 1% (vol/vol) TFA. Washing buffer 2: 20% (vol/vol) acetonitrile, 0.1% (vol/vol) TFA.  crItIcal Prepare these solutions before using them to avoid changes in the buffer composition. Elution buffer: ammonia solution 25% NH 4 OH in ultrapure water (pH 11.3). EQUIPMENT SETUP Agilent 1200 HILIC microHPLC The Agilent 1200 HPLC system is equipped with an autosampler, a UV detector with an 80-nl flow cell detecting at 210 nm, a fraction collector, a 40-µl loading loop and a standard six-inlet switch. All capillaries have 50-µm inner diameter and the instrument is operated in micromode with a maximum flow sensor of 20 µl min − 1 . See below for the fritless microcolumn packing protocol and details. See Microcolumn solvents RPLC-MS/MS equipment The EASY-nLC nanoHPLC connected to the LTQ Orbitrap XL mass spectrometer is equipped with a 100-µm inner diameter × 170 mm Kasil/formamide fritted nanocolumn packed with Reprosil-Pur C18-AQ (3 µm). The nanoHPLC gradient was 0-35% solvent B in solvent A (see REAGENT SETUP for solvent composition) for 55 min at a flow rate of 250 nl min − 1 . An MS scan (350-1,800 m/z) was recorded in the Orbitrap mass analyzer set at a resolution of 60,000 at 400 m/z, 1 × 10 6 automatic gain control target and 500-ms maximum ion injection time. The MS is followed by data-dependent collision-induced dissociation MS/MS scans on the five most intense multiply charged ions in the LTQ at 15,000 signal threshold, 20,000 automatic gain control target, 300-ms maximum ion injection time, 2.5 m/z isolation width, 30-ms activation time at 35 normalized collision energy and dynamic exclusion enabled for 30 s with a repeat count of 1. To ensure optimal instrument performance, we inject a 50-fmol tryptic digest of bovine serum albumin using a rapid gradient. 2| Prepare 500 µl of TSKgel Amide-80 HILIC slurry in a glass vial with a magnetic stirrer and bath and sonicate for 5 min. Place the vial into a vertical high-pressure column-packing bomb and insert the capillary through the vespel ferrule and into the vial. 3| Connect the vertical high-pressure column-packing bomb to compressed nitrogen and set the pressure to 100 bar. Allow the column material to pack against the Inline MicroFilter overnight or until the column is packed and dried.  crItIcal step It is important to pack the column until it is dry to prevent back-flow of the stationary phase once the pressure is released. 4| Turn off pressure and remove the column from the vertical high-pressure column-packing bomb. Membrane proteins extraction from mammalian cultured cells • tIMInG ~1.5 h 5| Wash cell pellets twice with ice-cold PBS (10 7 cells per condition) and spin down at 300g in centrifuge (MEGAFUGE 1.0R or similar model).  crItIcal step Steps 5-9 must be carried out at 4 °C. 6| Resuspend pellets in 1 ml of lysis buffer, mix in the ratio 1:1 and probe-tip sonicate (3 × 20 s). Incubate for 30 min with gentle agitation to introduce discontinuities into membrane vesicles and to remove the proteins loosely associated to artificial vesicles of microsomes and intact organelles such as mitochondria and lysosomes (i.e., the membrane proteins) 36 . 7| Transfer lysates to a 4-ml, thick-walled polycarbonate centrifuge tube and centrifuge in an ultracentrifuge at 150,000g for 1 h. 8| Remove the supernatant (cytoplasmic proteins).  crItIcal step After collecting the membrane fraction pellet, the lysis buffer incubation can be repeated two or three times to improve the purity of the membranes. 9| Wash the pellet gently with 500 µl of 500 mM NH 4 HCO 3 (pH 7.8).  pause poInt We recommend continuing with Step 9 immediately to minimize protein degradation. Lysates can otherwise be stored at − 80 °C. protein reduction, alkylation, tryptic digestion and phosphatase treatment • tIMInG overnight 10| Resolubilize the pellet containing membrane proteins in 50 µl of denaturing buffer using probe-tip sonication on ice to improve solubilization. 11| Determine protein amount by, e.g., fluorometer (Qubit Quantitation Platform, Invitrogen) or amino acid analysis. 12| Add DTT from a 1 M stock to 10 mM final concentration and incubate at 30 °C for 40 min.  crItIcal step Avoid higher temperatures to prevent urea-based carbamylation of primary amines. 13| Add IAA from a 0.5 M stock to 20 mM final concentration and incubate at room temperature (20-30 °C) for 40 min in the dark. 14| Add lysyl endopeptidase at a 1:100 enzyme/protein ratio for 3 h at room temperature. 15| Dilute the solution five times with 50 mM NH 4 HCO 3 (pH 7.8), to obtain a urea concentration < 1 M. 16| Add trypsin at an enzyme-to-substrate ratio of 1:50 to 1:100 and incubate overnight at 30 °C. 17| On digestion, freeze at − 80 °C and thaw the peptide mixture with bath sonication to inactivate the proteases or add protease inhibitors. 18| Treat the tryptic peptides with 20 U of alkaline phosphatase at 30 °C for 2 h to avoid copurification of phosphorylated peptides in the TiO 2 enrichment step.  crItIcal step Peptide mixtures must be dephosphorylated before enrichment of sialylated glycopeptides, as phosphopeptides have a high affinity for TiO 2 . However, it should be noted that not all the phosphorylation sites will be dephosphorylated with the phosphatase treatment.  pause poInt At this point, samples can be stored at − 80 °C for several weeks. preparing a suitable amount of tio 2 19| Prepare a TiO 2 bead slurry by weighing 10 mg of TiO 2 and adding 100 µl of acetonitrile in a 1-ml glass cylinder. Using magnetic stirring to keep the beads in suspension while pipetting, you should transfer 6 µl of the slurry to a low-binding 1.7-ml tube and dry it down in a vacuum centrifuge at room temperature for 5 min. The tube now contains 0.6 mg of TiO 2 .  crItIcal step When working with many samples, it is practical to prepare a large volume of bead slurry and divide this into smaller aliquots. Assuming that the slurry is well mixed, this will result in better reproducibility between samples. enrichment of sialylated glycopeptides with tio 2 • tIMInG ~3 h 20| Reduce the volume of the dephosphorylated peptide solution to ~100 µl by vacuum centrifugation. Add 1 ml of TiO 2 loading buffer and add this to 0.6 mg of TiO 2 per 100 µg of peptide mixture. Alternatively, adjust the 100-µl peptide solution to 1 ml using TiO 2 loading buffer.  crItIcal step For highly complex samples, the concentration of TFA should be kept at 5% (vol/vol) to avoid unspecific binding of nonsialylated peptides. 21| Incubate for 30 min on a mixer and pellet the beads by microcentrifugation (1,000g, room temperature) for 1 min. 22| Remove the supernatant by careful pipetting. The supernatant can be saved for further analysis.  crItIcal step The supernatant can be incubated with additional rounds of TiO 2 to increase the recovery of sialylated glycopeptides. The supernatant can also be saved for further analysis of non-modified peptides. 23| Add 400 µl of loading buffer to the beads. 24| Incubate for 1 min on the mixer (1,000g, room temperature) and pellet the beads by 1-min centrifugation in a microcentrifuge. 26| Add 400 µl of washing buffer 1 to the beads, vortex for 15 s and pellet by 1 min of microcentrifugation. 27| Remove the supernatant. 28| Add 400 µl of washing buffer 2. 29| Dry the beads for 5-10 min in a SpeedVac. 30| Incubate the beads with 200 µl of TiO 2 elution buffer solution for 20 min on the mixer (1,000g, room temperature) to elute the sialoglycopeptides. 31| Remove the TiO 2 beads by running the eluate over a P200 pipette tip plugged with C8 material using air pressure applied by a handheld 1-ml syringe. The P200 tip plugged with C8 material is made by stamping out a small plug from a 3M Empore C8 extraction disk using a HPLC syringe needle, and then placing it into the partially constricted end of the pipette tip. The C8 material will hold back the TiO 2 beads. 32| Wash the beads with 10 µl of 50% (vol/vol) acetonitrile to recover peptides that bind to the C8 material. 33| Lyophilize the sialoglycopeptides by vacuum centrifugation.  crItIcal step The dried sialoglycopeptides are now ready for enzymatic deglycosylation. Alternatively, they can be analyzed as intact glycopeptides in order to retrieve information about the heterogeneity of the glycan structures. It should be noted that several limitations exist in the data analysis of large-scale glycopeptide data. To check the purity of the enrichment, run a small aliquot in reversed-phase liquid chromatography coupled to MS/MS (RPLC-MS/MS) mode and detect the amount of three diagnostic ions (m/z 274.09 (Neu5Ac-H 2 O), 292.09 (Neu5Ac) and 657.24 (HexNAcHexNeu5Ac)).  pause poInt Lyophilized glycopeptides can be stored at − 20 °C for several weeks. ? trouBlesHootInG enzymatic deglycosylation • tIMInG overnight 34| Reconstitute the sialoglycopeptides in 40 µl of 50 mM NH 4 HCO 3 (pH 7.9), and add 1 U of PNGaseF.  crItIcal step We recommend the deglycosylation of mammalian N-linked glycopeptides with PNGaseF; however, the reaction is inhibited by the presence of α1,3-fucose on the peptide-linked GlcNAc, such as in many plant-derived N-linked glycopeptides. The use of PNGaseA, combined with fucosidases or glycosidases such as endoH and sialidase A in suitable conditions 37 , can overcome this complication. 35| Incubate at 37 °C overnight. 36| Add TFA at 0.1% (vol/vol) final concentration to terminate the deglycosylation reaction and check that pH is <3. 37| Desalt sample on a POROS Oligo R3 reversed-phase microcolumn. Add ~50 mg of a POROS Oligo R3 slurry onto a P200 tip plugged with C8 material (described above) and condition with 100 µl of 0.1% (vol/vol) TFA. Run the deglycosylated peptide mixture over the column and wash the column with 100 µl of 0.1% (vol/vol) TFA. 38| Elute the peptides with 70% (vol/vol) acetonitrile and 0.1% (vol/vol) TFA.  crItIcal step The flow-through from the POROS Oligo R3 reversed-phase microcolumn contains released N-glycans and can be saved for a further glycomic analysis to structurally characterize the sugars. 39| Lyophilize the peptides by vacuum centrifugation.  pause poInt Lyophilized peptides can be stored at − 20 °C for several weeks. HIlIc microHplc fractionation of deglycosylated peptides • tIMInG ~2 h 40| Reconstitute the sample in 41 µl of HILIC solvent B. Prepare the gradient as described in table 2 for peptide fractionation. Prepare the fraction collecting timeline described in table 3.  crItIcal step The sample needs to be reconstituted immediately before fractionation to prevent evaporation of acetonitrile, which can result in inefficient and nonreproducible retention of peptides. For high-resolution fractionation, it is important not to overload the column by exceeding its maximum capacity. For the HILIC microHPLC column described here, the capacity is approximately 20-50 µg. 41| Insert collection plate (i.e., 96 well) into the fraction collector. 42| Inject 40 µl onto the Agilent 1200 microHPLC instrument. See EQUIPMENT SETUP for details about configuration and settings. 43| Run the sample and collect fractions depending on the UV absorbance intensity and profile. Data analysis • tIMInG ~1 d to several weeks 46| Search the tandem mass spectra against an appropriate database, e.g., IPI Human (http://www.ebi.ac.uk/IPI/IPIhuman. html), using an appropriate search algorithm, e.g., MASCOT (http://www.matrixscience.com/). Set the variable modification of deamidation of asparagine to aspartic acid ( + 0.984 Da). Glycosylation sites are identified by the presence of a PNGaseFmediated deamidated asparagine within the N-linked glycosylation consensus motif (NXS/T/C, where X is any amino acid except proline). In this protocol, Proteome Discoverer software 1.2 (Thermo Scientific) was used to handle the data. See ANTICIPATED RESULTS for details regarding further data analysis that can be performed. These steps include removing redundancy and determining enrichment efficiency.  crItIcal step Other software platforms can be used to analyze the data. If a quantitative experiment is performed, choose the appropriate software depending on the chemical, enzymatic or label-free approach used ? trouBlesHootInG Troubleshooting advice can be found in table 4. • tIMInG Step 45, RPLC-MS/MS analysis: ~2 to 24 h (depending on the number of fractions analyzed) Step 46, Data analysis: ~1 d to several weeks The method described is a simple, robust and efficient method to enrich N-linked sialylated glycopeptides from different biological samples, such as biological fluids, tissues and cell cultures. To provide an example of the performance of this method, we have applied it to the study of the N-linked sialylated glycopeptides from enriched membrane proteins from HeLa cells. The strategy is illustrated in