Chemicals and solutions
Magnetic beads (1µm BcMag®Hydrazide-Modified Magnetic Beads) with hydrazide groups were purchased from Bioclone (San Diego). The glycerol free PNGase F solution was purchased from New England BioLabs. Water and acetonitrile (ACN) (J.T. Baker) were of HPLC quality. Unless otherwise stated, all other chemicals were purchased from Sigma Aldrich.
A single pool of human plasma (purchased from Bioreclamation Inc. New York) with a concentration of ca. 66 mg/ml was used for all experiments described in this study except for the study where the proteins identified by SPEG were compared to the proteins identified without SPEG. The latter samples were collected from five healthy individuals for which IRB approval had been obtained. An aliquot of 1mg of protein in 15 ul of plasma (denoted as 1 mg/15 ul) was either subjected to SPEG without further processing, or 10 mg/150 ul of the plasma was depleted of 14 abundant proteins using a MARS Hu-14 multiple affinity removal LC column (10 × 100 mm) according to instructions from the supplier (Agilent Technologies). The depleted material was concentrated using an Amicon Ultra-4 3 kDa MWCO filter (Millipore), pre-rinsed with 0.1% n-octyl-beta-d-glucopyranoside (Anatrace) in MARS A buffer, through centrifugation at 3950g for one hour. The 0.6 mg of depleted plasma material was then subjected to SPEG.
Optimized peptide-level capture SPEG
Plasma proteins were reduced, alkylated, digested with trypsin, and cis-diols of carbohydrate groups on the glycopeptides oxidized as previously described 21
except that the sample was heated in 8 M urea denaturation buffer at 37 °C rather than 60 °C. In addition, a different reverse phase cartridge and elution conditions were used for desalting of the digest before and after the oxidation step and prior to bead loading (details, below). Depleted plasma samples were buffer exchanged with 8 M urea/0.4 M ammonium bicarbonate using 3 kDa MWCO filters prior to proteolytic digestion.
For the magnetic beads approach, all glycopeptide capture and washing steps were conducted using the KingFisher magnetic particle processor (Thermo Scientific). KingFisher is constructed to work with 96-well plates covered by changeable plastic covers with 96 plastic pockets that go into each well. Up to eight 96-well plates can be placed on a rotating platform simultaneously and a magnet head with 96 magnets go into the corresponding plastic pockets to collect and transfer beads from one 96-well plate to another. For one sample, approximately two hours are saved when using magnetic beads in combination with KingFisher compared to manual processing of magnetic beads. When processing more than one sample, the increase in saved time would extrapolate linearly with the number of samples, with about 5–10 minutes added for every additional sample.
Magnetic hydrazide beads (4 mg of beads/mg of protein unless otherwise stated) were transferred to a 96 well plate compatible with the KingFisher processor and washed twice with 80% ACN/0.1% TFA. The beads were released into corresponding wells in a new 96-well plate that contained the digested and oxidized peptides, eluted in 400 ul of 80% ACN/0.1% TFA. The mixture of magnetic beads and oxidized peptides was incubated over night with vigorous shaking. After coupling of the glycopeptides, the beads were washed three times using KingFisher with each of the following solutions in this order: 80% ACN/0.1% TFA, 8 M urea/0.4 M ammonium bicarbonate/0.1% SDS, 100% N,N-Dimethylformamide (DMF, toxic) and 0.1 M ammonium bicarbonate. The washed beads were released in 100 ul 0.1 M ammonium bicarbonate, and 1.5 ul of PNGase F was added followed by incubation over night with shaking at 37 °C. The following settings on the Kingfisher were used for the steps described above: the magnetic heads where lowered into the sample 5 times during bead collection to ensure good recovery and transfer of beads between plates. For magnetic bead release and washing the magnets were removed from the plastic pockets followed by a 30 seconds fast speed shaking and 4.5 minutes medium speed shaking of the 96-well plates. Elution of peptides from the PNGaseF-treated beads and cleanup of the formerly glycosylated peptides was performed as previously described, using a 10 mg Oasis HLB cartridges instead of C18 SepPak, and 0.1% FA and 80% ACN/0.1% FA instead of 0.1% TFA and 80% ACN/0.1% TFA. Dried peptides were resuspended in 10 ul of 3% ACN/5% FA and 2 ul was analyzed by LC-MS/MS. While larger amounts of bead (up to 32 mg of beads/mg of protein) were found to provide somewhat higher glycocapture yield as described in the Results section, scaling the experiments to use these higher bead amounts during the method development phase would have increased the cost of the experiments significantly.
For the macroporous hydrazide resin approach, 50 ul of the 50% Affi-Prep® Hz Hydrazide Support slurry (BioRad) was washed once using 1 ml of deionized water and 5 minutes of shaking. Reduced, alkylated and oxidized plasma digests were loaded onto Oasis cartridges and peptides eluted in 400 ul of 80% ACN/0.1% TFA. The eluate was then coupled to the pre-washed beads and incubated over night with 1200 rpm shaking with an Eppendorf shaker. The unbound peptides were removed using the same washing strategy as described for the magnetic beads above, except that the wash steps were done manually. Each wash step consisted of 10 seconds of vortexing, followed by 5 minutes of shaking at 1200 rpm and centrifugation at 3000g. The elution and clean-up steps for formerly glycosylated peptides were the same as those used in the magnetic protocol, above. Dried peptides were resuspended in 10 ul of 3% ACN/5% FA and 2 ul was analyzed by LC-MS/MS.
Optimized protein-level capture SPEG
The undepleted plasma sample (15 ul/1 mg) was diluted to 40 ul with oxidation buffer (20 mM NaAc and 150 mM NaCl, pH 5.0), and oxidized with 15 mM sodium periodate (NaIO4) at room temperature (RT) in the dark with shaking for one hour. After oxidation, the excess sodium periodate was removed through a buffer exchange step with coupling solution using a 0.5 ml Zeba desalting spin column (Pierce). The oxidized protein mixture was then coupled to either magnetic hydrazide beads or macroporous hydrazide beads. The coupling solution consisted of 100 mM NaAc/1.5 M NaCl/0.2% CHAPS (w/v) when used in combination with the magnetic beads, whereas CHAPS was omitted for use with macroporous beads. The sample was diluted to 300 ul with coupling solution prior to coupling to the pre-washed beads.
For the protein-level coupling magnetic bead SPEG protocol, all washing steps were conducted using KingFisher with the same settings as for the peptide-level coupling. Four mg of magnetic beads (not optimized) was washed two times with coupling solution (100 mM NaAc/1.5 M NaCl/0.2% CHAPS (w/v)). The sample was resuspended in coupling solution, loaded onto the pre-washed beads and incubated over night at RT with shaking. Unbound proteins in the supernatant were saved for future analysis, while the beads with the bound glycoproteins were dissolved in denaturation buffer (8 M urea/0.4 M ammonium bicarbonate/0.1% SDS). Disulfides were reduced with 10 mM tris (2-Carboxyethyl) phosphine hydrochloride (Thermo Scientific) for one hour at RT and free thiols alkylated with 12 mM iodoacetamide for 30 minutes at RT. Unbound proteins were removed by washing 4-times with 1 ml of 8 M urea/0.4 M ammonium bicarbonate/0.1% SDS, with 5 minutes shaking for each wash step. Bound proteins were resuspended in 500 ul 0.1 M ammonium bicarbonate/0.2% CHAPS (w/v) before trypsin was added to a trypsin:protein ratio of 1:50, calculated from the amount of starting protein as determined by BCA. After over night incubation at 37 °C with shaking, the beads were washed three times using KingFisher with each of the following solutions in this order: 80% ACN/0.1% TFA, 8 M urea/0.4 M ammonium bicarbonate/0.1% SDS, 100% N,N-Dimethylformamide (DMF, toxic) and 0.1 M ammonium bicarbonate to remove the remaining non-glycopeptides. The washes were combined with the unbound peptide digest supernatant and saved for possible future analysis. Bound former glycosylation-site peptides were released by PNGase F and then desalted by Oasis cartridge as described in the peptide-level protocol above. Dried peptides were resuspended in 10 ul of 3% ACN/5% FA and 2 ul was analyzed by LC-MS/MS.
For the macroporous resin approach, 200 ul of the 50% resin slurry was washed once with 1 ml water during 5 minutes of shaking prior to adding the proteins in coupling solution. Coupling of protein to the beads, denaturation of proteins, reduction, alkylation and digestion were carried out as described for the magnetic bead approach above, except that the proteins were digested in 0.1 M ammonium bicarbonate. The rest of the procedure was performed as for the magnetic bead protein-level coupling approach described above, except that all washing steps were performed manually.
Quenching the oxidation reaction
We tested quenching of the sodium periodate oxidation step in the peptide-level capture protocol with sodium sulfite as an alternative to cleanup with the Oasis HLB cartridges. After one hour incubation with sodium periodate, sodium sulfite was added to a final concentration of 150 mM followed by 20 minute incubation with shaking at room temperature. After incubation, the solution was added directly to the pre-washed magnetic beads as previously described 20
. This procedure has not been incorporated in the optimized protocol as further explained in section “The influence of different coupling solutions on the SPEG performance”.
Basic pH reverse phase HPLC fractionation
Basic pH reverse HPLC was used to fractionate digested plasma samples using a Narrow-Bore 2.1 × 150 mm capillary reverse phase column (Agilent: ZORBAX) packed with 3.5 µm beads, coupled to an Agilent 1100 HPLC system. The mobile phases were as follows: mobile phase A (20 mM Ammonium Formate in water, 2% ACN, pH 10) and mobile phase B (90%ACN/10% 20 mM Ammonium Formate, pH 10). The gradient was 0–5 mins 0% B, 5–55 mins 50% B, 55–57 mins 100% B, 57–61 mins 100% B, 61–80 mins 0% B at a constant flow rate of 0.200 ml/min. A total of 12 factions were collected in a linear fashion from 0–70 minutes. Fractions 1 and 2 and fractions 11 and 12 were combined, giving a total of 10 fractions to be analyzed by LC-MS/MS.
All the LC-MS/MS analyses were done using an Agilent nanoflow HPLC system (Agilent, Palo Alto, CA). A PicoFrit column (New Objective, Woburn, MA) with an inner diameter of 75 µm packed with 12–14 cm of ReproSil-Pur C18 3 µm particles, was directly interfaced to an LTQ-FT mass spectrometer (Thermo Fisher, Waltham, MA) equipped with a custom nanoelectrospray ionization source. Analyses were of 90 min total duration, using the following mobile phases: mobile phase A (0.1% FA) and mobile phase B (0.1% FA/90% ACN). The gradient used was as follows: hold at 3% B at 0.6 µl/min from 0–13 min then reduce flow to 0.2 µl/min from 13–15 min. From 15–18 min 3–18% B, from 18–68 min 18–60% B, from 68–73 minutes 60–100% B. From 73–81 min hold at 100% B at a flow 0.6 µl/minute 81–82.5 min then ramp from 100%-3% B, and re-equilibrate column at 3% B from 82.5–100 min. For the MS method, nine scan events were conducted. The mass spectrometer was set to do one full FTMS scan at 100,000 resolution in profile mode followed by 8 data-dependent MS/MS scans at low-resolution in centroid mode in the LTQ on the top 8 most abundant peptide precursor ions. For the experiments described in sections “Comparison of proteins identified with and without glycocapture”, “Glycoproteins spiked into plasma” and “SPEG comparison between undepleted plasma and plasma depleted by MARS Hu-14” in the Results section, 3 MS/MS scans of the top 3 most abundant peptide precursors were acquired with an isolation width of 2 m/z. Charge state screening was enabled along with monoisotopic precursor selection and non-peptide monoisotopic recognition to prevent triggering of MS/MS on precursor ions with unassigned charge or a charge state of 1. Normalized collision energy was set to 35 with an activation Q of 0.25 and activation time of 30 ms. Dynamic exclusion parameters included a repeat count of 2, a repeat duration of 20 sec, and an exclusion duration of 30 sec.
MS/MS data was searched against the International Protein Index (IPI) database version 3.32 using the Spectrum Mill software package v4.0 beta (Agilent Technologies, Santa Clara, CA). The search parameters were: a maximum of two missed cleavages, precursor mass tolerance 0.035 Da, a product mass tolerance 0.7 Da and carbamidomethylation of cysteines as the fixed modifications. Allowed variable modifications were oxidized methionines and deamidation of asparagine. Identities interpreted for individual spectra were automatically designated as valid by applying the scoring threshold criteria provided below to all spectra derived from a particular experiment in a two step process. First, protein mode was used which requires 2 or more matched peptides per protein and while allowing a range of medium to excellent scores for each peptide. Second, peptide mode was applied to the remaining spectra allowing for excellent scoring peptides that are detected as the sole evidence for particular proteins. Protein mode thresholds: protein score >25, peptide (score, Scored Percent Intensity, delta rank1 – rank2) peptide charge +2: (>8, >65%, > 2) peptide charge +3: (>9, >65%, > 2) peptide charge +4: (>9, >70%, > 2) peptide charge +2: (>6, >90%,> 1). Peptide mode thresholds for all charge states: >13, >70, > 2, respectively.
The above criteria yielded a false discovery rate of <1% as estimated by target-decoy based searches using reversed sequences. In addition to the criteria described above, for a peptide to be assigned as a glycopeptide and matched to a glycoprotein, it also had to contain the consensus sequence motif for N-glycosylation. Furthermore, the precursor mass and relevant fragments for the former glycosylation-site peptide had to be shifted lower in mass by 0.9840 Da relative to the Asn-containing peptide. This is due to the conversion of the attachment-site Asn to Asp upon release of the carbohydrate by PNGase F. These additional criteria are filtering options that can be selected by the user in Spectrum Mill.
Extracted Ion Chromatograms (XICs)
The peak area for the XIC of each precursor ion in the intervening high-resolution MS1 scans of the data-dependent LC-MS/MS runs was calculated automatically by the Spectrum Mill software using narrow windows around each individual member of the isotope cluster. Peak widths in both the time and m/z domains are dynamically determined based on MS scan resolution, precursor charge and m/z subject to quality metrics on the relative distribution of the peaks in the isotope cluster vs. theoretical.
Calculation of CV for SPEG pipeline reproducibility study
The intraday and interday reproducibility for the SPEG process in both the peptide- and protein-level coupling modes were calculated using “protein XIC” values which are the sum of the observed XIC values of the constituent peptides for each specific protein. Intraday variation was determined from the variation in protein XIC values for five process replicate samples (a dataset) run on two different days (generating two data sets). The CV for each protein XIC value was calculated if the protein was observed in a minimum of two of the five replicates run on the same day. The median intraday CV of all proteins in a dataset was determined from the CVs of the protein XIC values. The median CVs for each of the two datasets is reported as an average value (). To determine the interday variation, the two intraday dataset were combined, and one CV for the XIC values for each protein over the runs was calculated if the protein was identified in two or more of the ten process replicates. The median CV was extracted from the resulting list of CVs ().
Calculated median CV values for the SPEG pipeline using either depleted or undepleted plasma with peptide-level coupling or undepleted plasma with protein-level coupling.
Calculation of SPEG pipeline performance
The principal criteria we used to evaluate the different SPEG pipelines and the effect of changes in the sample processing methods were the observed specificity for glycopeptide or glycoprotein enrichment and the total number of glycopeptide and glycoprotein identified. The definition of a glycopeptide in this context was that it had to be identified with the glyco-motif, and had to have a mass difference corresponding to deamidation. The definition of a glycoprotein was a protein that contained one or more identified glycopeptides. The specificity of the glycopeptide capture was defined as the percentage of unique glycopeptides among the total number of unique peptides identified. The specificity of glycoprotein capture was calculated from the percentage of unique proteins identified that contained identified glycopeptides.