A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

This procedure isolates specifically glycosylated Glycopeptides from human plasma. Plasma is digested with trypsin to yield a mixture of peptides and glycopeptides glycopeptides standards. Positive and negative controls for lectin chromatography are spiked into the plasma.

The sample is subjected to high pressure liquid chromatography based lectin affinity chromatography. The lectins used bind diff fuco and sialic acid respectively, peptides and irrelevant glycopeptides elute from the column in the flow through fraction. Acetic acid is used to elute the bound fraction from the column.

This is enriched for specifically modified glycopeptides. Hi, I'm Penny Drake from the laboratory of Susan Fisher in the Department of Obstetrics Gynecology and Reproductive Sciences and the San Lamore mass spectrometry core facility at the University of California San Francisco. And I'm Eric Johansen, also from the Sandler Moore Mass spectrometry core facility and the Fisher Lab.

Today we will show you a procedure for performing lectin chromatography to enrich glycopeptides from human plasma and other complex mixtures. We use this procedure in our laboratory as a general tool that has numerous applications. For instance, it plays an important role in our breast cancer biomarker discovery workflow.

So let's get started. To begin this procedure, wear a mask to protect against inhalation of the beads. Then weigh out 100 milligrams of porro beads and transfer to a clean einor tube.

Approximately 300 microliters of beads are needed to pack one column. Additionally, weigh out unconjugated lectin and transfer to a clean einor tube. The lectins SNA and A A L are used.

Proceed to add PBS to the lectin to form a five to 20 milligram per milliliter solution. Reserve 25 microliters of this pre conjugated solution. Next, wash the porro speeds by adding one milliliter of PBS and centrifuging in a micro centrifuge at maximum speed.

For three minutes, remove the supernatant and repeat the wash. Now work in the fume hood and transfer the remaining lectin solution to the washed porro speeds. And add sodium sano boro hydride to a final concentration of 50 millimolar.

Note that sodium sano boro hydride is toxic and waste must be disposed of appropriately. Secure closed tube with perfil and place on a rocker to react overnight at room temperature After the overnight binding of the lectin to the porro speeds, pellet the porro speeds.Again. Remove the supernatant and save as the post conjugation solution to block the remaining reactive sites on the beads.

First, wash the beads by centrifuging with one milliliter of quenching buffer made up of one molar CL at pH 7.4. Then add one milliliter of quenching buffer and sodium sano hydride to a final concentration of 50 millimolar. Place the tube on a rocker and incubate at room temperature for 30 minutes.

Quenching is now complete. Centrifuge the beads. As before.

Remove the supernatant and proceed to wash the beads five times with one milliliter of one molar sodium chloride each time and discard the supernatant. Perform a protein concentration assay on the pre conjugated and post conjugated lectin solutions and use the difference to determine the amount of protein that was conjugated to the porro speeds. Target lectin concentrations are between two to 20 milligrams per milliliter calculated as the amount of protein conjugated per bead volume.

The conjugated porro beads are now ready for packing into a column. To pack the beads into a column, assemble the packing system made up of two columns supported on a metal ring stand. The upper column serves as a reservoir for the packing material.

The packing system consists of from bottom to top pressure restrictor. End column coupler one frt the target column, end column coupler, two column connector, end column coupler three, the reservoir column, end column coupler four and end fitting. Next, resuspend the 300 microliters of porro speeds in 600.

Microliters of buffer A For packing one column, transfer the beads into the upper reservoir column. Add buffer A until it reaches the top of the column. Gently place the end fitting onto the top of the column.

Trying to avoid air bubbles in the column. Connect the end fitting of the upper column to the HPLC system. Pack the column by flowing buffer A or PBS through the system.

Start with a flow rate of 0.5 milliliters per minute. Increase the flow rate by 0.5 milliliters per minute each minute until either four milliliters per minute or the maximum pressure has been reached. Continue at the maximum possible flow rate until at least 35 milliliters of buffer have passed through the column.

When finished, turn off the pumps and allow the pressure on the column to drop below 20 PSI. Now that the column is packed, gently disassemble the packing system starting from the top. When reaching the end column coupler two, remove it carefully.

Using a razor blade gently wipe away the packed material extruding from the top of the column. Do not apply pressure to the beads while doing this. Disengage the packed column from the ring stand.

Place a new frit into a new end coupler and turn the packed column over to connect this with the FR plus end coupler on the open column end. The column is now ready for the HPLC run. When not in use, the column can be stored in PBS with 0.5%sodium azide at four degrees Celsius for up to six months.

The details of programming your HPLC will vary according to your setup and the specifics of the manufacturer's software. Here we use a microme paradigm MG four HPLC on this machine. Methods are built and accessed under the setup tab at the top of the screen program.

The method shown, which is also described in detail in the accompanying written protocol. If using an autos sampler program it following the written protocol as well. Use buffer A as before and buffer B for running the HPLC and to keep the buffers as well as the columns at room temperature.

Program the UV detector to read a two and 80 nanometers from zero to 19 minutes and 50 seconds. Adjust the Y axis to detect low absorbance levels in the range of zero to 50 absorbance units. Next, prepare the samples.

The test samples are derived from human plasma depleted of the 14 most abundant proteins using a Mars column. The depleted plasma samples and two standards. Human lactoferrin and yeast invertase are each individually tripsin digested and de salted as previously described.

To neutralize the samples have neutralization buffer ready in the sample vials that will collect EIT for the paradigm MG four one vial collects the flow through and three, collect the eluate. It will require approximately 1.5 milliliters of buffer to neutralize one milliliter of eit. The target neutralized pH is 7.0 to 8.0.

Make sure to test the final pH with pH indicator paper. Desalt the samples as described later in this protocol. And resuspend the sample in one milliliter of buffer A for best results.

Perform three experimental replicates so that data may be compared and combined. To prepare samples for three replicates, add three picaMoles of trys trypsin, digested invertase, and one microliter of HPLC purified lectin specific lactoferrin to 30 microliters of Mars depleted trypsin, digested plasma equivalents or pe. This amounts to approximately 50 micrograms of plasma protein used for each replicate add buffer A to the sample to reach a final volume of 330 microliters or 110 microliters per experimental Replicate using either the A a L or the SNA lectin column first run two blank methods injecting only buffer A.Ensure that the lectin column corresponds to the lectin specific lactoferrin used as a standard and previously purified chromatograph.

The three plasma samples injecting a blank in between each injected sample for a total of seven HPLC runs. Fractions from blank runs need not be collected. Again, make sure to neutralize the samples by having neutralization buffer present in the sample vials that collect eluate after desalting and P-N-G-A-F digestion, the samples will be ready for mass spectrometric analysis.

Use one one milliliter waters oasis, H-L-B-S-P-E cartridge for each of the HBLC fractions from the lactoferrin standard and the experimental samples to desalt. Start by attaching the number of required cartridges to a vacuum manifold. Wet each cartridge with one milliliter of 80%acetonitrile in 1%Formic acid.

The vacuum gauge on the manifold should read five to 20 inches. Mercury equilibrate the cartridges with 1.5 milliliters of 0.1%formic acid. Again, the vacuum gauge on the manifold should read five to 20 inches.

Mercury load the entire volume of one sample onto one cartridge. Now the vacuum gauge on the manifold should read two to 2.5 inches. Mercury and the flow rate should not exceed one milliliter per minute.

Wash the cartridges three times with one milliliter of 0.1%formic acid using a single labeled collection tube for each cartridge. Elute the glycopeptides with one milliliter of 80%Acetonitrile in 0.1%Formic acid neutralize the EIT by adding 60 microliters of 0.5 molar ammonium bicarbonate to each collection tube. The target neutralized pH is 7.0 to 8.0.

Make sure to test the final pH with pH indicator paper. Concentrate the eluded peptides to approximately 50 microliters by using a vacuum centrifuge such as thermo savant speed vac for approximately two hours at 35 degrees Celsius. Again, test the sample pH after concentrating to ensure it is between 7.0 to 8.0 If needed, add 50 millimolar ammonium bicarbonate to increase the pH.

If the final sample volume is larger than 100 microliters, concentrate the sample by vacuum centrifugation until it is between 50 to 100 microliters. Finally, remove N-linked carbohydrates from the peptides by adding 250 units glycerol free peptide n glycosate F or P-N-G-A-F to each sample tube and incubate at 37 degrees Celsius overnight. Following the overnight incubation with P-N-G-S-F, you zip tip pipette tips containing a reverse phase C 18 matrix to desalt the samples before beginning.

Make two droplets, one of 0.1%formic acid and one of 80%acetonitrile plus 0.1%formic acid on a piece of paraform for washing and eluding Droplets are used to prevent cross-contamination of peptides between samples and new droplets are prepared for each sip tipped sample. Start by wetting a zip tip with acetyl nitrile solutions. Equilibrate the zip tip by pipetting up and down with 10 microliters of 0.1%formic acid three times.

Load the sample by pipetting up and down 10 times. Then wash the tip with 10 microliters of 0.1%formic acid five times. Finally, elute the sample twice with 80%acetonide trial in 1%Formic acid collect eluted peptides in an einor tube.

Reduce the sample volume to less than two microliters by concentration in a vacuum centrifuge and finish by resus suspending the samples in 20 microliters of 0.1%formic acid. The samples are now ready for analysis by L-C-M-S-M-S. The porro conjugation typically yields on bead lectin concentrations in the range of two to 20 milligrams per milliliter, which is optimal for affinity chromatography.

Lectin chromatography of tryin digested Mars depleted human plasma typically enriches glycopeptides in the bound fraction. Our workflow identifies N linked glycopeptides, which contain the unlinked consensus sequence NXS or NXT, where X is any residue except proline. PNGF treatment removes the unlinked glycans and deaminate the asparagine residue to which glycans were attached.

Dation results in a one Dalton mass shift of the de glycosylated peptide or de glycopeptide from the unmodified peptide. Thus DEG glycopeptides are identified as peptides containing the N-linked consensus sequence where the asparagine residue within the consensus sequence is deam observed as a mass difference of plus one compared to an unmodified disparaging residue. Here two example spectra illustrate this concept.

The top spectrum shows the fragmentation of an unmodified peptide containing the EnLink consensus sequence. The bottom spectrum shows fragmentation of the same peptide with the deaminated asparagine residue within the EnLink consensus sequence. The circled molecular ions from the Y seven, Y eight and B three fragments of the deamidated peptide are increased in mass by one Dalton as compared to their counterparts in the unmodified peptide.

This mass shift indicates that the peptide in the bottom spectrum contains a deam asparagine residue. Typically using a QStar elite 1000 to 1300 total peptides will be identified in the flow through fraction and 200 to 400 peptides will be identified in the bound fraction. On average, the flow through fraction from a a l or SNA chromatography contains two to 4%de glycopeptides, whereas 30 to 50%of the peptides recovered in the bound fraction are de glycopeptides.

We use the term deg glycopeptides rather than glycopeptides as the de glycosylated deamidated molecules are observed during mass spectrometric analysis. However, we propose that the majority of DEG glycopeptides in our samples were previously unlinked glycopeptides that were deg glycosylated and deaminated by PN GF treatment. The enrichment of de glycopeptides in our bound sample supports this hypothesis and allows us to use detection of D glycopeptides as a proxy for the glycopeptides captured by our lectin affinity chromatography protocol.

Two or more distinct invertase d glycopeptides should be observed in the flow through fraction and two or more distinct lactoferrin de glycopeptides in the bound fraction. We've just shown you how to prepare lectin columns and perform lectin affinity chromatography in an HPLC format. When doing this procedure, it's important to start out with high quality glycopeptide samples to monitor the pH of your samples throughout the experiment and to confirm the quality of your chromatography with glycopeptide standards.

So that's it. Thanks for watching and good luck with your experiments.