The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

This protocol streamlines the identification of bacterial glycop%eptides, enabling researchers to identify novel glycosylation events, as well as the differences in the glycans used for glycosylation between strains. The main advantage of this technique is that it enables non-glycosylation experts to identify potential glycosylation events in bacterial proteome samples. Open searching is a generally applicable approach that can be used for the identification of a range of modifications, not just glycosylation, and can be applied to any proteome sample.

To prepare STB-RPS stop-and-go extraction tips, use a 14-gauge blunt needle to excise three STB-RPS discs from a 47-square-millimeter STB-RPS membrane for binding 50 micrograms of peptide. For larger peptide amounts, increase the number of discs accordingly. Prior to using the SDB-RPS stage tips, wash the tips by adding 150 microliters of 100%acetonitrile and spinning the buffer by centrifugation or gently applying pressure using a syringe.

Next, wash the tips with 150 microliters of 30%methanol containing 1%TFA, then equilibrate the tips with 150 microliters of 90%isopropanol containing 1%TFA. After equilibration, load the proteome samples onto the STB-RPS stage tips by centrifugation or using a syringe. Then, wash the tips with 150 microliters of 90%isopropanol containing 1%TFA followed by 150 microliters of 1%TFA.

To elute the peptides from the STB-RPS stage tips, use 150 microliters of 5%ammonium hydroxide and 80%acetonitrile added to a single tip and collect the eluted samples in individual tubes by gently applying pressure using a syringe. Alternatively, if eluting via centrifugation, use 150 microliters at 5%ammonium hydroxide in 80%acetonitrile added to all stage tips and collect in a clean plate. Dry the eluted peptides by vacuum centrifugation at 25 degrees Celsius.

Next, to enrich the glycopeptide samples, prepare a zwitterionic hydrophilic interaction liquid chromatography, or ZIC-HILIC, stage tips. Excise one C8 disc from a 470-square-millimeter C8 membrane using a 14-gauge blunt needle and pack the disc into a P-200 tip to create a frit. Then, add approximately five millimeters of ZIC-HILIC material resuspended in 50%acetonitrile onto the frit by gently applying pressure using a syringe.

Before using the tips, equilibrate the resin with 20 bed volumes of ZIC-HILIC elution buffer, leaving 10 microliters of volume above the resin to ensure that the resin does not go dry. Then, sequentially wash the resin with 20 bed volumes of ZIC-HILIC preparation buffer followed by 20 bed volumes of ZIC-HILIC loading or wash buffer. Next, resuspend the digested and dried peptides in the ZIC-HILIC loading buffer to a final concentration of four micrograms per microliter.

Vortex briefly for one minute to ensure the samples are resuspended then spin them for one minute at 2000 times G and 25 degrees Celsius. After spinning, load the resuspended peptide sample onto the conditioned ZIC-HILIC column, then wash the column three times with 20 bed volumes of the ZIC-HILIC loading buffer by gently applying pressure using a syringe. Finally, elute the glycopeptides with 20 bed volumes of the ZIC-HILIC elution buffer into a 1.5-milliliter tube by gently applying pressure using a syringe, then dry the eluent by vacuum centrifugation at 25 degrees Celsius.

For LC-MS analysis, resuspend the samples in Buffer A*to a final concentration of one microgram per microliter, then load the samples onto an HPLC-UPLC coupled to an MS to enable the separation and identification of glycopeptides. Monitor the resulting MS data collection, ensuring the data is being collected with the desired parameters. To analyze the proteome and glycopeptide-enriched samples, open FragPipe and click on the Workflow tab.

From the Workflow pull-down menu, select the Open search option and click Add files to import the data files to be searched into FragPipe. Then, click the Database tab followed by Download to launch the download manager for downloading proteome databases from UniProt using a UniProt accession number. Within the download manager, click the Add decoys and contaminants option to incorporate decoy and contaminant proteins into this database.

Next, click the MSFragger tab, then within the Peak Matching box, increase the Precursor mass tolerance from the default 500 dalton to 2, 000 dalton to identify large modifications. Click the Run tab to define the location of the outputs of FragPipe, then click the Run button to begin the search. With high-confidence glycopeptides assigned, identify commonly observed glycan-associated ions to improve the identification of glycopeptides.

Next, click the MSFragger tab and add the determined delta masses of the observed glycans into the Variable modifications and Mass offsets sections with individual masses separated with a slash. Then, add the glycan-associated fragment masses of these glycans into the Glyco/Labile Mods section of MSFragger. Finally, upload all MS data associated with proteomic studies to centralized proteomic repositories, such as the PRIDE or MassIVE repositories.

Open searching of the Acinetobacter baumannii strain AB307-0294 revealed two dominant delta masses corresponding to 648.25 and 692.28 daltons. The ACICU capsule is known to be composed of a tetrasaccharide K unit corresponding to a predicted mass of 843.31 dalton. This capsule structure is consistent with the most frequently observed delta mass within the ACICU.

Open searching analysis of the strain D1279779 revealed the presence of multiple delta masses consistent with 203.08, 794.31, and 1588.62 daltons. Using the interactive peptide spectral annotator, glycopeptides identified across three A.baumannii strains were assessed revealing potential glycan-associated ions. Glycan-focused searches led to a notable increase in the total number of glycopeptide/peptide spectral matches identified across all three A.baumannii strains, corresponding to a 37%increase in ACICU, a 117%increase in AB307-0294, and a 363%increase in D1279779.

For individual MS events, the inclusion of glycan-specific information typically increases the observed Hyperscore, albeit this increase is highly glycan-dependent. When undertaking ZIC-HILIC enrichment, ensure that the resin always remains wet, as loss of solvent may compromise the isolation of glycopeptides. Data on differences in glycosylation can be used to inform mutagenesis studies or identify gene classes responsible for glycan biosynthesis.

Using open database searching, we've begun to characterize glycan diversity across bacterial genera. This has shown that bacterial glycosylation is both more more common and far more diverse than we once thought.