Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be responsible for the large-scale epidemic globally. The SARS-CoV-2 S protein is highly conserved and involved in multiple processes, including receptor recognition and viral attachment. The viral S protein is modified by glycosylation which may be implicated in immune evasion from the host immune system by shielding the protein surface from detection by antibodies, affecting the ability of the host to mount an effective adaptive immune response. Here, we demonstrate the utility of Cyclic IMS (cIMS) for in-depth glycopeptide characterisation using the multi-pass feature to separate co-eluting glycoforms related to the SARS-CoV-2 S1 protein.
The SARS-CoV-2 S1 protein, which contains a His Tag (S1N-C52H3) and is expressed from human 293 (HEK293) cells, was used as an example protein to illustrate the benefits of the multi-pass functionality. The sample was reduced, alkylated and digested with trypsin prior to LC-MS analysis. The corresponding peptides were separated using one-dimensional liquid chromatographic (LC) configured as trap and elute and utilising a 75 micron analytical column. 20ng of digest was loaded and separated over a 30min gradient. The LC was coupled to an IMS oa-QTof MS and data were obtained using an ion mobility enabled DIA method, HDMSE and targeted HDMSMS. Data were processed using a variety of informatic tools in order to provide annotated glycopeptide identifications.
Initial assessment of the digest was conducted using HDMSE, which comprised of a single pass of the cyclic device. The resulting data were processed using ProteinLynx Global Server and searched against a sequence specific database. Based on the initial, single pass data and observation of typical oxonium ions within the fragment ion spectra, glycopeptides at m/z 969.08 (3+) and 1262.9 (3+) were selected as candidates for further investigation using quadrupole isolation and multi-pass ion mobility. In order to allow multi-pass acquisitions, the cIMS settings were configured with mobility separate times derived from the instrument control software pass calculator and by measuring the ion behaviours for 1 and 2 passes. A total of five passes of the cyclic device was sufficient to show the emergence of multiple glycoforms for the ion at m/z 1262.9 and it was found that the fragment spectra corresponding to this species had the characteristic sialic acid linkage (m/z 657.2). Interpretation of the glycopeptide sequences was conducted using the GlycReSoft software package, in addition to manual interpretation as further validation.