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De Novo Sequencing of SARS-CoV-2 and influenza monoclonal antibodies by mass spectrometry using HCD and EThcD fragmentation and Supernovo software

ABSTRACT: In this study, we used electron-transfer/higher energy collision dissociation (EThcD) fragmentation to develop a mass-spectrometric (MS) method for distinguishing between Ile and Leu in Bovine Serum Albumin (BSA) and a SARS-CoV-1 anti-receptor binding domain (RBD) Spike monoclonal antibody (mAb). Supplemental activation normalized collision energy (SA-NCE) values were optimized. Combinations of multiple enzyme digestions, coupled with higher-energy collisional dissociation (HCD) and EThcD fragmentation, were used to increase coverage of all amino acid residues of the antibody. The data was searched with Supernovo™, which generated a complete de novo sequence for the test mAb and two additional SARS-CoV-2 human mAbs, giving correct sequences for the variable regions and selection between Ile and Leu residues. We then used the method on an additional set of twenty-five anti-hemagglutinin (HA) influenza antibodies of unknown sequence and determined high confidence sequences for >99% of the complementarity determining regions (CDRs). Importantly, the recombinant expression of these mAbs verified their binding and specificity to the HA influenza antigen. Unique glycosylation sites were also identified for eight antibodies. Our findings indicate this methodology results in almost complete antibody sequence coverage with >90% of residues determined with high confidence. Ile/Leu residues can be differentiated and glycosylation sites can be reliably identified.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood

SUBMITTER: Peter Faull

LAB HEAD: Maria Person

PROVIDER: PXD030094 | Pride | 2022-06-03

REPOSITORIES: Pride

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Project description:The haematopoietic cytokine thrombopoietin (Tpo) is the primary regulator of megakaryocyte and platelet numbers and is required for maintenance of the haematopoetic stem cell compartment. Tpo is a heavily glycosylated, hepatocyte-derived cytokine which functions by binding to its receptor (TpoR) on target cells and thereby activating intracellular signalling cascades that induce their proliferation and/or differentiation. In addition to its role in signal propagation, TpoR is expressed on the surface of platelets, where it contributes to regulation of Tpo levels by sequestering circulating cytokine. TpoR belongs to the homodimeric Class I cytokine receptor family but is unusual due to a duplication of the Cytokine binding Homology Region (CHR). Almost thirty years after initial discovery of TpoR, the structure of the human Tpo:TpoR interaction was recently reported. Here we determine the structure of extracellular portion of the murine Tpo:TpoR signalling complex using single particle cryo-EM. The structure reveals that Tpo:TpoR forms a largely symmetrical 1:2 complex. The cytokine cross-links the same site on the membrane-distal CHR of both receptor chains using opposing surfaces and with significantly different affinities. This orients the two membrane-proximal CHRs such that they contact one another adjacent to the plasma membrane. The potential cytokine-binding site in CHR2 is glycosylated and does not interact with Tpo. A large insertion in CHR1 that is unique to Tpo forms a partially structured loop that is disulphide bonded to CHR2 and, in one receptor chain, contacts cytokine. Biochemical analyses indicate that the glycosylated C-terminal domain of Tpo does not influence receptor binding. We demonstrate that the therapeutic TpoR agonist Romiplostim binds to the same site on the receptor as does cytokine. Our study characterises the Tpo/TpoR interaction structurally and biochemically to allow for the future development of potent TpoR agonists for therapeutic use.

Dataset Information

De Novo Sequencing of SARS-CoV-2 and influenza monoclonal antibodies by mass spectrometry using HCD and EThcD fragmentation and Supernovo software

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