FLAG-KRAS4B as a Model System for KRAS4B Proteoform and PTM Evaluation by Mass Spectrometry.

Prior analysis of intact and modified protein forms (proteoforms) of KRAS4B isolated from cell lines and tumor samples by top-down mass spectrometry revealed the presence of novel posttranslational modifications (PTMs) and potential evidence of context-specific KRAS4B modifications. However, low endogenous proteoform signal resulted in ineffective characterization, making it difficult to visualize less abundant PTMs or perform follow-up PTM validation using standard proteomic workflows. The NCI RAS Initiative has developed a model system, whereby KRAS4B bearing an N-terminal FLAG tag can be stably expressed within a panel of cancer cell lines. Herein, we present a method for combining immunoprecipitation with complementary proteomic methods to directly analyze N-terminally FLAG-tagged KRAS4B proteoforms and PTMs. We provide detailed protocols for FLAG-KRAS4B purification, proteoform analysis by targeted top-down LC-MS/MS, and validation of abundant PTMs by bottom-up LC-MS/MS with example results.

SARS-CoV-2 IgG Spike antibody levels and avidity in natural infection or following vaccination with mRNA-1273 or BNT162b2 vaccines.

Certain aspects of the immunogenicity and effectiveness of the messenger ribonucleic acid (mRNA) vaccines (mRNA-1273 and BNT162b2) developed in response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic are still uncharacterized. Serum or plasma samples from healthy donor recipients of either vaccine (BNT162b2 n = 53, mRNA-1273 n = 49; age 23-67), and individuals naturally infected with SARS-CoV-2 (n = 106; age 18-82) were collected 0-2 months post-infection or 1- and 4 months after second dose of vaccination. Anti-Spike antibody levels and avidity were measured via an enzyme-linked immunosorbent assay (ELISA). Overall, vaccination induced higher circulating anti-Spike protein immunoglobulin G (IgG) antibody levels and avidity compared to infection at similar time intervals. Both vaccines produced similar anti-Spike IgG concentrations at 1 month, while mRNA-1273 demonstrated significantly higher circulating antibody concentrations after 4 months. mRNA-1273 induced significantly higher avidity at month 1 compared to BNT162b2 across all age groups. However, the 23-34 age group was the only group to maintain statistical significance by 4 months. Male BNT162b2 recipients were approaching statistically significant lower anti-Spike IgG avidity compared to females by month 4. These findings demonstrate enhanced anti-Spike IgG levels and avidity following vaccination compared to natural infection. In addition, the mRNA-1273 vaccine induced higher antibody levels by 4 months compared to BNT162b2.