Transporters belonging to the r esistance- n odulation- d ivision (RND) superfamily of proteins are invariably present in the genomes of Gram-negative bacteria and are largely responsible for the intrinsic antibiotic resistance of these organisms. The numbers of genes encoding RND transporters per genome vary from 1 to 16 and correlate with the environmental versatilities of bacterial species. Pseudomonas aeruginosa strain PAO1, a ubiquitous nosocomial pathogen, possesses 12 RND pumps, which are implicated in the development of clinical multidrug resistance and known to contribute to virulence, quorum sensing, and many other physiological functions.

Background/Objectives: Multiple Myeloma (MM) is a hematologic malignancy caused by clonally expanded plasma cells that produce a monoclonal immunoglobulin (M-protein), a personalized biomarker. Recently, we developed an ultra-sensitive mass spectrometry method to quantify minimal residual disease (MS-MRD) by targeting unique M-protein peptides. Therapeutic antibodies (t-Abs), key in MM treatment, often lead to deep and long-lasting responses. However, t-Abs can significantly decrease the total polyclonal immunoglobulin (Ig) levels which require supplemental IgG infusion. Here, we demonstrate the simultaneous monitoring of M-proteins, t-Abs, and polyclonal Ig-titers using an untargeted mass spectrometry assay, offering a comprehensive view of therapy response. Methods: Sera collected between 2013 and 2024 from four patients and cerebrospinal fluid (CSF) from one patient who received various t-Abs were analyzed with MS-MRD. M-protein sequences were obtained with a multi-enzyme de novo protein sequencing approach. Unique peptides for M-proteins and t-Abs were selected based on linearity, sensitivity, and slope coefficient in serial dilutions. Ig constant regions were monitored using isotype-specific peptides. Results: The MS-MRD multiplex analysis provided detailed information on drug concentrations and therapy response kinetics. For example, in two patients with refractory disease over five lines of therapy, the MS-MRD analysis showed that the deepest responses were achieved with bispecific t-Ab (teclistamab) treatment. M-protein and t-Ab were also detectable in the CSF of one patient with MS-MRD. Conclusions: This proof-of-concept study shows that the multiplex monitoring of the M-protein, any t-Ab combination, and all Ig-isotypes within one mass spectrometry run is feasible and provides unique insight into therapy response kinetics.