Introduction
Endogenous antibodies, or immunoglobulins (Igs), are pivotal for the immune system's ability to identify and neutralize foreign threats. Despite their abundance in body fluids, the enormous variability in sequence and concentration of these molecules presents significant analytical challenges. Recent advances in protein-centric analysis utilizing Orbitrap-based systems have facilitated the detailed profiling of human antibody repertoires by analyzing fragment antigen-binding (Fab) regions. However, effective Fab profiling requires optimized separation techniques and rapid mass spectrometric analysis to differentiate hundreds of Fabs with nearly identical masses but potentially diverse biochemical properties.
Here, we demonstrate for the first time that timsTOF mass spectrometer coupled to nanoLC can be effectively employed to analyze antibody repertoires from minimal amounts of serum.

Methods
IgG antibodies were affinity-purified using CaptureSelect FcXL resins and digested with the IdgE hinge-cleaving protease. This process released the Fabs from the remaining fragment crystallizable (Fc) region attached to the bead. Prior to Fab profiling, LC runs with 7 recombinant monoclonal Fab mixture were performed to compare the sensitivity of UHPLC system versus the nanoLC, whereby the nanoLC provided approx. 50-fold increase in intensity. Further, Fab fragments from serum samples were profiled on both MS platforms with identical nanoLC and separation methods. LC-MS data were analyzed by Thermo BioPharma Finder or Bruker DataAnalysis with in-house implemented sliding window algorithm, followed by MaxEnt deconvolution.

Results
Advancement in the analysis and profiling of the circulating antibody population in human serum samples are presented, marking the first-time use of a Bruker timsTOF HT mass spectrometer. It was observed that injections of 1-2 µg into the nanoLC-timsTOF system induced a noise-like signal deteriorating phenomenon in the 1000−2000 m/z range. Consequently, sample amounts were reduced (~56-110ng), resulting in a notable sensitivity enhancement, leading to the detection of hundreds of distinct IgG1 clones. Notably, the timsTOF HT data yielded >1.5 times more identified clones compared to the Orbitrap Eclipse. This increase was primarily due to the identification of clones with abundance lower than 1%. On the other hand, the 100 most abundant clones from both platforms demonstrated high identity overlap, reinforcing confidence in our proposed workflow.

Our data reveal that the timsTOF mass spectrometer can effectively track antibody repertoires, matching the quality of prior workflows. Its high sensitivity allows for detailed Fab profiling from just 50 nL of serum, which is a substantial decrease in sample consumption from the 1-2 µL typically required in previous studies.