First SaD Then HAPPY: Towards Blood Serum Antibody Analysis from Dried Serum Spots with ITEM-SIX Mass Spectrometry.

The HAPPY (Harvested Antibodies are Precipitated, Phase separated, and Yoked) protocol allows to rapidly prepare nanoESI mass spectrometry-compatible solutions of blood serum-borne polyclonal immunoglobulins that had been eluted from dried serum spots (DSS). DSS are serum protein containers through which proteins from blood serum can be stored and shipped at room temperature. We subjected DSS-eluted antibody-containing solutions directly to mass spectrometric ITEM-SIX (Intact Transition Epitope Mapping-Serological Inspection by Epitope EXtraction) analyses for determining whether or not the specifically targeting antibody of interest is present in the originating blood serum or not. The proof-of-principle study and assay development were achieved with spiking in monoclonal antiHis-tag or polyclonal antiTRIM21 antibodies with known epitopes into a model blood serum. Inducing immune complex formation was successful upon adding epitope peptide mixtures as multiplex probes when the polyclonal immunoglobulin-containing electrospray-compatible solution originated from blood serum into which the antibody of interest had been spiked in. Presence of an immune complex with targeted epitope specificity was assured by ITEM-SIX mass spectrometry. Upon ionizing and desolvating by nanoESI all peptides and proteins, including formed immune complexes, are simultaneously transitioned into the gas phase and subsequently separated by the mass analyzer. Immune complexes are then dissociated under controlled conditions within the mass spectrometer's collision cell, and detection of the complex-released epitope peptide from the probe is proof of the presence of the immune complex in the investigated antibody-containing solution. Consequently, detection of the complex-released epitope peptide by ITEM-SIX is also proof of the presence of a specific antibody in the originating blood serum. We show that not all epitope peptides are equally well suitable as probes, and investigations of background interferences are recommended for inclusion/exclusion assessment of a probe's peptides. In sum, seroconversion analysis by multiplex ITEM-SIX mass spectrometry has now become in immediate reach.