Hydrogen/deuterium exchange mass spectrometry is a well-established tool of structural biology, providing dynamic insights into the functioning of isolated protein systems. One of the often-mentioned advantages is the "no size limit" feature, allowing to study larger protein assemblies. Here we employed HDX-MS to get detailed insight into the heterogeneous complex assemblies of haptoglobin and follow its interaction with hemoglobin. Haptoglobin is one of the abundant plasma proteins and is responsible for protection again oxidative stress arising from freely circulating hemoglobin. It exists in three major phenotypes, depending on the combination of alleles 1 and 2 (1-1, 2-1 or 2-2). The phenotype 1-1 forms dimers, while 2-1 and 2-2 are characterized by higher order oligomers (dimer, trimer up to at least pentadecamer) with different architecture. The existing high-resolution structures however used only the homogeneous, fully dimeric 1-1 phenotype and employed its stabilization via addition of other interacting proteins. Here all three phenotypes were studied in the free form in solution and their interaction with hemoglobin was followed for various molar ratios. Next, the role of N-glycosylation was probed and the whole study was taken to proteome wide level by following the interaction in crude as well as albumin/Ig depleted serum. Once the serum HDX was established, interactions of additional serum proteins with small molecule ligands were followed and compared with the experiments done on isolated proteins. The present study demonstrates how HDX-MS can be taken to more complex levels and how it provides insights into the protein interactions on the level of individuals.

Financial support from the MEYS/EU OP JAK project Talking microbes - CZ.02.01.01/00/22_008/0004597 is gratefully acknowledged.