Proteomic and network analysis of human serum albuminome by integrated use of quick crosslinking and two-step precipitation

Abstract

Affinity- and chemical-based methods are usually employed to prepare human serum albuminome; however, these methods remain technically challenging. Herein, we report the development of a two-step precipitation (TSP) method by combined use of polyethylene glycol (PEG) and ethanol. PEG precipitation was newly applied to remove immunoglobulin G for albuminome preparation, which is simple, cost effective, efficient and compatible with downstream ethanol precipitation. Nonetheless, chemical extraction using TSP may disrupt weak and transient protein interactions with human serum albumin (HSA) leading to an incomplete albuminome. Accordingly, rapid fixation based on formaldehyde crosslinking (FC) was introduced into the TSP procedure. The developed FC-TSP method increased the number of identified proteins, probably by favouring real-time capture of weakly bound proteins in the albuminome. A total of 171 proteins excluding HSA were identified from the fraction obtained with FC-TSP. Further interaction network and cluster analyses revealed 125 HSA-interacting proteins and 14 highly-connected clusters. Compared with five previous studies, 55 new potential albuminome proteins including five direct and 50 indirect binders were only identified by our strategy and 12 were detected as common low-abundance proteins. Thus, this new strategy has the potential to effectively survey the human albuminome, especially low-abundance proteins of clinical interest.

Macromolecular crowding effects on the kinetics of opposing reactions catalyzed by alcohol dehydrogenase

“…3 ) and lysozyme appear to be a result of chemical interactions with YADH, as judged by an effect even at 25 g/L. Over 200 proteins have been documented to interact with serum albumins [ 104 , 105 ], and thus it is reasonable to believe that YADH will interact with BSA, thereby altering its kinetics.…”

Protein-aggregating ability of different protoporphyrin-IX nanostructures is dependent on their oxidation and protein-binding capacity

“…S7 ). Since albumin also binds to a vast range of proteins in plasma [the “albuminome” ( 70 , 71 , 72 )], PP-IX binding could lead to disruption of components in the “albuminome.” In fact, oxidized human serum albumin is reported to differ significantly as compared with its unoxidized counterpart with respect to ligand binding and antioxidant properties ( 73 ).…”

A next generation setup for pre-fractionation of non-denatured proteins reveals diverse albumin proteoforms each carrying several post-translational modifications

“…The three apparently different molecular weights of plasma albumin could not be explained by PTM, since MS data does not provide strict stoichiometry. Nevertheless, besides PTM it is also possible that apparently large size proteoforms result from complex formation with members of the “albuminome” 55 . We unfortunately cannot discriminate complex formation from co-elution, since non-covalent, low affinity complexes might be dissolved after 2D-AEC.…”