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The pharmaceutical industry’s interest in monoclonal antibodies (mAbs) and their derivatives has spurred rapid growth in the commercial and clinical pipeline of these effective therapeutics. The complex micro-heterogeneity of mAbs requires in-depth structural characterization for critical quality attribute assessment and quality assurance. Currently, mass spectrometry (MS)-based methods are the gold standard in mAb analysis, primarily with a bottom-up approach in which immunoglobulins G (IgGs) and their variants are digested into peptides to facilitate the analysis. Comprehensive characterization of IgGs and the micro-variants remains challenging at the proteoform level. Here, we used both top-down and middle-down MS for in-depth characterization of a human IgG1 using ultra-high resolution Fourier transform MS. Our top-down MS analysis provided characteristic fingerprinting of the IgG1 proteoforms at unit mass resolution. Subsequently, the tandem MS analysis of intact IgG1 enabled the detailed sequence characterization of a representative IgG1 proteoform at the intact protein level. Moreover, we used the middle-down MS analysis to characterize the primary glycoforms and micro-variants. Micro-variants such as low-abundance glycoforms, C-terminal glycine clipping, and C-terminal proline amidation were characterized with bond cleavages higher than 44% at the subunit level. By combining top-down and middle-down analysis, 76% of bond cleavage (509/666 amino acid bond cleaved) of IgG1 was achieved. Taken together, we demonstrated the combination of top-down and middle-down MS as powerful tools in the comprehensive characterization of mAbs.

Protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase; it forms diverse heterotrimeric holoenzymes that counteract kinase actions. Using a peptidome that tiles the disordered regions of the human proteome, we identified proteins containing [LMFI]xx[ILV]xEx motifs that serve as interaction sites for B′-family PP2A regulatory subunits and holoenzymes. The B′-binding motifs have important roles in substrate recognition and in competitive inhibition of substrate binding. With more than 100 novel ligands identified, we confirmed that the recently identified LxxIxEx B′α-binding motifs serve as common binding sites for B′ subunits with minor variations, and that S/T phosphorylation or D/E residues at positions 2, 7, 8 and 9 of the motifs reinforce interactions. Hundreds of proteins in the human proteome harbor intrinsic or phosphorylation-responsive B′-interaction motifs, and localize at distinct cellular organelles, such as midbody, predicting kinase-facilitated recruitment of PP2A-B′ holoenzymes for tight spatiotemporal control of phosphorylation at mitosis and cytokinesis. Moroever, Polo-like kinase 1-mediated phosphorylation of Cyk4/RACGAP1, a centralspindlin component at the midbody, facilitates binding of both RhoA guanine nucleotide exchange factor (epithelial cell transforming sequence 2 (Ect2)) and PP2A-B′ that in turn dephosphorylates Cyk4 and disrupts Ect2 binding. This feedback signaling loop precisely controls RhoA activation and specifies a restricted region for cleavage furrow ingression. Our results provide a framework for further investigation of diverse signaling circuits formed by PP2A-B′ holoenzymes in various cellular processes.

Mass spectrometry (MS)-based phosphoproteomics remains challenging due to the low abundance of phosphoproteins and substoichiometric phosphorylation. This demands better methods to effectively enrich phosphoproteins/peptides prior to MS analysis. We have previously communicated the first use of mesoporous zirconium dioxide (ZrO(2)) nanomaterials for effective phosphopeptide enrichment. Here, we present the full report including the synthesis, characterization, and application of mesoporous titanium dioxide (TiO(2)), ZrO(2), and hafnium dioxide (HfO(2)) in phosphopeptide enrichment and MS analysis. Mesoporous ZrO(2) and HfO(2) are demonstrated to be superior to TiO(2) for phosphopeptide enrichment from a complex mixture with high specificity (99%), which could almost be considered as a "purification", mainly because of the extremely large active surface area of mesoporous nanomaterials. A single enrichment and Fourier transform MS analysis of phosphopeptides digested from a complex mixture containing 7% of alpha-casein identified 21 out of 22 phosphorylation sites for alpha-casein. Moreover, the mesoporous ZrO(2) and HfO(2) can be reused after a simple solution regeneration procedure with comparable enrichment performance to that of fresh materials. Mesoporous ZrO(2) and HfO(2) nanomaterials hold great promise for applications in MS-based phosphoproteomics.