Protein complexes exhibit great diversity in protein membership, post-translational modifications and noncovalent cofactors, enabling them to function as the actuators of many important biological processes. The exposition of these molecular features with current methods lacks either throughput or molecular specificity, ultimately limiting the use of protein complexes as direct analytical targets in a wide range of applications. Here, we apply native proteomics, enabled by a multistage tandem mass spectrometry approach, to characterize 125 intact endogenous complexes and 217 distinct proteoforms derived from mouse heart and human cancer cell lines in discovery mode. The native conditions preserved soluble protein–protein interactions, high-stoichiometry noncovalent cofactors, covalent modifications to cysteines, and, remarkably, superoxide ligands bound to the metal cofactor of superoxide dismutase 2. The data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses mass spectrometry as a bridge to structural biology.

Top-down proteomics is capable of identifying and quantitating unique proteoforms through the analysis of intact proteins. We extended the coverage of the label-free technique, achieving differential analysis of whole proteins <30 kDa from the proteomes of growing and senescent human fibroblasts. By integrating improved control software with more instrument time allocated for quantitation of intact ions, we were able to collect protein data between the two cell states, confidently comparing 1577 proteoform levels. To then identify and characterize proteoforms, our advanced acquisition software, named Autopilot, employed enhanced identification efficiency in identifying 1180 unique Swiss-Prot accession numbers at 1% false-discovery rate. This coverage of the low mass proteome is equivalent to the largest previously reported but was accomplished in 23% of the total acquisition time. By maximizing both the number of quantified proteoforms and their identification rate in an integrated software environment, this work significantly advances proteoform-resolved analyses of complex systems.

Mutations of the gene are found in human cancers with high frequency and result in the constitutive activation of its protein products. This leads to aberrant regulation of downstream pathways, promoting cell survival, proliferation, and tumorigenesis that drive cancer progression and negatively affect treatment outcomes. Here, we describe a workflow that can detect and quantify mutation-specific consequences of KRAS biochemistry, namely linked changes in posttranslational modifications (PTMs). We combined immunoaffinity enrichment with detection by top-down mass spectrometry to discover and quantify proteoforms with or without the Gly13Asp mutation (G13D) specifically in the KRAS4b isoform. The workflow was applied first to isogenic colorectal cancer (CRC) cell lines and then to patient CRC tumors with matching genotypes. In two cellular models, a direct link between the knockout of the mutant G13D allele and the complete nitrosylation of cysteine 118 of the remaining WT KRAS4b was observed. Analysis of tumor samples quantified the percentage of mutant KRAS4b actually present in cancer tissue and identified major differences in the levels of C-terminal carboxymethylation, a modification critical for membrane association. These data from CRC cells and human tumors suggest mechanisms of posttranslational regulation that are highly context-dependent and which lead to preferential production of specific KRAS4b proteoforms.

Top-down proteomics studies intact proteoform mixtures and offers important advantages over more common bottom-up proteomics technologies, as it avoids the protein inference problem. However, achieving complete molecular characterization of investigated proteoforms using existing technologies remains a fundamental challenge for top-down proteomics. Here, we benchmark the performance of ultraviolet photodissociation (UVPD) using 213 nm photons generated by a solid-state laser applied to the study of intact proteoforms from three organisms. Notably, the described UVPD setup applies multiple laser pulses to induce ion dissociation, and this feature can be used to optimize the fragmentation outcome based on the molecular weight of the analyzed biomolecule. When applied to complex proteoform mixtures in high-throughput top-down proteomics, 213 nm UVPD demonstrated a high degree of complementarity with the most employed fragmentation method in proteomics studies, higher-energy collisional dissociation (HCD). UVPD at 213 nm offered higher average proteoform sequence coverage and degree of proteoform characterization (including localization of post-translational modifications) than HCD. However, previous studies have shown limitations in applying database search strategies developed for HCD fragmentation to UVPD spectra which contains up to nine fragment ion types. We therefore performed an analysis of the different UVPD product ion type frequencies. From these data, we developed an ad hoc fragment matching strategy and determined the influence of each possible ion type on search outcomes. By paring down the number of ion types considered in high-throughput UVPD searches from all types down to the four most abundant, we were ultimately able to achieve deeper proteome characterization with UVPD. Lastly, our detailed product ion analysis also revealed UVPD cleavage propensities and determined the presence of a product ion produced specifically by 213 nm photons. All together, these observations could be used to better elucidate UVPD dissociation mechanisms and improve the utility of the technique for proteomic applications.

The inhibition of ornithine aminotransferase (OAT), a pyridoxal 5′-phosphate-dependent enzyme, has been implicated as a treatment for hepatocellular carcinoma (HCC), the most common form of liver cancer, for which there is no effective treatment. From a previous evaluation of our aminotransferase inhibitors, (1S,3S)-3-amino-4-(perfluoropropan-2-ylidene)cyclopentane-1carboxylic acid hydrochloride (1) was found to be a selective and potent inactivator of human OAT (hOAT), which inhibited the growth of HCC in athymic mice implanted with human-derived HCC, even at a dose of 0.1 mg/kg. Currently, investigational new drug (IND)-enabling studies with 1 are underway. The inactivation mechanism of 1, however, has proved to be elusive. Here we propose three possible mechanisms, based on mechanisms of known aminotransferase inactivators: Michael addition, enamine addition, and fluoride ion elimination followed by conjugate addition. On the basis of crystallography and intact protein mass spectrometry, it was determined that 1 inactivates hOAT through fluoride ion elimination to an activated 1,1′-difluoroolefin, followed by conjugate addition and hydrolysis. This result was confirmed with additional studies, including the detection of the cofactor structure by mass spectrometry and through the identification of turnover metabolites. On the basis of this inactivation mechanism and to provide further evidence for the mechanism, analogues of 1 (19, 20) were designed, synthesized, and demonstrated to have the predicted selective inactivation mechanism. These analogues highlight the importance of the trifluoromethyl group and provide a basis for future inactivator design.