Advantages of Extended Bottom-Up Proteomics Using Sap9 for Analysis of Monoclonal Antibodies

Despite the recent advances in structural analysis of monoclonal antibodies with bottom-up, middle-down, and top-down mass spectrometry (MS), further improvements in analysis accuracy, depth, and speed are needed. The remaining challenges include quantitatively accurate assignment of post-translational modifications, reduction of artifacts introduced during sample preparation, increased sequence coverage per liquid chromatography (LC) MS experiment, and ability to extend the detailed characterization to simple antibody cocktails and more complex antibody mixtures. Here, we evaluate the recently introduced extended bottom-up proteomics (eBUP) approach based on proteolysis with secreted aspartic protease 9, Sap9, for analysis of monoclonal antibodies. Key findings of the Sap9-based proteomics analysis of a single antibody include: (i) extensive antibody sequence coverage with up to 100% for the light chain and up to 99-100% for the heavy chain in a single LC-MS run; (ii) connectivity of complementarity-determining regions (CDRs) via Sap9-produced large proteolytic peptides (3.4 kDa on average) containing up to two CDRs per peptide; (iii) reduced artifact introduction (e. g., deamidation) during proteolysis with Sap9 compared to conventional bottom-up proteomics workflows. The analysis of a mixture of six antibodies via Sap9-based eBUP produced comparable results. Due to the reasons specified above, Sap9-produced proteolytic peptides improve the identification confidence of antibodies from the mixtures compared to conventional bottom-up proteomics dealing with shorter proteolytic peptides.

Middle-down approaches for mass spectrometry-based protein identification and characterization

Kristina Srzentic

Mass spectrometry (MS) has emerged over the last two decades as the analytical technique of choice in systems-level protein studies, known as proteomics. Two are the MS-based approaches generally applied to proteomics: bottom-up (BU), which relies on the proteolytic digestion of proteins into short (~10 amino acids) peptides, and top-down (TD), where proteolysis is omitted, intact proteins are detected and fragmented in gas phase. Both methods present advantages as well as drawbacks. Here, we sought to establish a complete platform to put forward a third MS-based proteomic approach; middle-down (MD). It implies protein digestion as in BU, but aims to generate large peptides which size approaches the one of small intact proteins that are readily analyzed in TD. This novel domain aims to account for the shortcoming of both classical approaches. Until now, the main reasons behind the limited use of MD proteomics have been the lack of easy-to-use cleaving agents capable of producing peptides in the desired 3-15 kDa mass range with high specificity, limitations in MS and allied instrumentation, and the absence of dedicated bioinformatics tools for processing of acquired data. The latter greatly impedes the next milestone in MD proteomics â large-scale analysis. MD can potentially combine the analysis of large portions of proteins carrying set of biologically-relevant modifications â allowing exploring proteins of molecular weight or complexity incompatible with current TD capabilities â with the high-throughput hallmark of BU proteomics. Here, we first in silico evaluated the potential target amino acid residues to produce peptides in the MD mass range within the proteomes of different organisms. This bioinformatics work was followed by an experimental study based on synthetic MD-sized peptides, aimed at determining the optimal MS and tandem MS parameters for large peptide characterization. Next, we pursued two distinct ways of performing MD proteolysis: i) the use of an enzyme and ii) the use of a chemical reagent. We selected the protease Sap9 as a target enzyme for MD, which we fully characterized and successfully applied to the study of a mixture of monoclonal antibodies, where it showed an advantage over traditional BU in terms of reduced introduction of artifacts to the sample, allowing the post-translational modification investigation and unambiguous antibody identification. The chemical cleavage way we addressed via judicious protocol optimization for hydrolysis at the N-terminal side of cysteine with NTCB reagent. We also advanced MD protocols by generation of large (~50 kDa) subunits of monoclonal antibodies through the use of papain and another more specific novel protease, GingisKHAN, combined with new MS signal processing and data analysis capabilities. The developed workflow improved mapping of the connectivity of cysteines involved in inter- and intra-molecular disulfide bridges in antibodies. To summarize, we demonstrated that MD approach to mass spectrometry and proteomics is a powerful, yet underdeveloped, complement to BU and TD. This work has benchmarked MD for targeted protein analysis. In the near future, with advancements of the field, we envision its growing use for large-scale complex proteome analysis.

EPFL2016

Practical Considerations for Improving the Productivity of Mass Spectrometry-based Proteomics

Luca Fornelli, Anton Kozhinov, Unige Anna Laskay, Kristina Srzentic, Yury Tsybin, Oxana Upir

Mass spectrometry (MS) is currently the most sensitive and selective analytical technique for routine peptide and protein structure analysis. Top-down proteomics is based on tandem mass spectrometry (MS/MS) of intact proteins, where multiply charged precursor ions are fragmented in the gas phase, typically by electron transfer or electron capture dissociation, to yield sequence-specific fragment ions. This approach is primarily used for the study of protein isoforms, including localization of post-translational modifications and identification of splice variants. Bottom-up proteomics is utilized for routine high-throughput protein identification and quantitation from complex biological samples. The proteins are first enzymatically digested into small (usually less than ca. 3 kDa) peptides, these are identified by MS or MS/MS, usually employing collisional activation techniques. To overcome the limitations of these approaches while combining their benefits, middle-down proteomics has recently emerged. Here, the proteins are digested into long (3-15 kDa) peptides via restricted proteolysis followed by the MS/MS analysis of the obtained digest. With advancements of high-resolution MS and allied techniques, routine implementation of the middle-down approach has been made possible. Herein, we present the liquid chromatography (LC)-MS/MS-based experimental design of our middle-down proteomic workflow coupled with post-LC supercharging.

Schweizerische Chemische Gesellschaft2013

Multiplexed Middle-Down Mass Spectrometry as a Method for Revealing Light and Heavy Chain Connectivity in a Monoclonal Antibody

Daniel Ayoub, Luca Fornelli, Natalia Gasilova, Anton Kozhinov, Laure Menin, Konstantin Nagornov, Kristina Srzentic, Yury Tsybin

Pairing light and heavy chains in monoclonal antibodies (mAbs) using top-down (TD) or middle-down (MD) mass spectrometry (MS) may complement the sequence information on single chains provided by high-throughput genomic sequencing and bottom-up proteomics, favoring the rational selection of drug candidates. The 50 kDa F(ab) subunits of mAbs are the smallest structural units that contain the required information on chain pairing. These subunits can be enzymatically produced from whole mAbs and interrogated in their intact form by TD/MD MS approaches. However, the high structural complexity of F(ab) subunits requires increased sensitivity of the modern TD/MD MS for a comprehensive structural analysis. To address this and similar challenges, we developed and applied a multiplexed TD/MD MS workflow based on spectral averaging of tandem mass spectra (MS/MS) across multiple liquid chromatography (LC)-MS/MS runs acquired in reduced or full profile mode using an Orbitrap Fourier transform mass spectrometer (FTMS). We first benchmark the workflow using myoglobin as a reference protein, and then validate it for the analysis of the 50 kDa F(ab) subunit of a therapeutic mAb, trastuzumab. Obtained results confirm the envisioned benefits in terms of increased signal-to-noise ratio of product ions from utilizing multiple LC-MS/MS runs for TD/MD protein analysis using mass spectral averaging. The workflow performance is compared with the earlier introduced multiplexed TD/MD MS workflow based on transient averaging in Orbitrap FTMS. For the latter, we also report on enabling absorption mode FT processing and demonstrate its comparable performance to the enhanced FT (eFT) spectral representation.

AMER CHEMICAL SOC2018

Middle-down approaches for mass spectrometry-based protein identification and characterization

Kristina Srzentic

EPFL2016