MASH Suite Pro: A Comprehensive Software Tool for Top-Down Proteomics

Top-down mass spectrometry (MS)-based proteomics is arguably a disruptive technology for the comprehensive analysis of all proteoforms arising from genetic variation, alternative splicing, and posttranslational modifications (PTMs). However, the complexity of top-down high-resolution mass spectra presents a significant challenge for data analysis. In contrast to the well-developed software packages available for data analysis in bottom-up proteomics, the data analysis tools in top-down proteomics remain underdeveloped. Moreover, despite recent efforts to develop algorithms and tools for the deconvolution of top-down high-resolution mass spectra and the identification of proteins from complex mixtures, a multifunctional software platform, which allows for the identification, quantitation, and characterization of proteoforms with visual validation, is still lacking. Herein, we have developed MASH Suite Pro, a comprehensive software tool for top-down proteomics with multifaceted functionality. MASH Suite Pro is capable of processing high-resolution MS and tandem MS (MS/MS) data using two deconvolution algorithms to optimize protein identification results. In addition, MASH Suite Pro allows for the characterization of PTMs and sequence variations, as well as the relative quantitation of multiple proteoforms in different experimental conditions. The program also provides visualization components for validation and correction of the computational outputs. Furthermore, MASH Suite Pro facilitates data reporting and presentation via direct output of the graphics. Thus, MASH Suite Pro significantly simplifies and speeds up the interpretation of high-resolution top-down proteomics data by integrating tools for protein identification, quantitation, characterization, and visual validation into a customizable and user-friendly interface. We envision that MASH Suite Pro will play an integral role in advancing the burgeoning field of top-down proteomics

Top-Down Structural Analysis of an Intact Monoclonal Antibody by Electron Capture Dissociation-Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry

Top-down electron capture dissociation (ECD) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was performed for structural analysis of an intact monoclonal antibody (IgG1kappa (κ) isotype, ∼148 kDa). Simultaneous ECD for all charge states (42+ to 58+) generates more extensive cleavages than ECD for an isolated single charge state. The cleavages are mainly localized in the variable domains of both heavy and light chains, the respective regions between the variable and constant domains in both chains, the region between heavy-chain constant domains C_H2 and C_H3, and the disulfide bond (S–S)-linked heavy-chain constant domain C_H3. The light chain yields mainly N-terminal fragment ions due to the protection of the interchain disulfide bond between light and heavy chain, and limited cleavage sites are observed in the variable domains for each chain, where the S–S spans the polypeptide backbone. Only a few cleavages in the S–S-linked light-chain constant domain, hinge region, and heavy-chain constant domains C_H1 and C_H2 are observed, leaving glycosylation uncharacterized. Top-down ECD with a custom-built 9.4 T FTICR mass spectrometer provides more extensive sequence coverage for structural characterization of IgG1κ than does top-down collision-induced dissociation (CID) and electron transfer dissociation (ETD) with hybrid quadrupole time-of-flight instruments and comparable sequence coverage for top-down ETD with orbitrap mass analyzers

Top-Down Structural Analysis of an Intact Monoclonal Antibody by Electron Capture Dissociation-Fourier Transform Ion Cyclotron Resonance-Mass Spectrometry

Top-down electron capture dissociation (ECD) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was performed for structural analysis of an intact monoclonal antibody (IgG1kappa (κ) isotype, ∼148 kDa). Simultaneous ECD for all charge states (42+ to 58+) generates more extensive cleavages than ECD for an isolated single charge state. The cleavages are mainly localized in the variable domains of both heavy and light chains, the respective regions between the variable and constant domains in both chains, the region between heavy-chain constant domains C_H2 and C_H3, and the disulfide bond (S–S)-linked heavy-chain constant domain C_H3. The light chain yields mainly N-terminal fragment ions due to the protection of the interchain disulfide bond between light and heavy chain, and limited cleavage sites are observed in the variable domains for each chain, where the S–S spans the polypeptide backbone. Only a few cleavages in the S–S-linked light-chain constant domain, hinge region, and heavy-chain constant domains C_H1 and C_H2 are observed, leaving glycosylation uncharacterized. Top-down ECD with a custom-built 9.4 T FTICR mass spectrometer provides more extensive sequence coverage for structural characterization of IgG1κ than does top-down collision-induced dissociation (CID) and electron transfer dissociation (ETD) with hybrid quadrupole time-of-flight instruments and comparable sequence coverage for top-down ETD with orbitrap mass analyzers

Effective Protein Separation by Coupling Hydrophobic Interaction and Reverse Phase Chromatography for Top-down Proteomics

One of the challenges in proteomics is the proteome’s complexity, which necessitates the fractionation of proteins prior to the mass spectrometry (MS) analysis. Despite recent advances in top-down proteomics, separation of intact proteins remains challenging. Hydrophobic interaction chromatography (HIC) appears to be a promising method that provides high-resolution separation of intact proteins, but unfortunately the salts conventionally used for HIC are incompatible with MS. In this study, we have identified ammonium tartrate as a MS-compatible salt for HIC with comparable separation performance as the conventionally used ammonium sulfate. Furthermore, we found that the selectivity obtained with ammonium tartrate in the HIC mobile phases is orthogonal to that of reverse phase chromatography (RPC). By coupling HIC and RPC as a novel two-dimensional chromatographic method, we have achieved effective high-resolution intact protein separation as demonstrated with standard protein mixtures and a complex cell lysate. Subsequently, the separated intact proteins were identified by high-resolution top-down MS. For the first time, these results have shown the high potential of HIC as a high-resolution protein separation method for top-down proteomics

Three Dimensional Liquid Chromatography Coupling Ion Exchange Chromatography/Hydrophobic Interaction Chromatography/Reverse Phase Chromatography for Effective Protein Separation in Top-Down Proteomics

To address the complexity of the proteome in mass spectrometry (MS)-based top-down proteomics, multidimensional liquid chromatography (MDLC) strategies that can effectively separate proteins with high resolution and automation are highly desirable. Although various MDLC methods that can effectively separate peptides from protein digests exist, very few MDLC strategies, primarily consisting of 2DLC, are available for intact protein separation, which is insufficient to address the complexity of the proteome. We recently demonstrated that hydrophobic interaction chromatography (HIC) utilizing a MS-compatible salt can provide high resolution separation of intact proteins for top-down proteomics. Herein, we have developed a novel 3DLC strategy by coupling HIC with ion exchange chromatography (IEC) and reverse phase chromatography (RPC) for intact protein separation. We demonstrated that a 3D (IEC-HIC-RPC) approach greatly outperformed the conventional 2D IEC-RPC approach. For the same IEC fraction (out of 35 fractions) from a crude HEK 293 cell lysate, a total of 640 proteins were identified in the 3D approach (corresponding to 201 nonredundant proteins) as compared to 47 in the 2D approach, whereas simply prolonging the gradients in RPC in the 2D approach only led to minimal improvement in protein separation and identifications. Therefore, this novel 3DLC method has great potential for effective separation of intact proteins to achieve deep proteome coverage in top-down proteomics

Online Hydrophobic Interaction Chromatography–Mass Spectrometry for Top-Down Proteomics

Recent progress in top-down proteomics has led to a demand for mass spectrometry (MS)-compatible chromatography techniques to separate intact proteins using volatile mobile phases. Conventional hydrophobic interaction chromatography (HIC) provides high-resolution separation of proteins under nondenaturing conditions but requires high concentrations of nonvolatile salts. Herein, we introduce a series of more-hydrophobic HIC materials that can retain proteins using MS-compatible concentrations of ammonium acetate. The new HIC materials appear to function as a hybrid form of conventional HIC and reverse phase chromatography. The function of the salt seems to be preserving protein structure rather than promoting retention. Online HIC-MS is feasible for both qualitative and quantitative analysis. This is demonstrated with standard proteins and a complex cell lysate. The mass spectra of proteins from the online HIC-MS exhibit low charge-state distributions, consistent with those commonly observed in native MS. Furthermore, HIC-MS can chromatographically separate proteoforms differing by minor modifications. Hence, this new HIC-MS combination is promising for top-down proteomics

Specific Enrichment of Phosphoproteins Using Functionalized Multivalent Nanoparticles

Analysis of protein phosphorylation remains a significant challenge due to the low abundance of phosphoproteins and the low stoichiometry of phosphorylation, which requires effective enrichment of phosphoproteins. Here we have developed superparamagnetic nanoparticles (NPs) whose surface is functionalized by multivalent ligand molecules that specifically bind to the phosphate groups on any phosphoproteins. These NPs enrich phosphoproteins from complex cell and tissue lysates with high specificity as confirmed by SDS-PAGE analysis with a phosphoprotein-specific stain and mass spectrometry analysis of the enriched phosphoproteins. This method enables universal and effective capture, enrichment, and detection of intact phosphoproteins toward a comprehensive analysis of the phosphoproteome