Motivation Native top-down proteomics (nTDP) integrates native mass spectrometry (nMS) with top-down proteomics (TDP) to provide comprehensive analysis of protein complexes together with proteoform identification and characterization. Despite significant advances in nMS and TDP software developments, a unified and user-friendly software package for analysis of nTDP data remains lacking. Results We have developed MASH Native to provide a unified solution for nTDP to process complex datasets with database searching capabilities in a user-friendly interface. MASH Native supports various data formats and incorporates multiple options for deconvolution, database searching, and spectral summing to provide a “one-stop shop” for characterizing both native protein complexes and proteoforms. Availability and implementation The MASH Native app, video tutorials, written tutorials and additional documentation are freely available for download at https://labs.wisc.edu/gelab/MASH_Explorer/MASHSoftware.php. All data files shown in user tutorials are included with the MASH Native software in the download .zip file. Supplementary information Supplementary data are available at Bioinformatics online.

Protein homeostasis (proteostasis) is crucial for proper cellular function, including the production of peptides with biological functions through controlled proteolysis. Proteostasis has roles in maintenance of cellular functions and plant interactions with the environment under physiological conditions. Plant stress continues to reduce agricultural yields causing substantial economic losses; thus, it is critical to understand how plants perceive stress signals to elicit responses for survival. As previously shown in Arabidopsis thaliana, thimet oligopeptidases (TOPs) TOP1 (also referred to as organellar oligopeptidase) and TOP2 (also referred to as cytosolic oligopeptidase) are essential components in plant response to pathogens, but further characterization of TOPs and their peptide substrates is required to understand their contributions to stress perception and defense signaling. Herein, label-free peptidomics via liquid chromatography-tandem mass spectrometry was used to differentially quantify 1111 peptides, originating from 369 proteins, between the Arabidopsis Col-0 wild type and top1top2 knock-out mutant. This revealed 350 peptides as significantly more abundant in the mutant, representing accumulation of these potential TOP substrates. Ten direct substrates were validated using in vitro enzyme assays with recombinant TOPs and synthetic candidate peptides. These TOP substrates are derived from proteins involved in photosynthesis, glycolysis, protein folding, biogenesis, and antioxidant defense, implicating TOP involvement in processes aside from defense signaling. Sequence motif analysis revealed TOP cleavage preference for non-polar residues in the positions surrounding the cleavage site. Identification of these substrates provides a framework for TOP signaling networks, through which the interplay between proteolytic pathways and defense signaling can be further characterized.

Native top-down proteomics (nTDP) integrates native mass spectrometry (nMS) with top-down proteomics (TDP) to provide comprehensive analysis of protein complexes together with proteoform identification and characterization. Despite significant advances in nMS and TDP software developments, a unified and user-friendly software package for analysis of nTDP data remains lacking. Herein, we have developed MASH Native to provide a unified solution for nTDP to process complex datasets with database searching capabilities in a user-friendly interface. MASH Native supports various data formats and incorporates multiple options for deconvolution, database searching, and spectral summing to provide a one-stop shop for characterizing both native protein complexes and proteoforms. The MASH Native app, video tutorials, written tutorials and additional documentation are freely available for download athttps://labs.wisc.edu/gelab/MASH_Explorer/MASHNativeSoftware.php. All data files shown in user tutorials are included with the MASH Native software in the download .zip file.

Proteostasis is essential for proper cellular function, including the production of peptides with biological functions via the breakdown of proteins/peptides through controlled proteolysis. Proteostasis contributes to the maintenance of cellular functions and plant‐environment interactions under physiological conditions. Plant stress continues to reduce agricultural yields resulting in significant economic losses; thus, it is crucial to understand how plants perceive stress signals to elicit responses for survival. As previously shown in Arabidopsis thaliana, thimet oligopeptidases (TOPs) are essential components in plant response to oxidative stress triggered by pathogens. However, further characterization of TOP1 (also referred to as organellar oligopeptidase), TOP2 (also referred to as cytosolic oligopeptidase) and their peptide substrates is required to understand their contributions to stress perception and defense signaling. Label‐free peptidomics via liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) was used to differentially quantify 1111 peptides, originating from 369 proteins, between the Arabidopsis Col‐0 wild type and top1top2 knock‐out mutant. This revealed 350 peptides as significantly more abundant in the mutant, representing accumulation of these potential TOP substrates. Ten direct substrates were validated using in vitro enzyme assays and electrospray ionization mass spectrometry (ESI‐MS) with recombinant TOPs and synthetic candidate peptides. The TOP substrates discovered are derived from proteins involved in photosynthesis, biogenesis, glycolysis, protein folding, and antioxidant defense, implicating TOP involvement in processes beyond defense signaling. Identification of these substrates provides a framework for TOP signaling networks, through which the interplay between proteolytic pathways and defense signaling can be further characterized.

Top-down mass spectrometry (MS)-based proteomics has become a powerful tool for analyzing intact proteins and their associated post-translational modification (PTMs). In particular, membrane proteins play critical roles in cellular functions and represent the largest class of drug targets. However, the top-down MS characterization of endogenous membrane proteins remains challenging, mainly due to their intrinsic hydrophobicity and low abundance. Phospholamban (PLN) is a regulatory membrane protein located in the sarcoplasmic reticulum and is essential for regulating cardiac muscle contraction. PLN has diverse combinatorial PTMs and their dynamic regulation has significant influence on cardiac contractility and disease. Herein, we have developed a rapid and robust top-down proteomics method enabled by a photocleavable anionic surfactant, Azo, for the extraction and comprehensive characterization of endogenous PLN from cardiac tissue. We employed a two-pronged top-down MS approach using an online reversed-phase liquid chromatography tandem MS (LC-MS/MS) method on a quadrupole time-of-flight (Q-TOF) MS and a direct infusion method via an ultrahigh-resolution Fourier-transform ion cyclotron resonance (FTICR) MS. We have comprehensively characterized the sequence and combinatorial PTMs of endogenous human cardiac PLN. We have shown the site-specific localization of phosphorylation to Ser16 and Thr17 by MS/MS for the first time and the localization of S-palmitoylation to Cys36. Taken together, we have developed a streamlined top-down targeted proteomics method for comprehensive characterization of combinatorial PTMs in PLN toward better understanding the role of PLN in cardiac contractility.