The top-down approach to proteomics offers compelling advantages due to the potential to provide complete characterization of protein sequence and post-translational modifications. Here we describe the implementation of 193 nm ultraviolet photodissociation (UVPD) in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa is achieved with UVPD including the unambiguous localization of a single residue mutation and several protein modifications on Pin1 (Q13526), a protein implicated in the development of Alzheimer’s disease and in cancer pathogenesis. The 5 nanosecond, high-energy activation afforded by UVPD exhibits far less precursor ion-charge state dependence than conventional collision-based and electron-based dissociation methods.

Many top-down proteomics experiments focus on identifying and localizing post-translational modifications and other potential sources of "mass shift" on a known protein sequence. A simple application to match ion masses and facilitate the iterative hypothesis testing of PTM presence and location would assist with the data analysis in these experiments. ProSight Lite is a free software tool for matching a single candidate sequence against a set of mass spectrometric observations. Fixed or variable modifications, including both post-translational modifications and a select number of glycosylations, can be applied to the amino acid sequence. The application reports multiple scores and a matching fragment list. Fragmentation maps can be exported for publication in either PNG or SVG format. ProSight Lite can be freely downloaded from http:// prosightlite.northwestern.edu, installs and updates from the web, and requires Windows 7 or higher. KeywordsTop-down proteomics; proteomics software; MS Analysis; proteoform characterization Top-down proteomics describes the study of intact proteins with mass spectrometry [1,2]. Traditional bottom-up proteomics experiments are marked by the use of an enzyme, typically trypsin, to proteolyze intact proteins into more analytically manageable peptides (0.5-3 kDa) [3]. This proteolysis effects a loss of information between the ribosomallyexpressed pro-protein, which in eukaryotes often contains RNA splice variants, and the posttranslationally modified intact protein (termed a "proteoform") [4]. If a modification has been identified on two separate peptides, a typical bottom-up proteomics experiment cannot know whether those modification existed singly on two separate proteoforms or in tandem on a single proteoform. While more analytically challenging, top-down proteomics provides that full information [5]. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptAnalysis of top-down mass spectrometry data is separated into two main steps. First, the "analysis to infer mass (AIM)" step takes the complex m/z data provided by the mass spectrometer, performs deconvolution and deisotoping to provide neutral (or singly-charged) masses [6]. Second, those masses (with settings informed by experimental metadata) are matched against a hypothetical proteoform to determine the degree of matching. In this manuscript, we describe the release of ProSight Lite, a freely available application for targeted top-down proteomics data analysis. In a targeted top-down experiment, the user has generated a hypothesis (or set of hypotheses) for the identity of the proteoform under study (either through their experimental design or other, more complex 'discovery' experiments). The goal of a targeted top-down experiment is often to identify and localize posttranslational modifications (PTMs) on a known protein sequence. Offered as a partial replacement for our group's previously described software, ProSightPTM [7] and ProSightPTM2 [8], ProSight Lite describes a simple, i...

A new Orbitrap-based ion analysis procedure is shown to be possible by determining the direct charge for numerous individual protein ions to generate true mass spectra. The deployment of an Orbitrap system for charge detection enables the characterization of highly complicated mixtures of proteoforms and their complexes in both denatured and native modes of operation, revealing information not obtainable by traditional measurement of an ensemble of ions.

Top-down proteomics is emerging as a viable method for the routine identification of hundreds to thousands of proteins. In this work we report the largest top-down study to date, with the identification of 1,220 proteins from the transformed human cell line H1299 at a false discovery rate of 1%. Multiple separation strategies were utilized, including the focused isolation of mitochondria, resulting in significantly improved proteome coverage relative to previous work. In all, 347 mitochondrial proteins were identified, including ϳ50% of the mitochondrial proteome below 30 kDa and over 75% of the subunits constituting the large complexes of oxidative phosphorylation. Three hundred of the identified proteins were found to be integral membrane proteins containing between 1 and 12 transmembrane helices, requiring no specific enrichment or modified LC-MS parameters. Over 5,000 proteoforms were observed, many harboring post-translational modifications, including over a dozen proteins containing lipid anchors (some previously unknown) and many others with phosphorylation and methylation modifications. Comparison between untreated and senescent H1299 cells revealed several changes to the proteome, including the hyperphosphorylation of HMGA2. This work illustrates the burgeoning ability of top-down proteomics to characterize large numbers of intact proteoforms in a highthroughput fashion. Molecular & Cellular Proteomics

Targeted top-down (TD) and middle-down (MD) mass spectrometry (MS) offer reduced sample manipulation during protein analysis, limiting the risk of introducing artifactual modifications to better capture sequence information on the proteoforms present. This provides some advantages when characterizing biotherapeutic molecules such as monoclonal antibodies, particularly for the class of biosimilars. Here, we describe the results obtained analyzing a monoclonal IgG1, either in its ∼150 kDa intact form or after highly specific digestions yielding ∼25 and ∼50 kDa subunits, using an Orbitrap mass spectrometer on a liquid chromatography (LC) time scale with fragmentation from ion-photon, ion-ion, and ion-neutral interactions. Ultraviolet photodissociation (UVPD) used a new 213 nm solid-state laser. Alternatively, we applied high-capacity electron-transfer dissociation (ETD HD), alone or in combination with higher energy collisional dissociation (EThcD). Notably, we verify the degree of complementarity of these ion activation methods, with the combination of 213 nm UVPD and ETD HD producing a new record sequence coverage of ∼40% for TD MS experiments. The addition of EThcD for the >25 kDa products from MD strategies generated up to 90% of complete sequence information in six LC runs. Importantly, we determined an optimal signal-to-noise threshold for fragment ion deconvolution to suppress false positives yet maximize sequence coverage and implemented a systematic validation of this process using the new software TDValidator. This rigorous data analysis should elevate confidence for assignment of dense MS spectra and represents a purposeful step toward the application of TD and MD MS for deep sequencing of monoclonal antibodies.