A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large scale proteome analysis1 has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry (MS)2. This “bottom up” process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous2 characterization of alternative splice forms, diverse modifications (e.g., acetylation and methylation), and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species3. “Top down” interrogation of whole proteins can overcome these problems for individual proteins4,5, but has not been achieved on a proteome scale due to the lack of intact protein fractionation methods that are well integrated with tandem MS. Here we show, using a new four dimensional (4D) separation system, identification of 1,043 gene products from human cells that are dispersed into >3,000 protein species created by post-translational modification, RNA splicing, and proteolysis. The overall system produced >20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kilodaltons and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply-modified species in response to accelerated cellular aging (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database6, the data provide precise correlations to individual genes and proof-of-concept for large scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research7.

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.

ProSight PTM 2.0 (http://prosightptm2.scs.uiuc.edu) is the next generation of the ProSight PTM web-based system for the identification and characterization of proteins using top down tandem mass spectrometry. It introduces an entirely new data-driven interface, integrated Sequence Gazer for protein characterization, support for fixed modifications, terminal modifications and improved support for multiple precursor ions (multiplexing). Furthermore, it supports data import and export for local analysis and collaboration.

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...