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. Author manuscript; available in PMC: 2016 Apr 1.
Published in final edited form as: Proteomics. 2015 Apr;15(7):1235–1238. doi: 10.1002/pmic.201570050

ProSight Lite: Graphical Software to Analyze Top-Down Mass Spectrometry Data

Ryan T Fellers 1, Joseph B Greer 1, Bryan P Early 1, Xiang Yu 1, Richard D LeDuc 1, Neil L Kelleher 1,*, Paul M Thomas 1,*
PMCID: PMC4445472  NIHMSID: NIHMS662747  PMID: 25828799

Abstract

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.

Keywords: Top-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 ribosomally-expressed pro-protein, which in eukaryotes often contains RNA splice variants, and the post-translationally 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].

Analysis 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 post-translational 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, intuitive platform to characterize proteoforms and provide high-resolution graphical fragment maps, suitable for publication.

Application Workflow

As shown in Figure 1, ProSight Lite is intended to be used in an iterative fashion; users modify the candidate proteoform in silico to best match the experimental data. After the required tandem mass spectrometry data and proteoform hypothesis are entered into the application, a graphical fragment map is displayed (consists of the sequence with glyphs indicating matching fragment ions). The user is then free to apply a selection of PTMs (for example, selecting one of the set of defined glycosylations) or a custom mass to specific amino acid residues. One may also select to apply a fixed modification to all cysteine or methionine residues as needed. As changes are made, the fragment map, the matching fragment grid, scores, and other metrics update in real time.

Figure 1.

Figure 1

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ProSight Lite enables an iterative workflow to test hypotheses. 161×82mm (300 × 300 DPI)

When the user is satisfied with the resulting proteoform, the entire state of the application can be saved to a PCML file. This file contains all of the information required to completely reconstitute the result at a later time, making it the perfect format to exchange a fragment map with a collaborator. The PCML file is stored using a custom XML format, allowing a user to examine its contents using a simple text editor. In addition, a schema definition and example PCML file can be downloaded from SourceForge (http://sourceforge.net/projects/proteoform/).

The graphical fragment map can also be exported as either a portable network graphic file (PNG) or a scalable vector graphic file (SVG). PNG is a raster graphics file format that supports lossless data compression. This format is well-suited for a quick email or to post on a web site. SVG is an XML-based vector image format for infinitely scalable two-dimensional graphics. This format performs well when high resolution is needed for presentations or posters.

Input Dialogs

ProSight Lite has two dialogs for input and one main display. The experiment input dialog is used to input data from the mass spectrometer and sampling procedure such as the precursor and fragment ion masses. It also allows the user to define the fragmentation method, mass type (neutral vs. MH+), and a fragment matching tolerance. At present eight different fragmentation methods are supported with anywhere from two ion types (as in ETD, CID, etc.) to up to six ion types in UVPD. To enter a protein sequence, the user either enters the amino acid sequence or an accession number from UniProt (to perform an internet-based lookup). Users can also select to apply a fixed modification (also known as a static modification) to all cysteines or methionines via this dialog.

Main Window

Once the observed ions and protein sequence have been entered, the application will display the proteoform with its matching fragment ions overlaid (Figure 2). The right-hand panel is used to iteratively test hypotheses regarding the characterization of the proteoform. This is done by selecting any amino acid or either terminus with a left click on the fragment map and then selecting any post-translational modification (PTM), N-linked glycosylation, or custom mass shift in the modification pane on the right. When a modification has been selected, the background color of the selected residue or terminus will change color to match.

Figure 2.

Figure 2

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The ProSight Lite main window consists of four sections: (1) the menu bar, (2) the fragment map,(3) the modification pane, and (4) the matching fragments grid. These data are from a tandem experiment which fragmented intact ribonuclease B (P61823) containing a Mannopentaose-di-(N-acetyl-D-glucosamine) glycosylation, localized to Asn34

Each change to the sequence causes metrics and scores to immediately update at the upper right. The scores displayed are the P-score [9] and the Protein Characterization Score (PCS) [10]. The PCS uses shuffled protein sequences to create a decoy distribution to which the original (or forward) result is compared. We also display the percent of observed fragments that match (within tolerance) with any theoretical fragment ions.

The matching fragment grid just below the graphical fragment map displays detailed information about all matching ions. The name, ion type, ion number, and theoretical mass columns all refer to the mass and placement of a specific theoretical fragment ion. The observed mass corresponds to the fragment mass observed in the user’s experimental data. Lastly, the mass difference columns display how closely the theoretical mass matched to the observed mass.

N-Linked Glycosylation Support

ProSight Lite is aligned with UniCarbKB with regard to standardizing glycosylation searching and reporting. UniCarbKB is an initiative that aims to promote the creation of an online information storage and search platform for glycomics and glycobiology research[11]. At present a subset of six common N-linked glycosylations are supported; ProSight Lite will be updated in the near future to include access to all of the structures cataloged in UniCarbKB using a web service that is currently under construction [12]. Please see Figure 2 for an example of a localized Mannopentaose-di-(N-acetyl-D-glucosamine) (Man5) glycosylation.

Complex Fragmentation

ProSight Lite also supports many complex fragmentation types including ultraviolet photodissociation (UVPD)[13] and combined collisional and electron-based dissociations (EThcD[14], activated ion ECD[15], etc., creating both b/y and c/z• ions). In Figure 3, UVPD fragmentation results for RS28_HUMAN are shown with 6 different ion types, each with a unique color. Using the combined b/y and c/z• fragmentation method, it is possible, for example, to create a mixed fragment map displaying ETD fragments from one scan with CID fragments from another scan. In both cases, the P-score model is corrected to account for the additional theoretical fragment ions.

Figure 3.

Figure 3

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Graphical fragment map showing results from the ultraviolet photodissociation of RS28_HUMAN. 145×30mm (300 × 300 DPI)

ProSight Lite has been implemented in C# .NET 4.5 using Windows Presentation Foundation (WPF) and Visual Studio 2013. Most of the functionality (algorithms, custom displays, etc.) is provided by code written in C# .NET 4.0 and .NET 4.5.1 and also depends on a number of other .NET projects (MathNet.Numerics, DocumentFormat.OpenXML, GalaSoft.MvvmLight, SQLite, NHibernate, Fluent NHibernate, and Quadruple). Please see the application’s “About” dialog for specific versions and license information. ProSight Lite can be downloaded from http://prosightlite.northwestern.edu. Please address all questions or comments to pce@northwestern.edu.

Acknowledgments

The authors would like to thank Owen Skinner, Emma Doud, Caroline DeHart, and the rest of the Kelleher Research Group for their assistance in testing the application. We would also like to thank Drs. Joe Cannon and Jennifer Brodbelt from the University of Texas at Austin for providing us with example UVPD data and members of the UniCarbKB team for helpful discussions surrounding glycosylation support. This research is supported by Award No. P30DA018310 from the National Institute on Drug Abuse and Award No. R01GM067193 from the National Institute of General Medical Sciences.

ABBREVIATIONS

UVPD

ultraviolet photodissociation

SVG

scalable vector graphic

PNG

portable network graphic

XML

extensible markup language

WPF

Windows Presentation Foundation

Footnotes

The authors have declared no conflict of interest.

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