Identifying Proteins in a Top Down Standard with ProSightPC 4.1.

A simple mixture of five proteins is used by the National Resource for Translational and Developmental Proteomics to check the health of their mass spectrometers prior to running clinical samples.

The Top Down Standards mixture consists of the following proteins:

  • 1 pmol myoglobin (M5696)
  • 0.5 pmol trypsinogen (T1143)
  • 0.5 pmol carbonic anhydrase (C2624)
  • 0.1 pmol ubiquitin (U6253)
  • Superoxide dismutase – SOD1 – contaminant is present in carbonic anhydrase

An example RAW file containing data of Top Down Standard proteins can be downloaded and searched against a custom ProSightPC database to identify proteins in the standard’s mix.

A search can also be performed in ProSightPD as a quick exercise to check that ProSightPD is working correctly on your computer prior to attempting analyses of your own files.

Download the Standards .raw file and the Proteoform Database for Top Down Standards.

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TDCollider 1.0 Demo Data

The following data file is available for use with TDCollider 1.0:

golden_2016_06_02.tdReport

 

Identifying Proteins in a Pierce Intact Protein Standard with ProSightPD 1.1.

This tutorial will guide you through your first search with ProSightPD 1.1 for Proteome Discoverer 2.1 and ProSightPC 4.0. Specifically you will use the ProSightPD 1.1 nodes to search for proteins contained in the Pierce Intact Protein Standard. The tutorial assumes that Proteome Discoverer 2.1, ProSightPC 4.0, ProSightPD 1.1 and ProSightPD 1.1 with High Mass are already installed. If Proteome Discoverer 2.1 is not installed please contact your local Thermo Sales representative. If ProSightPC 4.0 is not installed, you can find the installer and installation instructions here. If ProSightPD 1.1 is not installed, you can find the installer and installation instructions here.

The Pierce Intact Protein Standard is a mixture of six recombinantly expressed proteins to check the health of mass spectrometers prior to running protein samples. This standard mixture will be commercially available soon through Thermo Fisher Scientific.

The Pierce Intact Protein Standard mixture consists of the following proteins:

  • IGF-I LR3 (Peprotech) (9 kDa)
  • Thioredoxin (12 kDa)
  • Protein G (21 kDa)
  • Carbonic Anhydrase II (29 kDa)
  • Protein AG (51 kDa)
  • Exo Klenow (68 kDa)

 

Follow these steps to perform your first search with ProSightPD 1.1 for Proteome Discoverer 2.1 and ProSightPC 4.0:

1. Ensure that Proteome Discoverer 2.1, ProSightPC 4.0, ProSightPD 1.1 and ProSightPD 1.1 with High Mass are installed.

2. Download the three Pierce Standard medium high raw files.

 

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3. Download the Medium High Pierce Standard pdAnalysis and the Pierce Intact Standard Proteoform Database.

 

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4. Follow the steps in this video to install the Proteoform Database for Pierce Intact Standards, and run the search.

 

Other ProSightPC & ProSightPD Demo Data

The following data files are available for testing various features of ProSightPC and ProSightPD. Example analyses can be found in the User’s Guide and Quick Start manual. Please see the other posts in Resources for TD standard files that can be run in ProSightPC and ProSightPD.

Files in a .zip folder:

ProSightPC Demo Data.zip

Low resolution file for ProSightPD analysis with ReSpect algorithm (will  not work properly in ProSightPC because MS1 data is not isotopically resolved):

Human_nuclear_extract_Top_Down_LTQ_FT_MS_MS.raw

Sample bottom up file:

PheATE_126kDa_Trypsin_Hi_Hi_Top3.raw

Two files with CID fragmentation data:

Ubiq_CID_857_50sc_100K_1_8559.616.raw

Ubiq_CID_857_50sc_100K_1_XT_00001_M_8559.616.raw

Using FASTA Files in ProSightPC and ProSightPD

High throughput searching is only possible if software can expect input files that are formatted in a predictable pattern. As the leader in High Throughput Top Down searching, Proteinaceous has developed its software to expect files that match the UniProt format.

Proteinaceous generally recommends the use of UniProt text files or XML files because they contain known PTM information. Using this known PTM information is what allows Proteinaceous software to perform the most sophisticated searching currently available.

Even though they do not contain nearly as much information, some users prefer to use fasta files. This is normally because they are easy to create and modify. Unfortunately, if a newly created or modified fasta file does not match the UniProt format it might not be as useable with Proteinaceous software.

The most common problem occurs when the description line does not follow the UniProt format. Fortunately, there is simple fix – reformat the description line to more closely match the UniProt format. The UniProt format includes >sp or >tr and two pipes(|). If the user changes the description line to include >sp or >tr, a unique accession for every entry and two pipes, the fasta file should work with both ProSightPC and ProSightPD.

UniProt entry:
>sp|P02144|MYG_HUMAN Myoglobin OS=Homo sapiens GN=MB PE=1 SV=2
MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASE
DLKKHGATVLTALGGILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKH
PGDFGADAQGAMNKALELFRKDMASNYKELGFQG

Example of non-conforming entry:

>MYG_HUMAN
MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASE
DLKKHGATVLTALGGILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKH
PGDFGADAQGAMNKALELFRKDMASNYKELGFQG

Example of fixed description line:
>sp|P02144|MYG_HUMAN
MGLSDGEWQLVLNVWGKVEADIPGHGQEVLIRLFKGHPETLEKFDKFKHLKSEDEMKASE
DLKKHGATVLTALGGILKKKGHHEAEIKPLAQSHATKHKIPVKYLEFISECIIQVLQSKH
PGDFGADAQGAMNKALELFRKDMASNYKELGFQG

Fixing fasta files can be challenging. If this solution does not solve the issue for you or if you are experiencing other issues please contact the development team.

Online Slide Decks

Several slide decks related to top down proteomics are available for free download. These include:

An overview of top down proteomics:

 

A short overview of ProSightPD:

 

An introduction to the ProSight World of Tools:

 

Identifying Proteins in a Top Down Standard with ProSightPD 1.1.

A simple mixture of five proteins is used by the National Resource for Translational and Developmental Proteomics to check the health of their mass spectrometers prior to running clinical samples.

The Top Down Standards mixture consists of the following proteins:

  • 1 pmol myoglobin (M5696)
  • 0.5 pmol trypsinogen (T1143)
  • 0.5 pmol carbonic anhydrase (C2624)
  • 0.1 pmol ubiquitin (U6253)
  • Superoxide dismutase – SOD1 – contaminant is present in carbonic anhydrase

An example RAW file containing data of Top Down Standard proteins can be downloaded and searched against a custom ProSightPC database to identify proteins in the standard’s mix.

A search can also be performed in ProSightPD as a quick exercise to check that ProSightPD is working correctly on your computer prior to attempting analyses of your own files.

Following these steps to do this analysis:

1: Ensure that both ProSightPC 4.1 and Proteome Discoverer 2.1 are installed on the computer on which you intend to run this exercise.

2: Download both the ProSightPD 1.1 Installer and the Quick Start Guide.

3. Follow the steps in the Quick Start Guide to install ProSightPD 1.1.

4. Download the Standards .raw file, the Proteoform Database for Top Down Standards, and the corresponding flat file.

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5. Follow the steps in this video to install the Proteoform Database for Top Down Standards, and run the search.

Learn more about our new ProSightPC and ProSightPD Bundle!

ProSightPC

Get more information and learn about purchasing our ProSightPC and ProSightPD Bundle from Thermo Scientific!

Use the ProSight Lite App

ProSightLite

Use the free ProSight Lite application available from Northwestern University for characterizing a single protein sequence. Download the software from here: http://prosightlite.northwestern.edu/.

Learn More About Top Down Proteomics

Consortium-for-Top-Down-Proteomics

Learn more about Top Down Proteomics from the Consortium for Top Down Proteomics website: http://www.topdownproteomics.org/