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The main advantages of the top-down approach include the ability to detect degradation products, sequence variants, and combinations of post-translational modifications.
Complex research endeavors such as the investigation of cell signaling pathways, disease state characterization, and biomarker discovery have all benefited from advances in mass spectrometry. Such pursuits often employ mass spectrometry-based bottom-up protein identification techniques. These same research endeavors would, in many cases, also benefit from high-throughput top-down mass spectrometric analyses. The main advantages of the top-down approach include the ability to detect degradation products, sequence variants, and combinations of post-translational modifications. In a Nature publication by Neil Kelleher and co-workers, over one thousand unique gene products were identified from human cells in a single experiment. This accounted for over three thousand differentially modified species. High-resolution mass spectrometry is not only essential to resolve co-eluting intact proteins but also to resolve the isotopic peaks of the highly charged large molecules for charge state determination and accurate mass determination. Additionally, due to the number of product ions generated during fragmentation of intact proteins, high-resolution mass analysis is required for accurate detection and assignment of product ions in the resultant complex MS/MS spectra.
For additional resources, search the Orbitrap Science Library
Overview
Workflow Overview for Top-Down Proteomics
High throughput top down proteomics involves identifying proteins in complex mixtures without prior digestion into their corresponding peptide species. For high throughput protein top down characterization, scientists can extract the proteins from their sample, fractionate using a variety of techniques including GELFREE™ fractionation by molecular weight or immunoprecipitation, and then analyze the resulting fractions by high resolution, accurate mass MS/MS on the Orbitrap-based platforms with online chromatography using data dependent acquisition methods with CID, HCD, and ETD fragmentation. The key benefits to high throughput top down analysis of complex mixtures are the ability to detect degradation products, sequence variants, and combinations of post-translational modifications.
NEW WEBSITE: The Consortium for Top Down Proteomics
LITERATURE HIGHLIGHT
Mapping intact protein isoforms in discovery mode using top-down proteomics
Tran JC, Zamdborg L, et al.
Nature. 2011 Oct 30;480(7376):254-8.
Sample Preparation
Sample Preparation Workflow for Top-Down Proteomics
Proper sample preparation is essential to the success of any high-throughput top-down workflow. Mass spectra for top-down proteomics samples have unique complexity challenges when compared to bottom-up peptide analyses. Each intact protein has a significantly higher number of charge states and isotopic peaks as well as more coeluting modified forms. This complicates detecting the spectra, particularly when multiple proteins elute at the same time. For these reasons, sample fractionation is necessary to improve the identification of intact proteins in complex samples.
Protein lysates of tissues or cells can be simplified using a variety of methods, including GELFREE™ fractionation which separates proteins according to molecular mass at high resolution in the solution phase (1). Removal of salts (before GELFREE fractionation) and detergents (following fractionation) prior to MS analysis will reduce both ion suppression and spectral interference resulting in improved data quality. The easy-to-use Thermo Scientific Pierce Zeba Spin Desalting Columns and Detergent Removal Spin Columns can quickly clean up samples with minimal protein loss (2). Alternatively, GELFREE fractions can be cleaned using a classical precipitation protocol (3).
References
Tran JC, Doucette AA.
Anal Chem. 2008 Mar 1;80(5):1568-73.
2. Efficient removal of detergents from proteins and peptides in a spin column format
Antharavally BS, Mallia KA, et al.
Anal Biochem. 2011 Sep 1;416(1):39-44.
Wessel D, Flügge UI.
Anal Biochem. 1984 Apr;138(1):141-3.
Additional Resources
NEW WEBSITE: The Consortium for Top Down Proteomics
Mapping intact protein isoforms in discovery mode using top-down proteomics
Tran JC, Zamdborg L, et al.
Nature. 2011 Oct 30;480(7376):254-8.
Eliuk S, Kellie J, et al.
Eliuk S.
Multiplexed size separation of intact proteins in solution phase for mass spectrometry
Tran JC, Doucette AA.
Anal Chem. 2009 Aug 1;81(15):6201-9.
Related Products
A Comprehensive Mass Spectrometry Sample Preparation Handbook
Zeba Spin Desalting Columns
Detergent Removal Spin Columns
Mass Spectrometry
Mass Spectrometry Workflow for Top-Down Proteomics
High-throughput top-down analyses are best performed on the hybrid Orbitrap mass spectrometers. The complex samples benefit from the highest available resolution, fast scan speeds, and multiple fragmentation techniques offered by these systems. With these benefits in mind, here are a few tips for creating Orbitrap-based acquisition methods (see downloadable methods for more details).
- With electrospray ionization, there are typically many different charge states per intact protein. A standard data-dependent method spends much time fragmenting the alternate charge states of the same protein. For intact protein mixture analyses, Orbitrap users can create a unique acquisition method that only selects a single, most-intense charge state per protein for MS/MS. This decreases redundant identifications and permits the sample to be probed deeper.
- Proteins fragment differently with each type of fragmentation. In some cases CID will offer more complete sequence coverage and in others ETD will be optimal. For data-dependent experiments, consecutive runs with differing fragmentation modes generally provide more information than replicate runs with a single fragmentation mode. Multiple fragmentation modes will maximize both the number of identifications and the fragment ion coverage over the length of the sequence, which assists in localization of sequence polymorphisms and post-translational modifications.
- For the analysis of samples fractionated by molecular weight (e.g. GELFREE fractionation), MS parameters such as resolution and number of microscans should be optimized based on the mass range of the fraction. This increases the quality of data and maximizes the number of spectra acquired.
Resources
Mapping intact protein isoforms in discovery mode using top-down proteomics
Tran JC, Zamdborg L, et al.
Nature. 2011 Oct 30;480(7376):254-8.
POSTER: Identification and characterization of intact proteins in complex mixtures using online frag...
ACCOMPANYING VIDEO POSTER
Eliuk S, Kellie J, et al.
Second T, Zabrouskov V, Makarov A.
Application Note 498
PRODUCTS
Orbitrap Elite
Orbitrap Velos Pro
LTQ Orbitrap XL
Q Exactive
Thermo Scientific provides UPLC/HPLC systems that perform at low nano, micro, and high flow rate regimes to meet a wide variety of experimental needs. Thermo Scientific EASY-nLC and Dionex UltiMate® 3000 RSLCnano LC systems use split-free designs to achieve exceptional stability and reproducibility and they easily couple to all Thermo Scientific mass spectrometers.
Data Analysis
Data Analysis Workflow for Top-Down Proteomics
Identification of intact proteins in complex mixtures faces a variety of challenges.
- The fragmentation of large molecules such as proteins creates complex spectra due to the number of fragment ions produced, each often present at multiple charge states.
- Co-isolation and fragmentation of multiple proteins detected in a single spectrum is very common, particularly for complex samples.
- A variety of processes including post-translational modifications, cleavages, disulphide bonds, single-nucleotide polymorphisms, all lead to a change in the precursor mass as well as complicating the fragment ion spectra.
- Low-abundance precursor ions may produce only a limited number of detectable fragment ions. Increasing the number of MS/MS microscans helps to improve the quality of spectra from low-abundance precursors.
All of these challenges can be met using the new Thermo Scientific ProsightPC 2.0 software (1). It is the first stand-alone software for the identification of intact proteins using high-resolution, accurate-mass MS and MS/MS data. It can process data generated using multiple fragmentation techniques including CID, HCD, and ETD. It is also the only proteomics software that allows the user to search their tandem MS data against proteome warehouses containing the known biological complexity present in UniProt.
For more information on ProSightPC 2.0 software, please visit the Thermo Scientific Proteomics Software Portal.
References
1. Web and database software for identification of intact proteins using "top down" mass spectrometry
Taylor GK, Kim YB, et al.
Anal Chem. 2003 Aug 15;75(16):4081-6.
Related Resources
Mapping intact protein isoforms in discovery mode using top-down proteomics
Tran JC, Zamdborg L, et al.
Nature. 2011 Oct 30;480(7376):254-8.
POSTER: Identification and characterization of intact proteins in complex mixtures using online frag...
ACCOMPANYING VIDEO POSTER
Eliuk S, Kellie J, et al.
Macek B, Waanders LF, et al.
Mol Cell Proteomics. 2006 May;5(5):949-58.
Second T, Zabrouskov V, Makarov A.
Application Note 498
LINKS
Read more about ProSightPC software for precision proteomics
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