Top-down Sequencing

A view from above

Many bottom-up proteomics research endeavors also benefit from high-throughput top-down mass spectrometry analysis. The main advantages of the top-down approach include detection of degradation products and sequence variants, which helps to solve inference issues (e.g., isoforms, proteoforms), and determination of post-translational modifications (PTMs) for protein stoichiometry and dynamics analysis.

High-resolution accurate mass (HRAM) MS is essential for top-down proteomics. HRAM MS resolves both co-eluting intact proteins as well as isotopic peaks of highly charged, large intact proteins, enabling both charge state and accurate mass determination. Additionally, due to the number of product ions generated during intact protein fragmentation, high-resolution mass analysis is required for accurate detection and assignment of product ion identities to the resulting, highly complex MS/MS spectra.

In addition to HRAM MS, novel high dynamic (HD) range electron transfer dissociation (ETD) allows fragmentation of a larger population of precursor ions, increasing detection limits and dynamic range. The higher efficiency of ETD HD experiments provides greater sequence coverage at faster acquisition rates, offering the possibility of new ETD experiments on a liquid chromatography (LC) time scale.

Top-down sequencing solutions

Reduce sample complexity

Successful top-down proteomics requires reduced sample complexity. Resolution of proteins from one another and from various proteoforms increases the probability of successful characterization by mass spectrometry. Thermo Scientific ProSwift RP-4H LC Columns are ideal for intact protein separation prior to MS analysis and are designed for microliter to nanoliter flow.

Achieve ultra high resolution of multiple proteins

Top-down proteomics challenges spectral detection because each intact protein has a higher number of charge states, isotopic peaks, and co-eluting modified forms than bottom-up peptides. Thermo Scientific UltiMate 3000 HPLC System and RSLCnano System, along with featured Thermo Scientific ProFlow technology, enable ultra high intact protein resolution in top-down workflows even when multiple proteins co-elute. These systems are designed for milliliter to nanoliter flow separations and easily integrate into MS systems for optimal resolution, sensitivity, and selectivity.

Obtain fast precursor isolation and fragment detection

Ultra high-resolution and accurate mass are critical for successful top-down analysis. The Thermo Scientific Orbitrap Fusion Lumos Tribrid Mass Spectrometer not only offers the highest resolving power of all Orbitrap-based instruments (up to 500,000 at m/z 200), but the high selectivity of its Advanced Quadrupole Technology also enables fast precursor isolation and fragment detection. Different fragmentation techniques (CID, HCD, ETD, and EThcD) during MSn analysis runs increase sequence coverage, delivering the highest quality data for protein isoform and PTM characterization.

Analyze and characterize proteomics data

Analyze top- and middle-down proteomics data and characterize PTMs with Thermo Scientific ProSightPC Software. This software processes results from multiple fragmentation techniques (ETD, EThcD, HCD, and CID) and HRAM MS/MS of top- or middle-down experiments. It is the only proteomics software that enables comparison of MS/MS data against proteome warehouses such as UniProt. Five different search modes help determine exact protein sequence plus PTMs and splicing. ProSightPC is available as a node within Thermo Scientific Proteome Discoverer Software.

Top-down sequencing workflow