Top-down proteomics

Bottom-up versus top-down proteomics

Top-down proteomics is a method of protein identification that uses an ion trapping mass spectrometer to store an isolated protein ion for mass measurement and tandem mass spectrometry analysis.^[1]^[2] Top-down proteomics is capable of identifying and quantitating unique proteoforms through the analysis of intact proteins.^[3] The name is derived from the similar approach to DNA sequencing.^[4] Proteins are typically ionized by electrospray ionization and trapped in a Fourier transform ion cyclotron resonance (Penning trap)^[5] or quadrupole ion trap (Paul trap) mass spectrometer. Fragmentation for tandem mass spectrometry is accomplished by electron-capture dissociation or electron-transfer dissociation. Effective fractionation is critical for sample handling before mass-spectrometry-based proteomics. Proteome analysis routinely involves digesting intact proteins followed by inferred protein identification using mass spectrometry.^[6] Top-down proteomics interrogates protein structure through measurement of an intact mass followed by direct ion dissociation in the gas phase.^[7]

Currently the most advanced instrument used to map proteoforms is the Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer. This high-performance instrument is able to give a quantitative analysis of proteoforms, identify them, and deeply characterize them with tandem MS/MS.^[8]

Advantages

The main advantages of the top-down approach include the ability to detect degradation products, sequence variants, and combinations of post-translational modifications.^[9]
In Top-down proteomics, researchers can feed intact full proteins directly into mass spectrometers, and are able to capture the distinct characteristics of each one-of-a-kind proteoform. In addition, Top-down proteomics allows for analysis of an entire protein molecule without digestion and also allows for low mass protein detection.^[8]
Top-down proteomics, when accompanied with polyacrylamide gel electrophoresis, can help to complement the bottom-up proteomic approach. Top-down proteomic methods can assist in exposing large deviations from predictions and has been very successfully pursued by combining Gel Elution Liquid-based Fractionation Entrapment Electrophoresis fractionation, protein precipitation, and reverse phase HPLC with electrospray ionization and MS/MS.^[10]
Characterization of small proteins represents a significant challenge for bottom up proteomics due to the inability to generate sufficient tryptic peptides for analysis. Top-down proteomics allows for low mass protein detection, thus increasing the repertoire of proteins known.^[11] While Bottom-up proteomics integrates cleaved products from all proteoforms produced by a gene into a single peptide map of the full-length gene product to tabulate and quantify expressed proteins, a major strength of Top-down proteomics is that it enables researchers to quantitatively track one or more proteoforms from multiple samples and to excise these proteoforms for chemical analysis.^[10]

Disadvantages

In the recent past, the top down approach was relegated to analysis of individual proteins or simple mixtures, while complex mixtures and proteins were analyzed by more established methods such as Bottom-up proteomics. Additionally protein identification and proteoform characterization in the TDP (Top-down proteomics) approach suffer from a dynamic range challenge where the same highly abundant species are repeatedly fragmented.^[3]
Although Top-down proteomics can be operated in relatively high output in order to successfully map proteome coverage at a large level, the rate of identifying new proteins after initial rounds reduces quite sharply.^[3]
Top-down proteomics interrogation can overcome problems for identifying individual proteins, but has not been achieved on a large scale due to a lack of intact protein fractionation methods that are integrated with tandem mass spectrometry.^[6]