Top Down proteomics: facts and perspectives

Abstract

The rise of the "Top Down" method in the field of mass spectrometry-based proteomics has ushered in a new age of promise and challenge for the characterization and identification of proteins. Injecting intact proteins into the mass spectrometer allows for better characterization of post-translational modifications and avoids several of the serious "inference" problems associated with peptide-based proteomics. However, successful implementation of a Top Down approach to endogenous or other biologically relevant samples often requires the use of one or more forms of separation prior to mass spectrometric analysis, which have only begun to mature for whole protein MS. Recent advances in instrumentation have been used in conjunction with new ion fragmentation using photons and electrons that allow for better (and often complete) protein characterization on cases simply not tractable even just a few years ago. Finally, the use of native electrospray mass spectrometry has shown great promise for the identification and characterization of whole protein complexes in the 100 kDa to 1 MDa regime, with prospects for complete compositional analysis for endogenous protein assemblies a viable goal over the coming few years.

Fig. 1

Comparison of Top Down and Bottom Up mass spectrometry [3]. In the traditional Bottom Up approach, intact proteins are digested into peptides prior to introduction into the mass spectrometer where they are then detected and fragmented. In Top Down mass spectrometry, the protein is ionized directly, allowing for improved sequence coverage and detection of PTMs.

Fig. 2

Diagram of the GELFrEE device [46]. A gel column is utilized to achieve electrophoretic separation of proteins, analogous to SDS-PAGE, which are then eluted into the liquid-phase for manual collection. The fractionation can then be visualized by running a portion of the fractions on a SDS-PAGE gel.

Fig. 3

Comparison of collisional dissociation (CID/HCD) and electron-based dissociation (ECD/ETD) of peptides and proteins. Collisional dissociation results in cleavage of the amide-bond, resulting in b- and y-type ions. Electron-based methods cleave of the N-C_α bond, resulting in c- and z^•- type ions.

Fig. 4

Search modes utilized within ProSightPC. Absolute mass searching (a) uses an intact mass window to generate candidates, whose theoretical fragments are compared within experimental data. The hash marks indicate a matching fragment ion within a set fragment tolerance. In this case the experimental data was matched to a proteoform within the database featuring a cleaved initial methionine with 131.20 Da mass shift (outlined and boxed in orange). In sequence tag searching (b), a tag (green and boxed in orange) is generated from fragmentation data which is then used to search for candidates within the database displaying the same tag. Biomarker searching (c) is similar to absolute mass searching, but searches all possible sequences of proteins in the database, in this case resulting in identification of a proteoform containing a large truncation of the N-terminus (outlined and boxed in orange).

Fig. 5

The charge state distribution of electrosprayed ubiquitin ions from denaturing (top) and native (bottom) solution conditions.