The emerging process of Top Down mass spectrometry for protein analysis: biomarkers, protein-therapeutics, and achieving high throughput

Abstract

Top Down mass spectrometry (MS) has emerged as an alternative to common Bottom Up strategies for protein analysis. In the Top Down approach, intact proteins are fragmented directly in the mass spectrometer to achieve both protein identification and characterization, even capturing information on combinatorial post-translational modifications. Just in the past two years, Top Down MS has seen incremental advances in instrumentation and dedicated software, and has also experienced a major boost from refined separations of whole proteins in complex mixtures that have both high recovery and reproducibility. Combined with steadily advancing commercial MS instrumentation and data processing, a high-throughput workflow covering intact proteins and polypeptides up to 70 kDa is directly visible in the near future.

Fig. 1

Schematic of the Bottom Up and Top Down approaches to protein identification. In the Bottom Up approach (A), enzymatic digestion is utilized to cleave intact proteins into peptides. Peptides are analyzed through tandem mass spectrometry, and protein identification can occur through identifying peptides matched in silico to a protein database by a search algorithm. Although low sequence coverages are typical, very high proteome coverages are common. Alternatively, in the Top Down approach (B), intact proteins are directly analyzed in the mass spectrometer (no enzymatic digestion). The resulting precursor and fragment masses are then matched to candidate sequences from a protein database. This enables the potential for full localization and characterization of post-translational modifications.

Fig. 2

Example work flow for Top Down proteomics. Total protein content from HeLa-S3 nuclei or cytosol is quantified and loaded onto a GELFrEE column. The GELFrEE device separates the protein samples according to molecular weight. Proteins of increasing molecular weight elute into solution-phase fractions, which can be visualized on a slab gel (top right). The solution-phase fractions are cleaned up to remove SDS before injection onto a µRPLC column for MS/MS. LC-MS/MS files are processed with ProSightPC 2.0, a software suite tailored for Top Down analysis in a high-throughput setting.

Fig. 3

Examples selected from an LC-MS/MS injection of fraction #3 from a GELFrEE run. A base-peak chromatogram is shown (A), with charge state distributions from selected retention times shown in B. In panel C, abundant charge states (above the arrows) were targeted for fragmentation. Fragmentation mass spectra for each protein are shown along with the corresponding identifications and E-values. A fragmentation map (D) results from the matching fragment ions of nucleoside diphosphate kinase B found in HeLa cells. The protein is N-terminally acetylated.

Fig. 4

Schematic of the Top Down identification process. From the intact mass and fragmentation data (top), three different search algorithms can identify and characterize the observed protein form. These algorithms function either by matching mass values from intact proteins (absolute mass), a subset of larger sequence (biomarker), or a series of unique amino acids (sequence tag) to a database sequence. In this depiction, the power of Top Down for identification of post-translational modifications is indicated by the phosphorylation (blue) and methylation (red) on this hypothetical protein.

Fig. 5

New LC-MS visualization software displays a map of masses detected as a function of LC retention time. Confocal microscopy images of live HeLa cells expressing markers for chromatin (red, histone 2B fused to red fluorescent protein) and plasma membrane (green, myristoylated/palmitoylated GFP) are shown (A). In panel B, two visualizations are shown for GELFrEE fractions of similar molecular weight from unsynchronized interphase and M-Phase-arrested HeLa cells. Total-ion chromatogram traces are overlaid in each map. Post-translational modifications (phosphorylations, shown in red) are detected (C) and highlighted in panel B (inset).