Decoding protein modifications using top-down mass spectrometry

Abstract

Top-down mass spectrometry is an emerging technology which strives to preserve the post-translationally modified forms of proteins present in vivo by measuring them intact, rather than measuring peptides produced from them by proteolysis. The top-down technology is beginning to capture the interest of biologists and mass spectrometrists alike, with a main goal of deciphering interaction networks operative in cellular pathways. Here we outline recent approaches and applications of top-down mass spectrometry as well as an outlook for its future.

Figure 1

The use of top-down MS for PTM detection: top-down characterization of a hypothetical protein and its PTMs from different cell states. (a) The protein is purified from asynchronous cells and subjected to intact MS analysis (blue trace) followed by MS/MS of individual protein forms (pink trace) to pinpoint PTM location (arrow). (b, c) The same protein target is purified from cell states of interest (for example, apoptosis (b) or mitosis (c)) and subjected to intact MS and MS/MS analysis for PTM determination. Ac, acetyl group; Me, methyl group (+14 Da); P_i, phosphate group (+80 Da).

Figure 2

Classical versus electron-based methods for fragmentation of protein and peptide ions in tandem mass spectrometry. (a) Most PTMs are stable during top-down fragmentation of proteins by either classical or electron-based MS/MS methods, which typically create b- and y- type or c- and z^•-type ions, respectively. (b) During classical fragmentation of small peptides generated during bottom-up analysis, some PTMs such as phosphorylation are not always stable, whereas in electron-based MS/MS methods they are. Ac, acetyl group; Me, methyl group; P_i, phosphate group.

Figure 3

The complexities of precisely characterizing eukaryotic proteins. (a) Top-down MS can distinguish between protein isoforms 1 and 2 (expressed from genes 1 and 2) with highly similar intact mass values based on differences in molecular mass and MS/MS fragmentation patterns. Protein forms 2 and 3 arise from alternative splicing of a single gene. The green lines indicate where MS/MS fragmentation is occurring. (b) Bottom-up techniques generate a mixture of peptides produced by proteolysis, making it difficult to determine to which protein each detectable peptide (red and blue) belongs.