In Theory
Analyzing a mixture of protein isoforms (proteoforms) with different combinations of PTMs, by bottom-up is usually not successful, as most of the information is lost upon trypsin digestion (Figure 1). On the contrary, in a top-down analysis, each proteoform is analyzed and fragmented individually, providing extra-information on the primary structure of the isoforms. Although early top-down analyses used collision induced dissociation (CID), alternative dissociation techniques based on electron transfer (ETD) or capture (ECD), are now providing complementary fragmentation profiles, significantly increasing protein sequence coverages. Furthermore, ETD and ECD, unlike CID, do not lead to the loss of certain labile PTMs such as phosphorylations, explaining their more and more common use in proteomics. Figure 1. The combination of post-translational modifications (green symbols), mutations (colored squares) and alternative splicing (dotted squares), can sometimes produce numerous protein isoforms of proteoforms. After digestion (bottom-up, left), it is impossible to know the origin of the trypsic peptides. In top-down (right), each proteoform is selected and fragmented individually.
In Practice
Protein mixtures (~ 2 pmol / 50ng) are desalted on C4 cartridges and separated on C4 nano-columns. The multicharged raw data (Figure 2, bottom) can be deconvoluted to obtain very accurate Molecular Weights with isotopic distributions (Figure 2, top).

Figure 2. MS spectra corresponding to a ~15 kDa protein.

For more complex mixtures, raw data can be automatically deconvoluted and 3D plots generated, showing the pattern of the most abundant proteins (Figure 3). Targeted top-down analysis can provide information on post-translational modifications (PTMs) and protein identification.

Figure 3. Separation of entire proteins after immunopurification.



Lesne J, Locard-Paulet M, Parra J, Zivković D, Menneteau T, Bousquet MP, Burlet-Schiltz O, Marcoux J. Conformational maps of human 20S proteasomes reveal PA28- and immuno-dependant inter-ring crosstalks. Nature Communications 11(1):6140.



Lesne J, Bousquet M-P, Marcoux J and Locard-Paulet M. Top-down and intact protein MS data visualization for proteoform analysis using VisioProt-MS. Bioinformatics and Biology Insights 13:1177932219868223.



Gervais V, Muller I, Mari PO, Mourcet A, Movellan K, Ramos P, Marcoux J, Guillet V, Javaid S, Burlet-Schiltz O, Czaplicki G, Milon A, Giglia-Mari G. Small molecule-based targeting of TTD-A dimerization to control TFIIH transcriptional activity represents a potential strategy for anticancer therapy. Journal of Biological Chemistry 293(39):14974-14988.


Locard-Paulet M, Parra J, Albigot R, Mouton-Barbosa E, Bardi L, Burlet-Schiltz O, Marcoux J. VisioProt-MS: interactive 2D maps from intact protein mass spectrometry. BioInformatics 35(4):679-681.



Parra J, Marcoux J, Poncin I, Canaan S, Herrmann JL, Nigou J, Burlet-Schiltz O, Rivière M. Scrutiny of Mycobacterium tuberculosis 19kDa antigen proteoforms provides new insights in the lipoglycoprotein biogenesis paradigm. Scientific Reports 7:43682.

Carel C, Marcoux J, Parra J, Réat V, Latgé G, Laval F, Burlet-Schiltz O, Demange P, Milon A, Daffé M, Tropis M, Renault M. Post-translational O-mycoloylation mediates protein targeting to the mycomembrane in C. glutamicum. Proceedings of the National Academy of Sciences 114(16) : 4231-4236.