Mapping intact protein isoforms in discovery mode using top-down proteomics

a b s t r a c tThe molecular architectures of intracellular signaling networks are largely unknown. Understanding their design principles and mechanisms of processing information is essential to grasp the molecular basis of virtually all biological processes. This is particularly challenging for human pathologies like cancers, as essentially each tumor is a unique disease with vastly deranged signaling networks. However, even in normal cells we know almost nothing. A few 'signalosomes', like the COP9 and the TCR signaling complexes have been described, but detailed structural information on their architectures is largely lacking. Similarly, many growth factor receptors, for example EGF receptor, insulin receptor and c-Met, signal via huge protein complexes built on large platform proteins (Gab, Irs/Dok, p130Cas[BCAR1], Frs families etc.), which are structurally not well understood. Subsequent higher order processing events remain even more enigmatic. We discuss here methods that can be employed to study signaling architectures, and the importance of too often neglected features like macromolecular crowding, intrinsic disorder in proteins and the sophisticated cellular infrastructures, which need to be carefully considered in order to develop a more mature understanding of cellular signal processing.

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“…Moreover, it can enable researchers to simultaneously observe thousands of protein isoforms and numerous modifications in a single sample [114].…”

Section: Some Methods For Investigating the Compositions And Architecmentioning

confidence: 99%

Beyond ‘furballs’ and ‘dumpling soups’ – towards a molecular architecture of signaling complexes and networks

2012

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“…5). In contrast to traditional bottom-up proteomics relying on trypsin digestion, the top-down MS approach consists of the analysis of entire proteins, enabling the identification of combinations of PTMs and the direct semiquantification of proteoforms (23)(24)(25). This approach is particularly relevant for the analysis of C. glutamicum porins that are rather hydrophobic with the presence of PTMs consisting of C32-C36 mycoloyl residues and that lack arginine and lysine residues for the generation of adequate tryptic peptides.…”

Section: Discussionmentioning

confidence: 99%

Identification of specific posttranslational O -mycoloylations mediating protein targeting to the mycomembrane

Carel

Marcoux

Réat

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum, a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum, we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O-mycoloylation, pyroglutamylation, and N-formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O-acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment.Corynebacteriales | O-acylation | sequence motif | top-down proteomics | NMR

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“…In contrast, when intact protein molecular ions are introduced in the MS, the strategy is called topdown (Tran et al, 2011;Catherman et al, 2014). In this case, the use of an ESI source requires coupling with a highly accurate mass analyzer for unique identification, to locate and characterize posttranslational modifications.…”

Section: Protein/peptide Analysismentioning

confidence: 99%