Unbiased Selective Isolation of Protein N-terminal Peptides from Complex Proteome Samples Using Phospho Tagging (PTAG) and TiO2-based Depletion

Dysregulated proteolysis represents a hallmark of numerous diseases. In recent years, increasing number of studies has begun looking at the protein termini in hope to unveil the physiological and pathological functions of proteases in clinical research. However, the availability of cryopreserved tissue specimens is often limited. Alternatively, formalin-fixed, paraffin-embedded (FFPE) tissues offer an invaluable resource for clinical research. Pathologically relevant tissues are often stored as FFPE, which represent the most abundant resource of archived human specimens. In this study, we established a robust workflow to investigate native and protease-generated protein N termini from FFPE specimens. We demonstrate comparable N-terminomes of cryopreserved and formalin-fixed tissue, thereby showing that formalin fixation/paraffin embedment does not proteolytically damage proteins. Accordingly, FFPE specimens are fully amenable to N-terminal analysis. Moreover, we demonstrate feasibility of FFPE-degradomics in a quantitative N-terminomic study of FFPE liver specimens from cathepsin L deficient or wild-type mice. Using a machine learning approach in combination with the previously determined cathepsin L specificity, we successfully identify a number of potential cathepsin L cleavage sites. Our study establishes FFPE specimens as a valuable alternative to cryopreserved tissues for degradomic studies. Molecular & Cellular Proteomics 15:

Section: Enrichment Of N-terminal Peptides From Ffpe Specimens-supporting

confidence: 55%

Formalin-Fixed, Paraffin-Embedded Tissues (FFPE) as a Robust Source for the Profiling of Native and Protease-Generated Protein Amino Termini

Lai

Weißer

Nilse

et al. 2016

“…Many will have suboptimal lengths or poor physiochemical properties for fragmentation and/or ionization. Several groups employ dimethylation or acetylation of ␣-and -amines prior to digestion to increase the lengths of Nand C-terminal peptides (35,37,40,47,49). This results in trypsin cleaving with ArgC specificity.…”

Section: Methods For Enriching Protein N Termini-n-terminalmentioning

confidence: 99%

“…The unbound N-terminal peptides enter the mass spectrometer directly, minimizing handling and consequent losses. Recently, Mommen et al developed a technique very similar to TAILS, except that instead of the hyperbranched polyglycerol aldehyde polymer, internal ␣-amines are phosphorylated via treatment with glyceraldehyde-3-phosphate and depleted via titanium dioxide chromatography (40).…”

Section: Methods For Enriching Protein N Termini-n-terminalmentioning

confidence: 99%

Proteolytic Post-translational Modification of Proteins: Proteomic Tools and Methodology

Rogers

Overall²

2013

140

Proteolytic processing is a ubiquitous and irreversible post-translational modification involving limited and highly specific hydrolysis of peptide and isopeptide bonds of a protein by a protease. Cleavage generates shorter protein chains displaying neo-N and -C termini, often with new or modified biological activities. Within the past decade, degradomics and terminomics have emerged as significant proteomics subfields dedicated to characterizing proteolysis products as well as natural protein N and C termini. Here we provide an overview of contemporary proteomics-based methods, including specific quantitation, data analysis, and curation considerations, and highlight exciting new and emerging applications within these fields enabling in vivo analysis of proteolytic events. Proteolysis involves the breakdown of proteins into smaller polypeptides or amino acids through the hydrolysis of peptide bonds by a protease. This represents a remarkably significant, but often underappreciated, post-translational modification (PTM) 1 in that is it irreversible yet also ubiquitous. Consequently, the functional sequence of a protein can very rarely be predicted from its transcript, as proteolysis products form new (neo-) N and C termini. These cleavage events, or proteolytic processing events, can result in activation, inactivation, completely altered protein function, and even excision of "neo-proteins" with growth factor activity from an extracellular matrix parent molecule, and they regulate a vast array of biological processes (1). These include DNA replication, cell cycle progression, cell proliferation, and cell death, as well as pathological processes such as inflammation, cancer, arthritis, and cardiovascular disease. For example, in protein synthesis and maturation, precise selective removal of the N-terminal methionine and the signal peptide is essential for correct protein maturation and secretion. In some proteins, scission of the chain forms a molecule with four termini when linked by disulfide bridges. Through the removal of signal, nuclear, and mitochondrial localization sequences and ectodomain shedding, proteases regulate protein localization, and in viral infection, via cleavage of pre-and pro-domains and polyprotein processing, inactive proteins are converted into their active form(s), are inactivated, or change receptor-binding affinity. Thus, proteolysis is involved in much more than the mere degradation and turnover of proteins, important though these processes are in homeostasis.Proteases exist in all orders of life and constitute one of the largest enzyme families in humans (2), and more than 30 drugs targeting these enzymes are currently approved for clinical use (3). However, in order to fully comprehend the cellular function(s) of a given protease, one must have knowledge of the proteins processed by that protease, as well as the functions of these substrates and specific processing events. This is currently far from the case, as half of all human proteases have no known substrates (4). Degradomic...

“…Quantitative proteomics have the advantage to detect phospho-peptides, sumoylated-peptides, ubiquitinated-peptides, and palmitoylated-peptides to name a few (134,135). As more researchers employ these techniques, greater understanding of post-translational modifications related to disease will arise.…”

Section: Importance Of Proteomics In Identifying Mechanismsmentioning

confidence: 99%

Molecular and Cellular Mechanisms of Axonal Regeneration After Spinal Cord Injury

Niekerk

Tuszynski

et al. 2016

Following axotomy, a complex temporal and spatial coordination of molecular events enables regeneration of the peripheral nerve. In contrast, multiple intrinsic and extrinsic factors contribute to the general failure of axonal regeneration in the central nervous system. In this review, we examine the current understanding of differences in protein expression and post-translational modifications, activation of signaling networks, and environmental cues that may underlie the divergent regenerative capacity of central and peripheral axons. We also highlight key experimental strategies to enhance axonal regeneration via modulation of intraneuronal signaling networks and the extracellular milieu. Finally, we explore potential applications of proteomics to fill gaps in the current understanding of molecular mechanisms underlying regeneration, and to provide insight into the development of more effective approaches to promote axonal regeneration following injury to the nervous system. Molecular & Cellular Proteomics