Protein carbamylation: In vivo modification or in vitro artefact?

Kollipara, Laxmikanth; Zahedi, René P.

doi:10.1002/pmic.201200452

Cited by 131 publications

(104 citation statements)

References 24 publications

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“…SUMO-Aβ(1-42) was simply isolated by Ni-NTA affinity chromatography on the basis of an N-terminal hexahistidine tag, whereby the use chilled buffers was sufficient for averting risks of carbamylation modifications from urea degradation (Kollipara and Zahedi 2013). Furthermore, refolding of the fusion protein was achieved rapidly by on-resin exchange into native buffer, with no loss in yield, purity or cleavage efficiency compared with refolding through 3 days of dialysis (data not shown).…”

Section: Resultsmentioning

confidence: 97%

A routine method for cloning, expressing and purifying Aβ(1–42) for structural NMR studies

2014

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Nuclear magnetic resonance (NMR) is a key technology in the biophysicist's toolbox for gaining atomic-level insight into structure and dynamics of biomolecules. Investigation of the amyloid-β peptide (Aβ) of Alzheimer's disease is one area where NMR has proven useful, and holds even more potential. A barrier to realizing this potential, however, is the expense of the isotopically enriched peptide required for most NMR work. Whereas most biomolecular NMR studies employ biosynthetic methods as a very cost-effective means to obtain isotopically enriched biomolecules, this approach has proven less than straightforward for Aβ. Furthermore, the notorious propensity of Aβ to aggregate during purification and handling reduces yields and increases the already relatively high costs of solid phase synthesis methods. Here we report our biosynthetic and purification developments that yield pure, uniformly enriched ¹⁵N and ¹³C¹⁵N Aβ(1-42), in excess of 10 mg/L of culture media. The final HPLC-purified product was stable for long periods, which we characterize by solution-state NMR, thioflavin T assays, circular dichroism, electrospray mass spectrometry, and dynamic light scattering. These developments should facilitate further investigations into Alzheimer's disease, and perhaps misfolding diseases in general.

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Section: Resultsmentioning

confidence: 97%

A routine method for cloning, expressing and purifying Aβ(1–42) for structural NMR studies

2014

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“…Two orthogonal protein denaturation conditions were used to control for incomplete protein solubilization and denaturation and to control for chemical artifacts such as carbamylation (54). Each sample was immediately transferred to a 2 ml microcentrifuge tube (with a screw cap containing an o-ring) containing ϳ100 l of 0.1 mm zirconia/silica beads (BioSpec Products, Bartlesville, OK, the beads will cause tubes with snap-caps to leak).…”

Section: Methodsmentioning

confidence: 99%

“…These peptides were purified by HPLC (typically Ն80% pure by UV), and simultaneously quantified by UV spectrophotometry using an incorporated trypsin-cleavable carboxyterminal "JPT-Tag." Amino-terminal native flanking residues were included during peptide synthesis (length ϭ three residues) to control for trypsin digestion efficiency and chemical artifacts such as aminoterminal carbamylation (54). Carboxy-terminal native flanking residues could not be incorporated because the JPT SPOT synthesis platform only allows incorporation of heavy Arg and Lys residues if they are already chemically bound to the JPT-Tag.…”

Section: Methodsmentioning

confidence: 99%

Targeted Proteomics-Driven Computational Modeling of Macrophage S1P Chemosensing

Manes

Angermann

Koppenol-Raab

et al. 2015

Molecular & Cellular Proteomics

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Osteoclasts are monocyte-derived multinuclear cells that directly attach to and resorb bone. Sphingosine-1-phosphate (S1P) 1 regulates bone resorption by functioning as both a chemoattractant and chemorepellent of osteoclast precursors through two G-protein coupled receptors that antagonize each other in an S1P-concentration-dependent manner. To quantitatively explore the behavior of this chemosensing pathway, we applied targeted proteomics, transcriptomics, and rule-based pathway modeling using the Simmune toolset. RAW264.7 cells (a mouse monocyte/macrophage cell line) were used as model osteoclast precursors, RNA-seq was used to identify expressed target proteins, and selected reaction monitoring (SRM) mass spectrometry using internal peptide standards was used to perform absolute abundance measurements of pathway proteins. The resulting transcript and protein abundance values were strongly correlated. Measured protein abundance values, used as simulation input parameters, led to in silico pathway behavior matching in vitro measurements. Moreover, once model parameters were established, even simulated responses toward stimuli that were not used for parameterization were consistent with experimental findings. These findings demonstrate the feasibility and value of combining targeted mass spectrometry with pathway modeling for advancing biological insight. Molecular & Cellular Proteomics 14: 10.1074/mcp.M115.048918, 2661-2681, 2015.Chemotaxis is defined as directed movement of a cell (or of an organism) resulting from stimulation by a chemokine or other chemotactic chemical. Eukaryotic cells employ intricate intracellular pathways to sense concentration differences of chemoattractants at their surface and move along such gradients using multiple synchronized cellular processes, including cell protrusion and adhesion at the leading end, de-adhesion at the trailing end, and mechanical force generation at both the leading and trailing ends (1-4).Activation of chemotactic receptors through chemoattractant concentration gradients results in nonuniform intracellular signaling responses that depend on numerous feedback mechanisms (5-7). Downstream, F-actin synthesis at the leading end causes the formation of cell protrusions (for example, filopodia, lamellipodia, and lamellae) that, in concert with actomyosin contraction, generates force at both the leading and trailing ends (8 -10). Chemotactic traction is produced by cell protrusions interacting with a confined environment and/or by adhesions that bind to the extracellular matrix and/or to cell adhesion molecules (11).Chemotaxis plays a major role in a wide range of physiological and pathophysiological processes. Sphingosine-1-phosphate (S1P), a phosphosphingolipid, mediates chemotaxis of many circulating cell types, including osteoclast precursors (OPs) (12)(13)(14). Osteoclasts are monocyte-derived multinuclear cells that directly attach to the bone matrix and resorb bone. They are solely responsible for bone resorption, and their misregulated activity has been imp...

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“…Another critical modification is the carbamylation of lysine residues and protein N-termini (+43.006 Da), which is a non-enzymatic PTM that has been related to protein aging. 5 It can be artificially introduced during sample preparation using urea as the protein denaturing agent. For in-solution tryptic digest with urea in Figure 5.…”

Section: Resultsmentioning

confidence: 99%