Profiling changes to natively-bound metals during Caenorhabditis elegans development

Abstract

Relatively little is known about the changing metalloproteome during early development. In this proof-of-concept study, we used size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) to examine the changing soluble metal-binding protein profiles for iron, copper and zinc during the development of the nematode, . Samples of eggs, larval stages and young adults were compared using an approach selected to ensure weak metal-ligand bonds were maintained. All three metals showed marked changes in associated proteins and total metal levels per protein mass, and the pattern of this change was unique to each metal. Additionally, to characterise the shifting metabolic needs throughout each life stage we examined changing levels of phosphorus in each developmental stage. The utility of this method can be further exploited through integration with existing proteomics workflows to identify and track the changes in metal-containing proteins during key stages of development.

Changes in ferrous iron and glutathione promote ferroptosis and frailty in aging Caenorhabditis elegans

“…Size exclusion chromatography-inductively coupled plasma-mass spectrometry was performed using an Agilent Technologies 1100 Series liquid chromatography system with a BioSEC 5 s column (5 μm particle size, 300 Å pore size, I.D. 4.6 mm, Agilent Technologies) and 7700x Series ICP-MS as previously described ( Hare et al, 2016b ). A buffer of 200 mM NH 4 NO 3 was used for all separations at a flow rate of 0.4 mL min −1 .…”

Recent Advances in Metalloproteomics

“…The nematode Caenorhabditis elegans is frequently used as a model organism in developmental biology. One SEC-ICP-MS-based study explored the changing metalloproteomic profiles for Fe, Cu, and Zn in eggs, four larval stages, and young adult worms [141]. Soluble metalloproteins were extracted using Tris-buffered saline containing an EDTA-free protease inhibitor cocktail.…”

Standardization approaches in absolute quantitative proteomics with mass spectrometry

“…Furthermore, the knowledge of the fixed metal/protein stoichiometry enables one to obtain information of protein quantities from the quantitative detection of the metallic heteroatom. Even though ICP‐MS‐based quantification of metalloproteins is more based on the use of isotope dilution (see section 3.2), it is possible to determine the quantity of the metallic heteroatom bound to the protein by means of external calibrations of known concentrations of metalloproteins . These studies are limited, nonetheless, by the stability and strength of the protein‐heteroatom union through the analytical procedure and measurement, because metal‐protein complex instability (especially during the LC separation step) can ruin the quantitative results .…”

“…69 In this regard, ICP-MS can be applied to Even though ICP-MS-based quantification of metalloproteins is more based on the use of isotope dilution (see section 3.2), it is possible to determine the quantity of the metallic heteroatom bound to the protein by means of external calibrations of known concentrations of metalloproteins. 73,74 These studies are limited, nonetheless, by the stability and strength of the protein-heteroatom union through the analytical procedure and measurement, because metal-protein | 721 complex instability (especially during the LC separation step) can ruin the quantitative results. 76 Consequently, the possibility to precisely detect heteroatoms covalently joined to the protein structure is of high interest.…”