Fractionation and identification of metalloproteins from a marine cyanobacterium

Abstract

Trace metals are essential for the growth of marine cyanobacteria, being required for key cellular processes such as photosynthesis and respiration. Despite this, the metalloproteomes of marine cyanobacteria are at present only poorly defined. In this study, we have probed the major cobalt, iron, manganese, and nickel-binding proteins in the marine cyanobacterium Synechococcus sp. WH8102 by using two different fractionation approaches combined with peptide mass fingerprinting. For the identification of intact metalloproteins, multidimensional native chromatography was used to fractionate the proteome, followed by inorganic mass spectrometry to identify metal-enriched fractions. This approach led to the detection of nickel superoxide dismutase together with its predicted cofactor. We also explored the utility of immobilized metal affinity chromatography (IMAC) to isolate subpopulations of proteins that display affinity for a particular metal ion. We conclude that low-resolution 2D liquid chromatography is a viable fractionation technique to correlate relatively low-abundance metal ions with their few cellular destinations (e.g. Ni), but challenges remain for more abundant metals with multiple destinations such as iron. IMAC has been shown as a useful pre-fractionation technique to screen for proteins with metal-binding capacity, and may become a particularly valuable tool for the identification of metal-trafficking proteins.

Protein fractionation and detection for metalloproteomics: challenges and approaches

“…WH8102 [5] revealed the presence of a putative nickel superoxide dismutase (Ni-SOD) in the fractions with the highest nickel concentrations. An earlier approach specifically designed to detect Ni-SOD in Synechococcus sp.…”

“…WH8102 were identified by ICP-MS, and major proteins in the respective fractions were identified by 1D SDS gel electrophoresis, followed by in-gel tryptic digestion and peptide mass fingerprinting (PMF) by MALDI-MS [5]. It should be noted that each fraction after these two separation steps still contained more than ten proteins that could be visualised and/or identified by PMF, and were likely to contain many more undetectable proteins at lower abundance.…”

“…In the accompanying technical note [5] we have described our IMAC-based approach for cell lysates of Synechococcus sp. WH8102.…”

“…WH8102 [5], we chose to combine size exclusion HPLC (1st dimension; isocratic elution with 20 mmol L −1 Tris-HCl pH 8.0, 50 mmol L −1 NaCl) with anion-exchange chromatography (2nd dimension; elution using a step-wise gradient of 0.1-2 mol L −1 NaCl with 20 mmol L −1 Tris-HCl pH 8.0 buffer). This order has the advantage that the anionexchange step effectively and rapidly concentrates the protein solutions.…”

“…We also include a summary of our own experiences with method development for metalloproteomics of marine cyanobacteria; experimental and technical details of this work are reported in Ref. [5] (published in this issue of Analytical and Bioanalytical Chemistry).…”