Protein fractionation and detection for metalloproteomics: challenges and approaches

Barnett, James P.; Scanlan, David J.; Blindauer, Claudia A.

doi:10.1007/s00216-012-5743-y

Cited by 62 publications

(51 citation statements)

References 116 publications

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“…A list of identified proteins in these selected fractions is given in Table 1. It is clear that one or more of these proteins may be the source of the measured metal ions and this is discussed in more detail in the accompanying critical review [16]. For the present discussion, of particular note is the identification of a putative nickel superoxide dismutase in the nickel containing fractions, consistent with the suggestion that this protein contains a nickel cofactor in Synechococcus sp.…”

Section: Resultssupporting

confidence: 52%

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Fractionation and identification of metalloproteins from a marine cyanobacterium

2012

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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.

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Section: Resultssupporting

confidence: 52%

“…Several other identified proteins (Table 2) were not predicted to bind metal ions, giving rise to further investigations as discussed in detail in the accompanying critical review [16].…”

Section: Resultsmentioning

confidence: 97%

Fractionation and identification of metalloproteins from a marine cyanobacterium

2012

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“…The value of natural products to drug discovery is still considerable, however, the protein target(s) of action of these fascinating compounds are largely unknown. Recently, MS-based proteomics has the potential to identify novel proteins that are related to the action mechanisms of natural products (Barnett et al, 2012;Schrattenholz et al, 2010;Zhang et al, 2013). Various types of biological networks are being constructed, including protein-protein interaction networks, and protein regulatory networks, to help drug discovery (Abu-Asab et al, 2011;Ge et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

Proteomics Analysis of Hepatoprotective Effects for Scoparone Using MALDI-TOF/TOF Mass Spectrometry with Bioinformatics

Zhang

Sun

et al. 2013

OMICS: A Journal of Integrative Biology

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Scoparone is an active ingredient of Yinchenhao (Artemisia annua L.), a well-known Chinese medicinal plant, and has been utilized in prevention and therapy of liver damage. However, the molecular drug targets associated with the pharmacological effects of scoparone are largely unknown. In the present article, we extend the previous research on Yinchenhao through a study of its active ingredient and thus the putative targets of scoparone. We employed two-dimensional gel electrophoresis, and all proteins expressed were identified by MALDI-TOF/TOF MS and database research. Protein-interacting networks and pathways were also mapped and evaluated. The possible protein network associated with scoparone was constructed, and contribution of these proteins to the protective effect of scoparone against the carbon tetrachloride-induced acute liver injury in rats are discussed herein. Hepatoprotective effects of scoparone on liver injury in rats were associated with regulated expression of six proteins which were closely related in our protein-protein interaction network, and appear to be involved in antioxidation and signal transduction, energy production, immunity, metabolism, and chaperoning. These observations collectively provide new insights on the molecular mechanisms of scoparone action against hepatic damage in rats.

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“…Although some metalloenzymes have been characterized, the majority still remain uncharacterized or unknown 6 . One of the major challenges in measuring metalloproteins is the requirement to maintain the native state of the protein 7 . Classical bottom-up proteomic techniques rely on the digestion of the proteins into peptides.…”

Section: Introductionmentioning

confidence: 99%

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Lothian

Roberts

2016

JoVE

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Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma -mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

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Protein fractionation and detection for metalloproteomics: challenges and approaches

Cited by 62 publications

References 116 publications

Fractionation and identification of metalloproteins from a marine cyanobacterium

Fractionation and identification of metalloproteins from a marine cyanobacterium

Proteomics Analysis of Hepatoprotective Effects for Scoparone Using MALDI-TOF/TOF Mass Spectrometry with Bioinformatics

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

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