Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Abstract

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.

Supranutritional Sodium Selenate Supplementation Delivers Selenium to the Central Nervous System: Results from a Randomized Controlled Pilot Trial in Alzheimer's Disease

“…A 20-μL injection of neat serum/CSF was resolved using a BioSEC3 150 Å, 4.6 × 300 mm size exclusion chromatography (SEC) column (Agilent Technologies) with a molecular weight range of 500 to 150,000 Da on an Agilent Technologies 1200 Series liquid chromatography (LC) system equipped with a Peltier-cooled (4°C) autosampler. A 200-mM ammonium nitrate buffer containing 10 μg/L cesium and antimony as online internal standards (see Lothian and Roberts [22]) was adjusted to pH 7.5-7.7 with 28% ammonium hydroxide and used as the isocratic mobile phase (0.4 mL/min flow rate) for all separations. The column was calibrated for molecular mass estimation using a BAnalyzed^refers to subjects who completed the protocol, where cognitive testing was completed at baseline and week 24, and where at least 1 biofluid sample was measured for selenium content standard mix of heteroatom and metal-containing proteins [23], and injections of sodium selenate (1.88 ppb) prepared in the chromatography buffer were used to estimate selenate retention time in serum and CSF samples.…”

Zinc Transporter-3 Knockout Mice Demonstrate Age-Dependent Alterations in the Metalloproteome

“…SEC-ICP-MS analysis was performed using the previously described method [15]. Whole brain tissue samples were homogenized by probe sonication (3 rounds of sonication for 15 seconds on ice, 40% amplitude) in 1 mL of homogenization buffer (Dulbecco's PBS with EDTA free proteinase inhibitor cocktails 2 and 3; 1:500; Roche) and centrifuged at 100,000× g for 30 minutes at 4 • C. The supernatant was collected, and both the pellet and supernatant were stored at −80 • C until further use.…”

Profiling changes to natively-bound metals during Caenorhabditis elegans development

“…Regardless, it is consistent with metalloprotein levels existing in a dynamic state throughout development, and also provides utility in identifying specic molecular weight ligands for further proteomic analysis as part of a more comprehensive analytical approach to metalloproteomics. 1,2,10 This may include use of subsequent orthogonal chromatography of collected mass fractions, protein identication via mass ngerprinting and use of genetic manipulation to ablate candidate metalloproteins. This latter point is particularly relevant for model system approaches using yeast, C. elegans and Drosophila melanogaster.…”

“…2 We have applied separation methods using native size exclusion chromatography (SEC) to preserve metal-protein bonds hyphenated to highly sensitive inductively coupled plasma-mass spectrometry (ICP-MS) to perform online detection of the metal status associated with specic molecular weights of biomolecules. 10 We have previously used this approach to improve the accuracy of transferrin saturation measurements in human serum, 11 prole the distribution of metalloproteins in cultured neurons and astrocytes 12 and examine major iron-binding proteins in adult C. elegans during normal biological ageing. 8 In this paper, we have applied this approach to begin to understand metalloprotein speciation in the developing C. elegans nematode.…”