Quantification of human plasma metalloproteins in multiple sclerosis, ischemic stroke and healthy controls reveals an association of haptoglobin-hemoglobin complexes with age

Abstract

Advanced analytical methods play an important role in quantifying serum disease biomarkers. The problem of separating thousands of proteins can be reduced by analyzing for a ‘sub-proteome’, such as the ‘metalloproteome’, defined as all proteins that contain bound metals. We employed size exclusion chromatography (SEC) coupled to an inductively coupled plasma atomic emission spectrometer (ICP-AES) to analyze plasma from multiple sclerosis (MS) participants (n = 21), acute ischemic stroke (AIS) participants (n = 17) and healthy controls (n = 21) for Fe, Cu and Zn-metalloproteins. Using ANOVA analysis to compare the mean peak areas among the groups revealed no statistically significant differences for ceruloplasmin (p = 0.31), α2macroglobulin (p = 0.51) and transferrin (p = 0.31). However, a statistically significant difference was observed for the haptoglobin-hemoglobin (Hp-Hb) complex (p = 0.04), being driven by the difference between the control group and AIS (p = 0.012), but not with the MS group (p = 0.13), based on Dunnes test. A linear regression model for Hp-Hb complex with the groups now adjusted for age found no statistically significant differences between the groups (p = 0.95), but was suggestive for age (p = 0.057). To measure the strength of association between the Hp-Hb complex and age without possible modifications due to disease, we calculated the Spearman rank correlation in the healthy controls. The latter revealed a positive association (r = 0.39, 95% Confidence Interval = (-0.05, 0.83), which suggests that either the removal of Hp-Hb complexes from the blood circulation slows with age or that the release of Hb from red blood cells increases with age. We also observed that the Fe-peak corresponding to the Hp-Hb complex eluted ~100 s later in ~14% of all study samples, which was not correlated with age or disease diagnosis, but is consistent with the presence of the smaller Hp (1–1) isoform in 15% of the population.

Recent Advances in Metalloproteomics

“…For metal quantitation, the overwhelming majority of LC-and CE-based studies involve ICP-MS [11,25,27], with a few examples for Inductively-Coupled Plasma Optical Emission Spectroscopy (ICP-OES) [58] and Graphite Furnace Atomic Absorption Spectrometry (GFAAS) [59]. For metal quantitation for 2D-GE, at least four methods are available: Laser-ablation ICP-MS (LA-ICP-MS) [60], X-ray fluorescence (XRF) [61][62][63], application of a metallochromic dye or fluorescent sensor [64], and autoradiography of proteins labelled with a radioactive metal ion.…”

“…Several studies by the Gailer team employed 1D SEC in FPLC format of plasma and serum. Building on previous metalloproteomic work [46,100], an approach that utilises the lability of metal-protein complexes to further understand plasma metal speciation was developed [58,101]. The analytical method involves SEC (Superdex TM 200 Increase 10/300 GL, Cytiva; 150 mM PBS pH 7.4) hyphenated to ICP-OES.…”

Recent Advances of Proteomics in Management of Acute Kidney Injury

“…In this way, ‘trapped’ proteases are exempted from molecular digestion of collagens and other large proteins/peptides, preventing conformational changes of small polypeptides entering into the ‘trap’. This so called ‘selectivity’ of α 2 M protein is based on the presence of a polypeptide ‘target’ region in the chemical structure of α 2 M which became attractive for most of the proteolytic peptidases [ 96 , 97 , 98 , 99 ]. In the past, most of the studies related to α 2 M role were focused in exploring hemostasis and thrombosis as critical conditions for patients in intensive care units (ICUs) [ 100 , 101 ].…”

Atomic spectrometry update: review of advances in elemental speciation

“…A group from the University of Calgary have used SEC-ICP-OES to determine the Fe, Cu and Zn-containing metalloproteins in human plasma samples from individuals with multiple sclerosis, acute ischemic stroke and healthy controls. 133 A Superdex™ 200 Increase 10/300 GL (300 × 10 mm id, 8 μm particle size) SEC column was used with a PBS eluent at pH 7.4 at a flow rate of 0.75 mL min −1 and a column temperature of 22 °C. To estimate the molecular weight of the detected metalloprotein peaks the SEC column was calibrated by injecting a size calibration standard protein mixture and measurement of the carbon emission line at a wavelength of 193.091 nm, to monitor elution of the proteins.…”